Solubilizing apirase, method and use

Abstract

The present invention provides pharmaceutical compositions, therapeutic uses, and methods useful for designing and therapeutically using solubilized apirase polypeptides, and for preventing and treating tissue damage. [Solution] A solubilized human apirase is provided having at least two modifications selected from a list consisting of N-terminal deletion, C-terminal deletion, and central modification.

Description

[Technical Field]

[0001] The present invention relates to the design and therapeutic use of solubilized apirase polypeptides, the prevention of tissue damage and This relates to pharmaceutical compositions and methods useful for treatment. [Background technology]

[0002] Apirase (ATP-diphosphatase, adenosine diphosphatase, ADPase) Or ATP diphosphohydrolase) is a nucleotide diphosphate and triphosphate (NDP and These are plasma membrane-binding enzymes with enzymatic activity against both NTPs and NDPs, with NDP acting as an intermediate. In the individual sequential phosphate release steps, NTPs are added to monophosphate nucleotides (NMPs). It breaks down water. It is present on the cell surface and hydrolyzes extracellular nucleotides (ecto-ATPas). Most of the enzymes e belong to this family. These enzymes convert both ATP and ADP into water. In terms of how it breaks down ATP, it differs from ATPases, which specifically hydrolyze ATP.

[0003] As surface antigen classification 39 (CD39, UniProt P49961 or SEQ ID NO: 1) Also known as the first known human apirase, ectonucleoside triphosphate diphosphate Hydrohydrolase-1 (gene: ENTPD1, ​​protein: NTPDase1) is a fine-grained enzyme. It is an enzyme localized on the cell surface and has a catalytic site that faces outward.

[0004] Among the known human CD39 family, member CD39L3 is related to CD39 and CD39. Ectoapirase (ecto ATPase) has biochemical activity with L1 (ecto ATPase). Known as ATPDase, specifically human CD39L3 is used for therapeutic purposes, for example. For example, in U.S. Patent No. 7,247,300B1 (which is incorporated herein by reference) As disclosed, or as included herein as Sequence ID No. 3, solubilized It is refined. [Overview of the project] [Means for solving the problem]

[0005] This disclosure, in particular, suggests that certain modifications of solubilized human apirases such as human CD39 are surprising. What should be important is that it yields an active protein, and its production is still safe and easy. This is based on the unexpected finding that...

[0006] According to a first aspect of the present invention, a list consisting of an N-terminal deletion, a C-terminal deletion, and a central modification. A solubilized human apirase having at least two modifications selected from is provided.

[0007] In one embodiment, solubilized human apirase undergoes N-terminal deletion, C-terminal deletion, and modification deletion. include.

[0008] In one embodiment, the central modification includes the deletion of one or more amino acids. In another embodiment, The central modification includes one or more amino acid point mutations, such as substitution mutations. In this embodiment, the central modification is the deletion of one or more amino acids and the point of one or more amino acids It is a combination of mutations, such as substitution mutations.

[0009] N-terminal deletions are found in 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40. , deletion of 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acids For example, the deletion of 30 to 50 amino acids from the N-terminus of the wild-type CD39 sequence according to SEQ ID NO: 1. It may be a deletion. In a preferred embodiment, the N-terminal deletion is 34, 37, 38 or 45 amino acids. amino acids.

[0010] The C-terminal deletion may be 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, or a deletion of 20 - 40 amino acids from the C-terminus of the wild-type CD39 sequence according to SEQ ID NO: 1. In a preferred embodiment, the C-terminal deletion is 22 or 29 out of 37 amino acids. There is.

[0011] The central deletion may be a deletion of 10, 11, 12, 13, 14 or 15 amino acids, such as a deletion of 10 - 15 consecutive amino acids from the wild-type CD39 sequence according to SEQ ID NO: 1. In a preferred embodiment, the central deletion is 12 amino acids, such as amino acids 193 - 204 with respect to the wild-type CD39 sequence according to SEQ ID NO: 1. In one embodiment, the solubilized human apyrase has K71E, N73Q, V95A, G102 D, Y104S, T106S, R113M, L149M, V151A, E173D, T2 29A, L254M, K258R, W263R, E276D, N292Q, R304G,

[0012] D, Y104S, T106S, R113M, L149M, V151A, E173D, T2 29A, L254M, K258R, W263R, E276D, N292Q, R304G, I319T, N327Q, A362N, F365S, N371Q, K405N, Y412 F, L424Q, H436D, I437N, F439S, G441D, N457Q, P4 63S and S469R, and contains 1, 2, 3, 4 or 5 point mutations with respect to the wild-type CD39 sequence according to SEQ ID NO: 1.

[0013] In one embodiment, the solubilized human apyrase has SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 32, A group consisting of Sequence ID No. 54, Sequence ID No. 56, Sequence ID No. 70, Sequence ID No. 76, and Sequence ID No. 78. Includes an array selected from.

[0014] In one embodiment, solubilized human apirase is sequence number 131, sequence number 133, sequence number Selected from the group consisting of number 135, sequence number 137, sequence number 139, and sequence number 141. Includes an array.

[0015] In one specific embodiment, the solubilized human apirase is 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 , Sequence ID 72, Sequence ID 215, Sequence ID 223, Sequence ID 211, Sequence ID 58 and Includes sequences selected from the group consisting of sequence number 229.

[0016] In one specific embodiment, the solubilized human apirase is 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 , Sequence ID 72, Sequence ID 215, Sequence ID 223, Sequence ID 211, Sequence ID 58 and It consists of sequences selected from the group consisting of sequence number 229.

[0017] In a preferred embodiment, solubilized human apirase is provided with SEQ ID NO: 58, SEQ ID NO: 72 and Includes sequences selected from the group consisting of column number 229.

[0018] In one embodiment, the solubilized human apirase contains SEQ ID NO: 58. In one embodiment, soluble Solubilized human apirase contains sequence number 72. In one embodiment, solubilized human apirase is sequence Includes number 229.

[0019] In a preferred embodiment, solubilized human apirase is provided with SEQ ID NO: 58, SEQ ID NO: 72 and It consists of sequences selected from the group consisting of column number 229.

[0020] In one embodiment, the solubilized human apirase is sequence number 58. Solubilized human apirase consists of Sequence ID No. 72. In one embodiment, solubilized human apirase is It consists of sequence number 229.

[0021] According to a second aspect of the present invention, the present invention relates to the treatment of apirase according to the first aspect of the present invention. A pharmaceutical composition containing an effective dose is provided, along with one or more pharmaceutically acceptable carriers.

[0022] In one embodiment, the pharmaceutical composition further comprises one or more additional active ingredients.

[0023] According to a third aspect of the present invention, an isolated apirate according to the first aspect for use as a pharmaceutical agent -ze will be provided.

[0024] According to a fourth aspect of the present invention, isolation according to the first aspect for use in the treatment of tissue damage Apirase is provided.

[0025] Tissue damage includes acute brain injury (stroke); acute multiple organ failure; and kidney or other solid organ transplantation. Post-transplant organ dysfunction; burn damage; radiation damage; trauma and / or low-acid injury. Acute injuries caused by venom, such as acute respiratory distress syndrome (ARDS) or lung injury; acute kidney injury. Harm, such as thoracic surgery (e.g., aortic valve replacement, coronary artery bypass surgery) or sepsis or transverse Scarmolysis or secondary acute kidney injury due to the toxic effects of antibiotics or other drugs, etc. It could be acute myocardial injury.

[0026] In another embodiment, a fourth aspect of the present disclosure relates to the treatment of acute kidney injury associated with cardiac surgery. This invention relates to an isolated apirase according to a first aspect of the present invention for use.

[0027] In another embodiment, a fourth aspect of the present disclosure relates to post-transplant organ dysfunction (DGF), acute Respiratory Distress Syndrome (ARDS), Acute Myocardial Infarction (AMI), Traumatic Brain Injury (TBI) / Acute This is called ischemic attack (AIS), ischemia-reperfusion injury (IRI), or multiple organ failure (MOF). For use in the treatment of those combinations, a first aspect of the present invention for isolation Regarding pyrase.

[0028] In one embodiment, a solubilizing agent is used for the treatment of acute kidney injury associated with cardiac surgery. Toapirase contains the amino acid sequence of SEQ ID NO: 58.

[0029] In one embodiment, a solubilizing agent is used for the treatment of acute kidney injury associated with cardiac surgery. Toapirase contains the amino acid sequence of SEQ ID NO: 72. In one embodiment, it is used in conjunction with cardiac surgery. Solubilized human apirase, used for the treatment of acute kidney injury, is SEQ ID NO: 229 Contains amino acid sequence.

[0030] In a further preferred embodiment, the present disclosure relates to the treatment of acute kidney injury associated with sepsis. This invention relates to the use of isolated apirase according to a first aspect of the present invention.

[0031] In one embodiment of the fourth aspect, for use in the treatment of acute kidney injury associated with sepsis Solubilized human apirase contains the amino acid sequence of SEQ ID NO: 58.

[0032] In one embodiment of the fourth aspect, for use in the treatment of acute kidney injury associated with sepsis Solubilized human apirase contains the amino acid sequence of SEQ ID NO: 72.

[0033] In one embodiment of the fourth aspect, for use in the treatment of acute kidney injury associated with sepsis Solubilized human apirase contains the amino acid sequence of SEQ ID NO: 229.

[0034] According to a fifth aspect of the present invention, a method for treating tissue damage in a human subject is provided. This involves administering a therapeutically effective dose of solubilized human apirase according to the first embodiment to the subject. This includes the following. One embodiment of the fifth aspect of the present invention provides the following to an object requiring such treatment. Cardiac hand This concerns methods for treating acute kidney injury associated with surgery.

[0035] Another embodiment of the fifth aspect of the present invention relates to the subject requiring such treatment. Post-organ transplant organs, comprising administering a therapeutically effective dose of isolated apirase according to embodiment 1. Dysfunction (DGF), acute respiratory distress syndrome (ARDS), acute myocardial infarction (AMI), trauma Traumatic brain injury (TBI) / acute ischemic attack (AIS) / ischemia-reperfusion injury (IRI) or multiple organ failure This concerns methods for dealing with combinations of these factors, often referred to as MOFs.

[0036] In one embodiment of the fifth aspect, a method for treating acute kidney injury associated with cardiac surgery is described. The solubilized human apirase used is SEQ ID NO: 58, SEQ ID NO: 72, or SEQ ID NO: 22. It contains a sequence of 9 amino acids.

[0037] One embodiment of a fifth aspect of the present invention relates to an object requiring such treatment, and the first aspect of the present invention A therapeutically effective dose of isolated apirase in the manner described above is administered to treat sepsis associated with sepsis. Regarding methods for treating acute kidney injury.

[0038] In one embodiment of the fifth aspect, a method for treating acute kidney injury associated with sepsis The solubilized human apirase used is SEQ ID NO: 58, SEQ ID NO: 72, or SEQ ID NO: 229. Contains amino acid sequences. Tissue damage includes acute brain injury (stroke); acute multiple organ failure; kidney or Post-transplant organ dysfunction following the transplantation of other solid organs; burn damage; radiation damage; Acute injury due to trauma and / or hypoxia, acute respiratory distress syndrome (ARDS), or lung injury, etc. Acute kidney injury, e.g., thoracic surgery (e.g., aortic valve replacement, coronary artery bypass surgery), or sepsis Secondary acute kidney disease due to rhabdomyolysis or toxic effects of antibiotics or other drugs Injuries, etc.; could be acute myocardial injury.

[0039] According to a sixth aspect of the present invention, isolation encoding any apirase according to the first aspect Nucleic acid molecules are provided.

[0040] According to the seventh aspect of the present invention, clonin comprising one or more nucleic acid sequences according to the sixth aspect A gene or expression vector is provided, wherein this vector is isolated according to a first embodiment. It is suitable for recombinant production of ze.

[0041] According to the eighth aspect of the present invention, one or more clones or expression vectors according to the seventh aspect Host cells containing the ter are provided.

[0042] According to a ninth aspect of the present invention, a step for producing apirase according to the first aspect is provided. This involves culturing host cells according to the eighth aspect, and purifying and recovering the apirase. This includes the following. [Brief explanation of the drawing]

[0043] [Figure 1] Figure 1 shows the sequence alignment. [Figure 2] Figure 2A shows the expression level of the supernatant containing human CD39 as measured by anti-APP Western blotting according to one embodiment. Figure 2B shows the expression level of the supernatant containing a cysteine ​​crosslinking-deleted human CD39 mutant as measured by anti-APP Western blotting according to one embodiment. [Figure 3] Figure 3 is a graph showing the results of the solid-phase ATPase assay for the CD39 mutant. [Figure 4] Figure 4 is a graph showing solid-phase ATP cleavage on HEK293 cells transformed with a human CD39 mutant according to one embodiment. [Figure 5] Figure 5 shows a schematic overview of a vector according to one embodiment. [Figure 6] Figure 6 shows an enzyme model based on the steady-state approximation. [Figure 7] Figure 7 shows the reaction rate data and model overview fitted to a protein according to one embodiment. [Figure 8] Figure 8 shows the reaction rate data and model overview fitted to a protein according to one embodiment. [Figure 9] Figure 9 shows the reaction rate data and model overview fitted to a protein according to one embodiment. [Figure 10A] Figure 10: Figure 10 is a schematic diagram of the experimental conditions. [Figure 10B] (As stated above.) [Figure 11] Figure 11 is a graph showing the AMP levels for proteins according to the embodiment. [Figure 12A] Figure 12: Figure 12 is a graph showing the in vivo results for proteins according to the embodiment. [Figure 12B] (As stated above.) [Figure 12C] (As stated above.) [Modes for carrying out the invention]

[0044] This disclosure, in particular, shows that certain modifications of solubilized CD39 surprisingly affect the active protein This is based on the unexpected finding that it provides a high quality and that its production is safe and easy.

[0045] As shown in the following specific examples, preferred embodiments include N-terminal deletion, C-terminal deletion and intermediate deletion. Solubilized human apira having at least two modifications selected from a list consisting of central deletions For example, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 32, SEQ ID NO: 54, SEQ ID NO: 56, Solubilization containing a sequence selected from the group consisting of sequence number 70, sequence number 76, and sequence number 78 Examples include human apirase.

[0046] The inventors believe that excessive modification increases the risk of increased immunogenicity and therefore the safety risk. Solubilized human apirate that retains activity and expresses its ability without introducing any embellishments. To obtain the enzyme, several different sequence modification strategies were tried. One sequence modification that was found to increase both efficiency and human apirase expression capacity was At the same time, it does not add an excessive risk of immunogenicity, and the deletion of the central section, so-called delta MIL, is not a factor. It was a (ΔMIL) modification.

[0047] To increase the expression of solubilized human apirase according to embodiments of the present invention, N-terminal expression We tested the tags. Although various N-terminal expression tags are known in this field, surprisingly, Not all tags worked. The inventors had not foreseen that several tags were not functional. They found that it worked.

[0048] These N-terminal tags are sequence numbers 131, 133, 135, and 135. It was 137, Sequence ID 139, or Sequence ID 141. As shown herein, Preferred tags are sequence number 133, sequence number 135, or sequence number 137.

[0049] Specific details are shown in Examples 9-13 below. However, the unpredictability of these examples Table 1 provides a summary of the comparisons to illustrate the properties.

[0050] [Table 1]

[0051] 1.Definition To facilitate the implementation of the present invention by those skilled in the art, the following terms are used throughout this description:

[0052] The terms “CD39” and “hCD39” are used synonymously throughout this disclosure. Unless otherwise indicated, human surface antimicrobial agents conforming to UniProt P49961 or Sequence ID No. 1. This refers to the original classification 39 (CD39).

[0053] The term "apirase" refers to human apirase unless otherwise specified. When used herein, "apirase" binds to the cell membrane as a wild-type protein. The apirase present has been modified so that it can no longer bind to the cell membrane. This means that it exists in a soluble state, that is, it is no longer anchored to the cell membrane.

[0054] The abbreviation "MIL" refers to the membrane interaction loop, which involves physical interaction across the cell membrane. In addition to the N-terminus and C-terminus which are anchored, wild-type (human) CD3 interacts with the cell membrane. 9. The central part of the protein is located. The term "DeltaMIL" or "ΔMIL" refers to the wild type. This refers to the deletion of the MIL sequence from (human) CD39.

[0055] The term "approximately" means, for example, + / - 10% with respect to a number x. When a term is used before a range of numbers or a list of numbers, it applies to each number in the series. For example, the phrase "approximately 1-5" should be interpreted as "approximately 1-approximately 5," or for example, "approximately The phrase "1, 2, 3, 4" should be interpreted as "approximately 1, approximately 2, approximately 3, approximately 4, etc." .

[0056] The word "substantially" does not exclude "completely," for example, the pair "substantially does not contain Y." The product may be completely Y-free. Where necessary, the term “substantially” is used in the definition of this disclosure. It can be omitted.

[0057] The term "comprising" is the same as "including". "Consisting" is included in "complementary" (complementary) X, for example. The composition (sing) may consist exclusively of X, or may include anything further, e.g. For example, X + Y.

[0058] "Identity" of native polypeptides and their functional derivatives is, at the maximum percentage. After aligning the sequences to achieve identity and introducing gaps where necessary, the sequences Conservative substitutions are not considered as part of identity, and the corresponding residues of the native polypeptide and This is defined herein as the percentage of identical amino acid residues in candidate sequences. Neither N-terminal extension nor C-terminal insertion is interpreted as reducing identity. Methods and computer programs for alignment are well known. Standard alignment For example, Altshul et al. (1990) J.Mol.B. Basic Local Ali as described by iol.,215:403 410) gnment Search Tool(BLAST);Needleman et a l.'s algorithm ((1970) J. Mol. Biol., 48:444 453); Alternatively, the algorithm of Meyers et al. ((1988) Comput.Appl Percent identity can be determined by Biosci., 4:11 17). The parameters are a gap penalty of 12 and a gap extension penalty of 4. Yes, it is a Blosum62 score matrix with a frame shift gap penalty of 5. It is possible. Percent identity between two amino acid or nucleotide sequences is also related to the gap length. The penalty is 12 and the gap penalty is 4. PAM120 weight limit Using a weight residue table, ALI The GN program (version 2.0) incorporates E. Meyers and The algorithm used is that of W. Miller ((1989) CABIOS, 4:11-17). It can be decided by that.

[0059] "Amino acids" refers to all naturally occurring L-α-amino acids, including D-amino acids. The phrase "amino acid sequence variant" means that, when compared to the sequence described herein, those amino acids This refers to molecules that have several differences in their acid sequence. For example, the proteins specified in this disclosure The amino acid sequence variant of the sequence still possesses apirase activity. The mutant is a substitution mutant (in which at least one amino acid residue is removed, and the polyp is removed according to the disclosure). (In which different amino acids are inserted at the same position in a molecule), insertion mutation Isotopes (one or more amino acids immediately after the amino acids at a specific position in the polypeptide according to this disclosure) (those inserted next to them) and deletion mutants (one or more amino acids that are polypeptide according to this disclosure) Includes (those removed in the chloride).

[0060] The terms "treatment" or "treat" are originally intended to be used in the context of the original text. In the details, the application or administration of apirase according to the present invention to the subject, or the subject or subject Application or administration of the pharmaceutical composition containing the apirase to the isolated tissue or cell line from which the apirase was derived. It is defined as having tissue damage and symptoms related to tissue damage, and the purpose is to By lowering extracellular ATP levels, tissue damage or some related tissue damage can be prevented. This refers to alleviating, relieving, or improving symptoms.

[0061] "Treatment" refers to the application of a pharmaceutical composition containing apirase to a target. Alternatively, a pharmaceutical mixture containing the apirase of the present invention may be administered or administered to isolated tissue or cell lines derived from the target. The application or administration of the substance is also intended, and the subjects have tissue damage or symptoms related to tissue damage. The purpose is to alleviate, relieve, or improve tissue damage or any symptoms associated with tissue damage. That is what it is.

[0062] The term "prevent" or "preventing" , referring to preventive or protective measures; this refers to the onset of disease, disorder and / or associated symptoms. Regarding delaying the onset of the disease or preventing its development.

[0063] When used herein, the subject means that such subject is biological, medical, or If you would benefit from such treatment in terms of quality of life, then you "need" the treatment. "

[0064] The term "pharmaceutically acceptable" means that it does not interfere with the effectiveness of the biological activity of the active ingredient. It means a non-toxic substance.

[0065] When used in this specification, the target compound is referred to as "administration". The terms "administering" or "administering" indicate that treatment is required. This means providing the compound of the present invention and its prodrug to one or more targets. Administration of these therapeutic agents "in combination" may involve simultaneous administration in any order and via any route of administration. This includes simultaneous and sequential administration.

[0066] As used herein, “therapeutic effective dose” means at least the amount of the disorder or recurrent disorder. To treat, prevent, prevent the onset of, cure, delay, or reduce the severity of a single symptom. To reduce, induce remission, or exceed the expected level without such treatment. Effective when administered as a single or multiple dose to patients (humans, etc.) to prolong survival, This refers to the dosage (amount) of pyrase. Where applicable, this term refers only to that component. When applied to a combination, this The terms, when combined, produce a therapeutic effect, whether administered sequentially or simultaneously. This refers to the combined dose or amount of the active ingredient.

[0067] The phrase "administration regimen" refers to a regimen used to treat a disease, for example, tissue This refers to the administration protocol used during the treatment of an injury.

[0068] The phrase "means for administration" refers to pre-filled syringes, vials and syringes. Injection pens, automatic infusion devices, intravenous (IV) infusion and bags, pumps, patch pumps, etc. This refers to any available tools for systemically administering drugs to a patient, including but not limited to those mentioned above. It is used for this purpose. Using such items, patients can self-administer drugs (i.e., by themselves). (To administer drugs for one's own benefit) or a physician may administer drugs. [Examples]

[0069] 2. Example 1: Membrane-free CD39 Wild-type human apirase CD39 (hCD39, UniProt P49961 or sequence) Number 1) consists of an N-terminal transmembrane domain (presumably aa17-37) and a central presumed membrane interaction domain. The subdomain (MIL estimated aa193~204) and the C-terminal transmembrane domain (estimated aa479~4) 99) It is naturally anchored in the cell membrane. Using mammalian host cells, the ability of CD39 CD is used to obtain membrane-free or soluble proteins in order to enable the expression of soluble mutants. Several elements of the 39 sequence were modified. Secretion reader and purification tag (SEQ ID NO: 13) 3) The natural leader sequence and N-terminal transmembrane region were replaced. Expression, purification and activation To optimize the sex parameters, the boundary of the extracellular domain of CD39 was altered. These are amino acid numbers 38-476 and 39-469 of SEQ ID NO: 1, respectively. (amino acids 46-461 and 46-476 of SEQ ID NO: 1). System By substituting the rhyn with alanine, or by creating a rhyn by disulfide crosslinking By shortening the p, the tendency for aggregation and enzyme activity of cysteine ​​and disulfide The effects of bridge construction were systematically evaluated (Sequence IDs 107, 109, 111, 113, and 115). The stretch of hydrophobic amino acids was described in the structural study of rat CD39 (Zebisch et al, J. Mol. Biol. (2012), 415, 288-306, wild type Rat CD39, Uniprot P97687 (shown in Sequence ID No. 2), this loop It is thought that it can interact with the cell membrane (MIL). The inventors believe that by sequence alignment Replacing our understanding of human CD39 sequences, loop deletions (CD39ΔMIL or EP) 28. A CD39 mutant was created that possesses functional CD3 (as shown in Sequence ID No. 4). The effects of MIL deletion (or delta / Δ) on the expression level and thermal stability of 9 were evaluated. .

[0070] As can be seen in Figure 1, which shows the sequence alignment of sequence numbers 1 and 4, CD3 To form 9ΔMIL (SEQ ID NO: 4), N-terminal amino acids 1-27 and C-terminal amino acids Acids 477-510 and central membrane interaction loop (MIL) amino acids 193-204 are wtC D39 (sequence number 1) was deleted.

[0071] The effects of different sequence modifications on thermal stability were studied. Furthermore, CHO cell expression yield and We studied the effects of different sequence modifications on monomer content.

[0072] (1) Method (a) Production of expression plasmids DNA encoding different hCD39 boundary mutants and membrane interaction loop (MIL) deletions The sequence includes codon optimality for Homo sapiens, as well as GeneArt (Life Tech) I placed an order with hnologies Inc. (Regensburg, Germany). The sequence encoding the CD39 variant was generated in a GeneArt-derived vector or internally. From that mutant, an expression vector suitable for secretion in mammalian cells was developed using standard molecular biology. Subcloning was performed using the appropriate technique. Cysteine ​​was modified by the modified oligonucleotide. Targeting the cysteine-to-alanine mutation present in cross-linking deletion mutants, the subsequent acetylcholine After the initial PCR step, the generated fragment is sub-clone into the same expression vector mentioned earlier. The expression vector elements were: promoter (cytomegalovirus) (CMV) enhancer-promoter), signal sequence to promote secretion, polya Denylation signals and transcription termination factors (bovine growth hormone (BGH) gene), episome Elements that enable replication and replication in prokaryotes (e.g., SV40 origin and Col E1 or other known in the art) and elements to enable selection (amplitude) Examples include syringin resistance genes and zeosin markers. (Shortened solubilized human CD39 bar) John's list is illustrated in Table 2, and the amino acid modifications are numbered based on Sequence ID No. 1.

[0073] [Table 2]

[0074] [Table 3]

[0075] (b) Microscale expression of hCD39 mutants As one of the preferred host cell lines for transient protein expression in the absence of serum, 293-6E cells for microscale experiments (as incorporated herein by reference) As disclosed in the publicly released pamphlet No. 2006096989A2, the following was selected. FuGene HD (Roche Applied Science) is used as a gene transfer reagent. Gene transfer was performed using Cat.No.04709705001. For cell introduction and proliferation, use V3 serum-free medium (Bioconcept, Cat. No. 293-6E cells were cultured in suspension culture using V3-K). Humidification was performed with 5% CO2. In the incubator, on an orbital shaker (100-120 rpm), Cornin Cells were grown in a shaking flask (Corning, Tewksbury, MA). Seed flask). Cells in seed culture are maintained in the exponential growth phase (5x10). 5 ~3x1 0 6 Cells should exhibit a >90% viability against gene transfer (cell density / mL). If the density is outside this range, a delay will occur after dilution, or the efficiency of gene transfer will decrease. One of these will happen.

[0076] For microscale (0.5 mL) gene transfer, aliquots of cells are seeded in culture. The cells were collected and placed in V3 serum-free medium at a density of 0.5 x 10⁶. 6 Adjusted to 14 μL of V 3. By diluting 0.5 μg of hCD39 expression plasmid in serum-free medium, D Prepare an NA solution (called Solution 1), then add 2.3 μL of FuGene HD solution to Solution 1. Diluted in 4 μL of V3 serum-free medium (Solution 2). Allow to steep at room temperature (RT) for 5-10 minutes. The liquid was left to warm. Then, solution 2 was added to solution 1 while gently mixing, and it was left at room temperature for another 5 minutes. The cells were left to warm for approximately 15 minutes. Next, a 48-well tissue culture plate (Corning, Tewks) was used. 0.5x10 sown in bury,MA) 6 Genetic information is transferred to 0.5 mL of cells at a rate of individual cells / mL. Add the mixed solution and use an orbital shaker (30) in a humidified incubator with 5% CO2. A plate was placed on top of the centrifuge (0 rpm). Centrifugation was performed at 4°C for 10 minutes at 4000 rpm. By performing this procedure, the culture was collected 3 days after gene transfer (Heraeus, Mu ltifuge 3 SR,Thermo Scientific,Rockford (IL). The collected cell supernatant was stored at 4°C until further processing.

[0077] (c) Western blot analysis of microscale expression supernatant To investigate the expression and correct formation of recombinant hCD39 mutants, microscale development Western blot analysis was performed on the supernatant. E-PAGE(trademark) loading slow Dilute 8 μL of supernatant in saturation solution (4x, Invitrogen, #EPBNF-01), E-Page48, 8% gel under non-reducing conditions (Invitrogen, #EP04808) I put it above. E-base mother device (Invitro Electrolyze the gel on the gen) for 23 minutes, and follow the manufacturer's instructions using the iBlot system. (Invitrogen) is used to transport proteins to a nitrocellulose membrane (Invitr Transferred to ogen IB301001 (run for 7 minutes). TBS / 0.05Twee After washing three times in n20 (TBST), gently agitate with 5% milk / TBST. Both membranes were incubated for 1 hour, followed by dilution of anti-APP mouse antibodies in 2% milk / TBST. A 4 μg / mL solution (used within Novartis for protein tagging) Along with producing antibodies against the peptide stretch of myloid precursor protein (APP) It was left to stand for 1 hour. After three further washing steps, the anti-massage was diluted in 2% milk / TBST. Us-IgG-alkaline phosphatase (Sigma-Aldrich, A5153-1M) The membrane was warmed with a 1:1000 dilution of L), washed again three times in TBST, and then T The BS rinsing stage was performed according to the manufacturer's instructions using SIGMAFAST® BCI. P(registered trademark) / NBT(Sigma-Aldrich,#B5655-25TAB) Use for 1-5 minutes to allow the signal to develop color, then rinse the film with water to stop the signal. Set.

[0078] (d) Solid-phase AxPase assay Pi Col for plate-captured hCD39 mutants from microscale expression supernatant orLock Gold Phosphate Detection System (Innova Biosciences, cat Using n.303-0030), ATPase, ADPase and AMPase activity The properties were determined (solid-phase Axpase assay). This method uses the solution recommended by the manufacturer. It was found that the sensitivity is lower compared to assays based on (liquid-phase Axpase assay). However, Ax is mediated by host cell enzymes that may be present in the microscale expression supernatant. It has the advantage of reducing Pase activity. 20 μL of anti-APP diluted in PBS 10 μg / mL antibody solution (for protein tagging within Novartis) Fabricated for the peptide stretch of amyloid precursor protein (APP) used in the production process. The antibody (processed) was placed in each well of a Maxisorp 384-well clear plate (Nunc). It was added and left to stand overnight at 4°C. After washing three times with TBST, it was gently stirred at room temperature. The wells were then blocked for 1 hour using 100 μL of 5% milk / TBST. After three further washing steps, 20 μL of serially diluted microscale in 2% milk / TBST The supernatant of the enzyme was added to the wells in sets of three, and the mixture was gently stirred while incubating at room temperature for 2 hours. Next, the wells were again filled four times with 100 μL of TBST and 80 μL of 50 mM Tris- Washed twice with Cl / 5mM MgCl2 pH 7.5. 50mM Tris-Cl / 5 mM MgCl2pH7.5(ATP:SIGMA A2383, ADP:SIGMA A2754) Three sets of 30 μL each of 80 μM adenosine phosphate solution diluted in A2754. It was added and left to stand at 37°C for 24 hours. 7.5 μL of Go prepared according to the manufacturer's instructions. The signal was allowed to develop color for 10 minutes using the ld reagent mixture, and 3 μL of stabilizer was used. The reaction was stopped using a TECAN Genios Pro instrument at 620nm. I read that it was absorbed at [location].

[0079] (2) Results (a) Boundaries, membrane interaction loop (MIL) deletions and systemic Impact of in-bridge deletion To evaluate the expression levels of different hCD39 mutants, 0.5 mL of 293-6E filtrate was used. In cells, a pair of corresponding expression plasmids were introduced for gene transfer, and the cells were collected 3 days after gene transfer. Western blotting (detection of anti-APP Ab) was performed on the supernatant. The results are shown in Figure 2A and This is illustrated in Figure 2B.

[0080] The results showed that compared to aa46, hCD39 expression levels starting at aa38 were higher. This indicates that the N-terminal boundary and MIL deletion do not appear to have a significant impact on expression levels. Under the conditions of hCD39(aa46-461), the first or fourth cysteine ​​crosslink is lost. Higher expression levels of hCD39 were also observed. hCD39(aa46-461)Δ We also used the MIL skeleton to confirm a higher expression level of the first cysteine ​​crosslink deletion.

[0081] Boundary for hCD39 activity, membrane interaction loop (MIL) deletion, and cysteine ​​crosslinking deficiency The impact of loss CD39 enzyme activity was measured in the supernatant sample described above by solid-phase AxPase assay. The results are illustrated in Figures 3 and 4 and Table 3.

[0082] [Table 4]

[0083] [Table 5]

[0084] [Table 6]

[0085] MIL deletion increases the proportion of functionally expressed CD39 recombinant protein. It is thought that the different boundaries have a significant impact on active hCD39 activity. This is not shown. The results show a strong decrease in ATPase activity in all cysteine ​​crosslink deletion mutants. This shows complete disappearance. Similar results were obtained using a solid-phase ADPase assay. Therefore, Surprisingly, the sequence modifications that increase both CD39 expression efficiency and CD39 expression capacity are This is a Delta MIL (ΔMIL) modification.

[0086] 3. Example 2: Expression Tag Various expression tags were tested to improve the expression characteristics of the candidate.

[0087] As shown in Table 4, different expression tags based on the N-terminal portion of IL-2 (SEQ ID NO: 13) 1) was tested. The expression tag 1-16aa by sequence number 131 was matched by Geneart. It was accomplished.

[0088] [Table 7]

[0089] As shown in Sequence ID No. 4, all expression tags were tested for CD39ΔMIL. All the constructs included APP tags and His tags.

[0090] As shown in Figure 5, the vector pRS5a was used for expression. The primer pair This is shown in Table 4.

[0091] The annealing temperature was 64°C in all cases.

[0092] 1 μL template DNA preservation solution, 25 μL Kapa Hifi Hotstart polymer -se (from kappa Biosystems / KK2602), 1.5 μL forward Mix the primer and 1.5 μL of reverse primer, and add H2O to a final volume of 50 μL. The PCR solution was prepared by adjusting the parameters.

[0093] PCR reactions were performed according to the schedule in Table 5.

[0094] [Table 8]

[0095] After the PCR reaction is complete, follow the manufacturer's instructions and apply Wizard® SV Gel. and PCR Clean-Up Kit, Promega, No.9282, 1 column, DNA extraction was performed using elution in 30 μL.

[0096] New England Biolabs (NEB) in CutSmart(R) buffer ), NruI-HF (NEB#R3192) and NotI-HF (NEB#R3189) The inserts and vectors were cleaved with enzymes supplied by [the company / organization]. The reaction time was 3 hours at 37°C. .

[0097] Rapid DNA dephosphorylation and ligation kit, following the producer's effective protocol. Using a dephosphorylation vector together with Fa.Roche, No.04898117001 I performed ligation overnight.

[0098] The following day, forward primer P270 (SEQ ID NO: 165) and reverse primer P2 For DNA-Miniprep and sequence analysis using 71 (SEQ ID NO: 166) I picked up Nee.

[0099] Furthermore, according to Table 6, several proteins known in this art to increase expression are... The qualitative arrangement was tested.

[0100] [Table 9]

[0101] The combinations tested and obtained are shown in Table 7.

[0102] [Table 10]

[0103] There were no prior art tags from Table 6 that resulted in the expression of the protein (data not shown). Prior art teaches that these sequences should increase expression, therefore this is It was unexpected.

[0104] 4. Example 3: Further Mutations To improve the characteristics of soluble CD39 and make it suitable for drug development, as shown in SEQ ID NO: 4 Further modifications were introduced in CD39ΔMIL and EP28. Different mutations and mutations were introduced. The naturally mutated mutants are shown in Table 8, and are similar to the wild-type CD39 as shown in Sequence ID No. 1. Numbering is done according to the position of the acid.

[0105] [Table 11]

[0106] [Table 12]

[0107] Two mutations in the active site lead to higher activity (365 and 412).

[0108] 5. Example 4: Removal of glycosylated sites Based on the EP14 mutant above, by introducing point mutations as shown in Table 9, glycosylation The effect of the ligation site was investigated, and according to the amino acid position of wild-type CD39 as shown in Sequence ID No. 1, Assign a number.

[0109] [Table 13]

[0110] (a) Materials and methods The expression vector pRS5a (Figure 5) was used for cloning. These are shown in Table 10. A primer was used as shown.

[0111] [Table 14]

[0112] Follow the manufacturer's instructions for PCR using QuikChange Lightning Site-directed Mutagenesis Kit(Agilent,No. .210519-5) was used.

[0113] The next day, a single colony was selected for DNA-Miniprep, and forward primer P Using 270 (SEQ ID NO: 165) and reverse primer P271 (SEQ ID NO: 166) Sequence analysis was performed.

[0114] To ensure the accuracy of the vector skeleton (induced by mutagenesis), use the following method: Then, the sequenced insertion fragment was cloned into a new vector skeleton of pRS5a (Figure 5). Ta.

[0115] This product contains APP_HIS-Tag, a concentrated storage solution containing 3.3 μg / μL of the expression tag sequence number. The vector was prepared using the vector skeleton of sequence number 36 along with 135.

[0116] Add 10 μg vector-DNA and 0.4 μL HindIII(1) to a final volume of 50 μL. 00U / μL, NEB), 2μL EcoRI(20U / μL, NEB), 5μL Cu By mixing tsmart buffer 10xconc.(NEB) and H2O, the vector The tar was digested. Digestion was carried out at 37°C for 3 hours.

[0117] Alkaline phosphatase, calf intestine (CIP, NEB, No. M0290L), 10 U / Dephosphorylation was performed using μL. Immediately after digestion, 3 μL of CIP was added to the digested vector. The mixture was added and incubated at 37°C for 30 minutes. The digested and dephosphorylated vector was fractionated with 0.8% TA. Place the vector on an E agarose gel and cut out a suitable band size of approximately 6100 bp. Wizard® SV Gel and PCR Clean-Up Kit, P Using romega, No.9282, 1 column, clean up with elution of 100 μL. A test was performed. OD260nm showed a concentration of 64 ng / μL.

[0118] 42.5 μL of DNA (approximately 3-5 μg of each DNA molecule), 5 μL of Cutsmart Buffer, 10xconc., NEB no B7204S, 0.4μL HindIII -HF, 100U / μL, NEB no.R3104S, 2μL EcoRI-HF, 2 Mix 0 U / μL of NEB no. R3103L and adjust the volume to 50 μL with H2O. The mutant insertion fragment was digested by this method. It was then digested in a PCR instrument at 37°C for 3 hours. The following procedure was performed: The digested insert was placed on a preparative 0.8% TAE agarose gel and prepared for approximately 1400°C. A reasonable band size was extracted from the bp vector. Wizard(registered trademark) SV G el and PCR Clean-Up Kit, Promega, No.9282, 1 color Cleanup was performed using a solution containing 30 μL of elution. OD260nm was 1-25n The concentration was shown in g / μL.

[0119] Rapid DNA Ligation Kit,No.K1423,Fa.Ther Using mo Scientific (vector:insertion ratio approximately 1:10) with Lige The procedure was performed. 4 μL of 5x ligation buffer was mixed with 1 μL of ligase and 2 μL of vector. - fragment, HindIII / EcoRI digestion, stock concentrate 64ng / μL, 13μL insert fragment pieces, HindIII / EcoRI digested, and mixed with 1-25 ng / μL of stock concentrate. RT We performed ligation for 10 minutes.

[0120] 30 minutes on ice with 80 μL of chemically competent XL1 Blue cells (Nova Transformation was performed by incubating a 10 μL ligation solution with rtis (FS / RL). A heat shock was performed at 42°C for 45 seconds in an Eppendorf incubator, followed by... It was then left on ice for 2 minutes. After that, 1 mL of 2YT medium was added, followed by Eppendorf. The samples were placed on an ORF shaker (800 rpm) at 37°C for 1.5 hours. Then, they were shaken at 7000 rpm for 3 hours. The cells were centrifuged for 1 minute and the colonies were seeded on LB / Carb / Gluc. Then pre The sample was left at 37°C overnight.

[0121] The next day, a single colony was selected for DNA-Miniprep, and forward primer P Using 270 (SEQ ID NO: 165) and reverse primer P271 (SEQ ID NO: 166) Sequence analysis was then performed.

[0122] After 7 days of expression, a valid sequence was introduced into HEK293 cells on a 200 mL scale. It was imported.

[0123] The following materials were used: Human embryonic kidney cells that constitutively express the SV40 large T antigen (HEK293-T), for example baATCC11268 Polyethyleneimine "MAX" MW40.000 (PEI) (Polyscience (s,Cat.24765), dissolved in H2O at RT, and adjusted to pH 7.05 with NaOH. . M11V3 serum-free medium (Bioconcept, CH, Cat: V3-K) DNA: Follow the protocol recommended by the supplier, using the Qiagen DNA Kit. Prepared using Midiprep-Kit (No. 12943).

[0124] All cell culture operations for transient gene transfer are performed using serum-free M11V3 medium. This procedure is performed using suspension-compatible HEK293-T cells.

[0125] In a humidified CO2 incubator with 5% CO2, an orbital shaker (115 rpm) is used. Cells are grown in a Corning shaking flask (Corning, USA) on top of the surface. Seed flask).

[0126] The cells used are in the exponential growth phase (5x10 5 ~3x10 6 Cell density / mL ), the survival rate was >90%.

[0127] Using counted cells, genetics can be studied on a small scale (here, 20 / 50 or 100 mL). Transfer cells and add the corresponding amount of cells in M11V3 medium (1.4 x 10⁶ cells). 6 Adjust to pieces / mL We used a cell suspension containing 36% of the final gene transfer volume.

[0128] Dilute 1 mg / L final volume DNA in 7% final volume M11V3 and mix gently. Prepare a DNA solution (Solution 1). Since the DNA is non-sterile filtered, Penc. / Strep was added to the transfected cells after feeding to prevent contamination of the culture. Next, dilute a 3 mg / L final volume PEI solution in 7% final volume M11V3 and mix gently (Solution 2). Incubate both solutions at room temperature (RT) for 5 - 10 minutes. Then, add Solution 2 to Solution 1 while mixing gently and incubate at RT for an additional 5 - 15 minutes. After incubation, add the transfection mixture to the cells and culture the cells for 4 hours (115 rpm, 37 °C, 5% CO2). Recover the supernatant 7 days after expression. Centrifuge at 4500 rpm for 15 min at 4 °C (Heraeus, Multifug e 3 S-R)

[0129] Clarify by sterile filtration through a 0.22 μm (Stericup filter, Thermo Scientific, Cat. 567-0020).

[0130] Send the supernatant for purification for further steps. Use a 1 mL sample of the supernatant for IPC on an Open Access APP-column. The sample vial was a glass crimp vial, 2 mL Agilent, catalog number 518

[0131] 2-0543 and cap: crimp 11 mm, catalog number 5040-4667. Use a 5 mL Histrap HP column (GE Life Sciences, Order

[0132] No. 17-5248-02) and follow the following protocol on an Aekta

[0133]

[0134]

[0135] Protein was purified using immobilized metal affinity chromatography (IMAC) on Pure or Aekta Avant (GE Healthcare). Specifications are shown in Table 11.

[0135]

Table 15

[0136] Buffers used were formulated according to Tables 12 and 13.

[0137]

Table 16

[0138]

Table 17

[0139] The resulting proteins were stored according to Table 14.

[0140]

Table 18

[0141] (b) Results and Interpretation There was no improvement in mutant with respect to analytical SEC yield and monomer peak. The expression-tagged parent protein (EP14) according to SEQ ID NO: 137 gave the best yield and the largest monomer peak in the analysis. The lowest yield and also the lowest monomer peak were obtained with mutant N371Q.

[0142] 6. Example 5: Combinations To try and further improve the properties, several of the mutations introduced in Example 3 above ​​The tsuka were combined according to Table 15 below. The mutations were wild-type, as shown in Sequence ID No. 1. The numbers are assigned according to the amino acid positions of CD39.

[0143] [Table 19]

[0144] (a) Materials and methods Primers were used as shown in Table 16.

[0145] [Table 20]

[0146] The PCR reaction was set up using the following pipetting scheme: 5 μL of 10x reaction buffer, 1μL ds-DNA-template (100ng / μL stock concentrate), 1.5 μL primer 1, 1.5 μL primer 2, 1μL dNTP mixture, 1.5 μL QuickSolution reagent, 35.5 μL H2O (relative to a final volume of 50 μL) and 1μL QuickChange Lightning Enzyme.

[0147] The PCR cycling parameters shown in Table 17 were used.

[0148] [Table 21]

[0149] Immediately after the reaction, add 2 μL of DpnI enzyme to each reaction, mix, and incubate at 37°C for 5 minutes. Ta.

[0150] Transformation into XL10-gold ultra-competent cells as follows The procedure was carried out. Cells were frozen and thawed on ice. 45 μL / transformation was used, and 2 μL B-ME was applied. It was added to each vial. Next, 3 μL of DpnI-digested PCR product was added, and 15 mL The sample was placed in a BD tube on ice for 30 minutes. Then, the sample was subjected to a heat shock treatment for 40 seconds. It was left on ice for 2 minutes. Next, 950 μL of SOC medium was added, followed by shaking incubator. The cells were incubated at 37°C for 1.5 hours. Finally, the cells were placed on an LB-carb plate. The seeds were sown and left overnight at 37°C. The following day, DNA-miniprep and sequence analysis were performed. I found a single colony.

[0151] As described in Example 4, a suitable sequence was introduced into HEK293 cells.

[0152] Using immobilized metal affinity chromatography (IMAC) as follows: The protein was purified. 95 mL of supernatant was used (approximately 4 mL of all supernatant was kept for analysis (IPC)). (To hold).

[0153] Materials used: Nickel-NTA-Agarose, Qiagen, Cat No. / ID:30 230, Poly-Prep Chromatography Column, empty y,BioRad,No.731-1550,IMAC A buffer pH7.4 (20mM (Contains NaPO4 buffer and 50 mM imidazole). IMAC B buffer pH 7.4 (Contains 20 mM NaPO4 buffer and 300 mM imidazole). TBS (Mill (Diluting 10x concentrate to 1x concentrate with iQ-Water). Ultracel-10 membrane Ren, 10K, Amicon Ultra-4 Centrif with UFC801096 Ugal Filter Unit.

[0154] Process stages: 1.Qiagen's 1mL Nickel-NTA-Agarose (=0.5mL Prepare the column using CV; Equilibriumized with 2.10CV IMAC A; 3. Loading 15 / 45 mL SN onto the column (recovering the flow-through); 4. Wash with 4.10CV IMAC A (recovered in a 15 mL Falcon tube); 5. Elution in 6.5 CV of IMAC B; 6. Determination of the concentration of eluted substances; 7.Amicon Ultra-4 Centrifugal Filter Uni Concentration of 3.5 mL sample to approximately 400 μL using t 10K; 8. Buffer solution exchange by adding TBS and centrifuging at 5000°C.

[0155] Analytical SEC was used for 40 μL of each sample, and protein gel was used for 12 μL of each sample. Then, the sample was analyzed.

[0156] The obtained protein was stored.

[0157] (b) Results and interpretation The results are shown in Table 18.

[0158] [Table 22]

[0159] Protease site: When matryptase was inserted, it was not produced or the yield was very low. There was a yield of approximately 40% at the site (however, with simultaneous gene transfer with furin plasmid) Therefore, only 50% of the DNA was used for gene transfer.

[0160] IL2-abbreviation: All abbreviations containing aa1-3 yield equivalent results, and only aa1-3 compared to the others yields the same result. It may be slightly lower, but this could be due to sample-to-sample variation. (Abbreviated aa4-1) In variant 2, protein expression is lost. Like all other EP- variants, IL2-star No difference was observed between t and hCD39-proteins in EP28 containing the TSS linker. It was not possible.

[0161] combination: Combining it with EP19(L424Q) did not lead to a significant improvement in protein expression. It was.

[0162] The combination with EP1(R113M) showed lower aggregation in the analytical SEC. NEG 726 was well expressed, but showed the lowest aggregation among all test samples (approximately 37%). EP The 14xEP17 combination did not lead to any further improvements (F365S (+Y412F).

[0163] 7. Example 6: Cloning of the final candidate For further testing, we expressed a selection of clinical candidates as shown in Table 19 below.

[0164] [Table 23]

[0165] The following primer was used:

[0166] [Table 24]

[0167] The PCR reaction was set up using the following pipetting scheme: 0.25 μL DMSO, 20ng vector 1.5μL insert (45ng / μL), 2μL 5xHF buffer, 0.1 μL Phusion pol, 0.08μL dNTP mixture 10-X μL ddH2O

[0168] The PCR cycling parameters shown in Table 17 were used.

[0169] [Table 25]

[0170] Immediately after the reaction, add 0.5 μL of DpnI enzyme to each reaction, mix, and incubate at 37°C for 2 hours. I placed it down.

[0171] Transformation into XL10-gold ultra-competent cells is performed as follows: The procedure was carried out. Cells were frozen and thawed on ice. 45 μL / transformation was used, along with 2 μL of B-ME. Next, 3 μL of DpnI-digested PCR product was added to each vial, and 15 mL The sample was placed in a BD tube on ice for 30 minutes. Afterward, the sample was subjected to a heat shock treatment for 40 seconds. Then, it was left on ice for 2 minutes. Next, 950 μL of SOC medium was added, followed by shaking the ink. The samples were incubated at 37°C for 1.5 hours. Finally, they were placed on an LB-carb plate. The cells were seeded and left overnight at 37°C. The following day, DNA-miniprep and sequence analysis were performed. I picked up a single colony.

[0172] To ensure the arrangement was correct, all constructs were placed on a new vector background. The construct was subcloned. For this purpose, all constructs were amplified by PCR and G4 An S-linker was inserted, and then digested with HindIII / EcoRI. The resulting protein Quality was preserved.

[0173] 8. Example 7: Preparation of comparative protein (1) Null mutation To create a negative control protein for in vivo experiments, half of the mutations were introduced into the parent human. This mutation was inserted into the CD39ΔMIL protein (EP28). The literature describes eliminating or reducing the enzyme activity of E. The mutation site is E. These are 174A and S218A.

[0174] The following primer was used:

[0175] [Table 26]

[0176] The PCR reaction was set up using the following pipetting scheme: 5 μL of 10x reaction buffer, 1 μL ds-DNA template (storage concentrate 100 ng / μL), 1.5 μL primer 1, 1.5 μL primer 2, 1μL dNTP mixture 1.5 μL QuickSolution reagent, 35.5 μL H2O (relative to a final volume of 50 μL) and 1μL QuickChange Lightning Enzyme.

[0177] The PCR cycling parameters shown in Table 17 were used.

[0178] [Table 27]

[0179] Immediately after the reaction, add 2 μL of DpnI enzyme to each reaction, mix, and incubate at 37°C for 5 minutes. Ta.

[0180] Transformation into XL10-gold ultra-competent cells is performed as follows: The procedure was carried out. Cells were frozen and thawed on ice. 45 μL / transformation was used, and 2 μL B-ME was added. It was added to each vial. Next, 3 μL of DpnI digested PCR product was added, and 15 mL B The sample was placed in a D-tube on ice for 30 minutes. Then, the sample was subjected to a heat shock treatment for 40 seconds. It was left on ice for 2 minutes. Next, 950 μL of SOC medium was added, followed by shaking incubator. The cells were incubated at 37°C for 1.5 hours. Finally, the cells were placed on an LB-carb plate. The seeds were sown and left overnight at 37°C. The following day, DNA-miniprep and sequence analysis were performed. I found a single colony.

[0181] The following protocol was used to transfer a suitable sequence into HEK293 cells.

[0182] Add 10 μg vector-DNA and 0.4 μL HindIII to a final volume of 50 μL. 100U / μL, NEB), 2μL EcoRI(20U / μL, NEB), 5μL C Digestion buffer was prepared using utsmart buffer 10xconc.(NEB) and H2O. The mixture was prepared. A digestion reaction was carried out at 37°C for 3 hours.

[0183] Immediately after digestion, a dephosphorylation reaction was performed on the digested vector mixture. Add phatase (10 U / μL, CIP, NEB, No. M0290L) (3 μL) It was left to stand at 37°C for 30 minutes.

[0184] To examine the sequence, digested and dephosphorylated vectors were subcloned.

[0185] The following protocol was used to transfer a suitable sequence into HEK293 cells.

[0186] Expression was performed for 7 days using the following materials: 1. Constituted SV40 large T antigen Human embryonic kidney cells that express (HEK293-T, ATCC11268) in a specific manner; 2. Polyethylene Lenymin "MAX" MW40,000 (PEI) (Polysciences, Cat .24765).

[0187] Carefully dissolve 1 g of PEI in 900 mL of cell culture grade water at room temperature (RT). Prepare the PEI solution by adding Na to it so that the final pH is 7.05. Neutralize with OH. Finally, adjust the volume to 1 L and pass the solution through a 0.22 μm filter. Filter, distribute in aliquots, and freeze at -80°C until further use. After freeze-thaw, Aliquots can be refreezed up to three times at -20°C, but they should not be stored for extended periods at -20°C. do not have.

[0188] M11V3 serum-free medium (Bioconcept, CH, Cat: V3-K).

[0189] Using suspension-compatible HEK293-T cells grown in serum-free M11V3 medium, Perform all cell culture procedures necessary for hypergenetic transfer.

[0190] For small-scale (<5L) gene transfer, use a humidified CO2 incubator with 5% CO2. Then, on an orbital shaker (100 rpm), a Corning shaking flask (Corn Growing cells (seed flask) in (USA).

[0191] Generally, cells during seed culture should be in the exponential growth phase (5x10 5 ~3x10 6 / mL cell density ), and the viability should be >90%. If the cell density is outside this range, either a lag phase will occur after splitting or the gene transfer efficiency will decrease.

[0192] For small-scale (here 2L) gene transfer, an aliquot of cells is taken from the seed culture and adjusted to 1.4x10 6 cells / mL at 36% of the final volume with M11V3 medium .

[0193] Dilute 1 mg / L final volume DNA in 7% final volume M11V3 and gently mix to prepare the DNA solution (Solution 1). To prevent contamination of the culture, this solution can be filtered through a 0.22 μ m filter (e.g., Millipore Stericup). Here, due to the small volume, sterile filtration is not performed. Next, dilute 3 mg / L final volume PEI solution in 7% final volume M11V3 and gently mix (Solution 2). Let both solutions stand at room temperature (RT) for 5 - 10 minutes. Then, add Solution 2 to Solution 1 while gently mixing and let stand at RT for another 5 - 15 minutes (PEI coats / condenses DNA into positively charged particles, which bind to anionic cell surface residues and are taken up by cells via endocytosis, so do not mix again during the standing time). After standing, add the gene transfer mixture to the cells and culture the cells for 4 hours (10 rpm, 37 °C, 6% CO2). to make it bind to anionic cell surface residues and be taken up by cells via endocytosis, so do not mix again during the standing time). After standing, add the gene transfer mixture to the cells and culture the cells for 4 hours (10 rpm, 37 °C, 6% CO2).

[0194] Finally, supply the culture with the remaining 50% final volume of M11V3 medium according to the following example. Inoculation volume: 1.4 x 10 cells 6 36 mL at a rate of 1 / mL.

[0195] Solution 1: 7 mL of M11V3 medium containing 100 μg plasmid DNA. Solution 2: 300 7 mL of M11V3 medium containing μg PEI (300 μL). Supply: 50mL M11V3, total 100mL.

[0196] Using immobilized metal affinity chromatography (IMAC) as follows: The protein was purified. 95 mL of supernatant was used (approximately 4 mL of all supernatant was kept for analysis (IPC)). (To hold).

[0197] Materials used: Nickel-NTA Agarose,Qiagen,Cat No. / ID:30 230,Poly-Prep Chromatography Column,empty y,BioRad,No.731-1550,IMAC A buffer pH7.4 (20mM (Contains NaPO4 buffer and 50 mM imidazole). IMAC B buffer pH 7.4 (Contains 20 mM NaPO4 buffer and 300 mM imidazole). TBS (Mill (Dilute 10x concentrate to 1x concentrate with iQ-Water). Ultracel-10 membrane , 10K, Amicon Ultra-4 Centrifu with UFC801096 Gal Filter Unit.

[0198] Process stages: 1.Qiagen's 1mL Nickel-NTA-Agarose (=0.5mL Prepare the column using CV; Equilibriumized with 2.10CV IMAC A; 3. Load 15 / 45 mL SN onto the column (collect the flow-through); 4. Wash with 4.10CV IMAC A (recovered in a 15 mL Falcon tube); 5.6.5 Elution in IMAC B of CV; 6. Determination of the concentration of eluted substances; 7.Amicon Ultra-4 Centrifugal Filter Uni Concentrate the 3.5 mL sample to approximately 400 μL using t 10K; 8. Add TBS and exchange the buffer solution by centrifugation at 5000°C.

[0199] Using an analytical SEC with 40 μL of each sample, and with 12 μL of each sample, The samples were analyzed using an protein gel.

[0200] The obtained protein was stored.

[0201] (2) plusMIL Overlap extension PCR using membrane interaction loop (aa193-204) to identify EP1 Cloning of 4aa1-3 was performed.

[0202] The following primers were used.

[0203] [Table 28]

[0204] The PCR reaction was set up using the following pipetting scheme: 1.2μL Phusion Hot Start Polymerase, 24μL 5xHF-buffer, 0.96μL 100mM dNTPs (25mM each dNTP), 0.6 μL Fw primer, 0.6 μL Rev primer, 92.64 μL DEPC H2O.

[0205] The PCR cycling parameters used were those specified in Table 17.

[0206] [Table 29]

[0207] Immediately after the reaction, add 2 μL of DpnI enzyme to each reactant, mix, and incubate at 37°C for 2 hours. did.

[0208] Transfer 2 μL of PCR product to a 96-well PCR plate and cool on ice to shape the product. A quality conversion was performed. 20 μL STELLAR chemical component. The bacteria were added and carefully mixed by pipetting in and out once. The sample was then placed on ice. Leave on the surface for 30 minutes, then leave in a PCR instrument at 42°C for 45 seconds, followed by 60 seconds on ice. Further incubation was performed. Finally, 90 μL of SOC medium was added and incubated at 37°C for 1 hour. All of the gene transfer mixture is seeded onto an LB-ampicillin or LB-carbencilin plate. They were allowed to grow overnight at 37°C.

[0209] The resulting protein EP14_plusM has an amino acid sequence according to SEQ ID NO: 155. The IL file was saved.

[0210] 9. Example 8: Enzyme activity The characteristics of the candidate prepared in the previous example were examined using an enzyme activity assay.

[0211] The following reagents were used: Pi-free buffer solution, phosphate-free physiological saline solution (140 mM Na). Cl, 5mM KCl, 1mM MgCl2, 2mM CaCl2, 10mM Hepe s, pH 7.4); and Pi-free buffer + 2% BSA, 20 mg / mL BSA; CD3 9. Phosphate-free physiological saline solution containing protein (according to Sequence ID No. 1); ATP.

[0212] A two-component CD39 solution was prepared at 2 μg / mL. 15 μL ATP preservation solution + 1185 μg A 1000 μM diploid ATP solution was prepared from a total of 1.2 mL of L buffer.

[0213] In a 48-well PCR plate filled to 120 μL final / well, 60 μL AT By mixing P with 60 μL of CD39, or with 60 μL of a control Pi-free buffer, The enzyme reaction was then tested. The final concentrations were 500 μM ATP and 1 μg / mL CD39. That was the case.

[0214] The samples were left at 37°C for 0, 5, 15, 30, 60, 90, and 150 minutes, respectively. Next, the samples were evaluated using either a Pi release assay or HPLC.

[0215] (1) Pi release assay (a) Materials and methods Reagents were prepared from a standard Pi detection kit according to the manufacturer's instructions.

[0216] A standard curve using Pi was created by dilution in water. Two sequential dilutions were prepared: 450 μL + 450 μL water. The standard curve concentration is 50 μM / 25 The concentrations were μM / 12.5μM / 6.25μM / 3.1μM / 1.5μM / 0μM.

[0217] Gold reagent mixture was prepared: 4 mL gold reagent + 40 μL accelerator (3 plates) (In contrast to) In a 96-well plate, the sample was diluted 1:10 in H2O (in water) Dilution: 10 μL sample + 90 μL H2O). 50 μL, 1:10 diluted sample, half of 96. The sample was distributed into each well of a region well plate (Corning, 3690). 12.5 μg Add the L Gold reagent mixture to each well (25% sample volume) and allow the samples to cool at room temperature for 10 minutes. The sample was kept warm. Absorption was read at 635 nm.

[0218] (b) Results and interpretation Table 26 shows the comparison results for the candidates.

[0219] [Table 30]

[0220] 500 μM ATP was added to the enzyme, and AT was measured over time by HPLC (as described below). Enzyme activity was measured by analyzing the concentrations of P, ADP, and AMP. The resulting reaction The rate curve was fitted to the model in Figure 6 to obtain the enzyme constant. The enzyme constant Kcat is... Regarding this, the enzymes are in the following order (low activity to high activity): EP28(wt), EP17, EP14, This shows EP15.

[0221] Figure 7 shows the reaction rate data and model fit for EP28. Figure 8 shows EP1 The reaction rate data and model fit for 4 are shown. Figure 9 shows the reaction rate for EP15. The degree data and model fit are shown.

[0222] Table 2 outlines the enzyme constants for EP28(wt), EP14, EP15, and EP17. As shown in 7, the three novel mutants show improved catalytic activity compared to the wild type (WT). Important Furthermore, the novel mutants have a catalytic rate constant (kcat) and catalytic efficiency (kcat / Km). A clear increase is observed. Reported ATP and ADP substrate concentrations during tissue injury and thrombosis are Since it exceeds the reported Km, this increase in kcat and kcat / Km is in vivo This could be rephrased as "higher activity."

[0223] [Table 31]

[0224] (2) HPLC validation assay (reaction rate and dose-response) (a) Materials and methods Candidates were tested using an HPLC validation assay. 70 μL of each sample was glassed for HPLC. I transferred it to a vial.

[0225] Candidate samples were prepared using a 5 mM preservation solution as shown in Table 28.

[0226] [Table 32]

[0227] Each 1000 μM 20 μL storage solution was mixed in an HPLC vial. 500 μM 20 μL + 1 mM + 20 μL H2O 100 μM 10 μL 1 mM + 90 μL H2O 10 μM 10 μL 100 μM + 90 μL H2O 1 μM 10 μL 10 μM + 90 μL H2O

[0228] CapPump (G1376A), Degasser (G1379A), ALS (G1 329A), Thermostat(G1330B, ColComp(G1316A) and Using the Agilent 1100 System with DAD (G1315A) HPLC separation was performed. Solvent A: 10 mM KH2PO4(04243, Riedel -de Haen) + 2mM TBA bromide, pH 7.0 (86857-10G-F, F (Luka) and solvent B: 10 mM KH2PO4 / ACN 1 / 1 + 2 mM TBA bromide Substance, pH 5.5. Column: Nucleodur 300-5 C18ec, 2 x 150m m, 5 μM, Macherey-Nagel 760185.20Batch E141 00258 36654055. Column temperature: 40°C, injection volume: 10 μL, flow rate: 0.3 The flow rate is mL / min, and the gradient is linear: 0-3': 0%B; 3-23': 0-95%B; 2 3-28':95%B, linear;28-29':95-0%B, linear;5'Post Time. DAD: 247nm and 259nm.

[0229] UPLC separation was performed using Waters UPLC Class I. Solvent A : 10mM KH2PO4 / 10mM K2HPO4¹ / ¹+2mM TBA bromide, pH 7.0. Solvent B: 10 mM KH2PO4 / ACN 1 / 1 + 2 mM TBA bromide pH 5.5. Column: Fortis Bio C18, 2.1 x 50 mm, 5 μm, d i2chrom BIO318-020301 SN H03161210-2. Column The temperature was 40°C, the injection volume was 10 μL, and the flow rate was 0.5 mL / min. The distribution is 0-1': 0%B; 1-8': 0-55%B, linear; 8-10': 55%B; 10 -11':55-0%B, linear; 14' stop time. DAD is 247nm and 259nm. That was the case.

[0230] (b) Results The results can be seen in Figures 7, 8, 9, and 11, which represent candidates for different embodiments. In contrast, reaction rate data and model fit are shown.

[0231] 10. Example 9: In vitro activity, initial screening Mammalian expression vector pRS5 without IL2-leader and without IL2-start In a_Leader_APP_His (Figure 5), the CD39 bar described in the previous example I cloned John from episodes 1 through 24.

[0232] (a) Materials and methods Small scale of EP-Hits in HEK293 (PEI-gene transfer) over 7 days Cell expression (20 / 50 mL scale) was performed, followed by IPC on APP-HPLC. (As stated above).

[0233] Proteins in 15 / 45 mL cell supernatants using a Ni-NTA column (0.5 mL CV) Purification; 6CV IMAC B buffer (20mM NaPO4-buffer, 300mM imida Elution at zole (pH 7.4); Concentration and rebuffering of purified protein in TBS, pH 7.4; Protein gel, analysis of proteins in analytical SEC; All mutants and three controls (parent hCD39-dMIL or EP28, IL2-star) Delivery with and without t, and 8M-version, IL2-start not included: TBS, p 90-200 μL of purified protein from H7.4.

[0234] (b) Results and interpretation The results are summarized in Table 29 below.

[0235] All samples were in TBS pH 7.4 and contained APP-(SEQ ID NO: 247) and His-Ta It contains g (sequence number 249).

[0236] Only the parent human CD39ΔMIL(EP28) has a 15-amino acid length IL2-start, a It has a1-15 (sequence number 133).

[0237] Pi release assay BOENKTH1-0252824, double det. 60 and Values ​​over a 180-minute period.

[0238] [Table 33]

[0239] [Table 34]

[0240] 11. Example 10: In vitro activity, precise screening In the second trial, a subset of 12 mutants was tested, but due to the IL-2 start... This enables larger expression scales.

[0241] (a) Materials and methods Mammalian expression vector pR with 15-amino acid-length IL2-start and aa1-15 S5a_Leader_APP_His (Sequence ID 133) (Figure 5). H over 7 days Small-scale expression of EP-Hits in EK293 (PEI-gene transfer) (50 / 100) Following the mL scale test, IPC was performed on APP-HPLC (as described above).

[0242] Protein analysis of 45 / 95 mL cell supernatant using a Ni-NTA column (0.5 mL CV) quality purification 6CV IMAC B buffer (20mM NaPO4-buffer, 300mM imidazole Elutes at pH 7.4. Concentration and rebuffering of purified protein in TBS, pH 7.4 Protein gel, analysis of proteins in SEC All mutants and controls (parent hCD39-dMIL or EP28, IL2-start) Delivery of aa1-15 (Sequence ID 133): 500 μL of purified protein in TBS, pH 7.4.

[0243] (b) Results and interpretation The results are summarized in Tables 30, 31, and 32 below.

[0244] [Table 35]

[0245] [Table 36]

[0246] [Table 37]

[0247] [Table 38]

[0248] 12. Example 11: In vivo activity, pK (a) Materials and methods In the case of in vivo PK, four conscious female C57BL / 6 mice were subjected to PBS buffer. The final concentration of 10 mg / kg of the compound was administered intravenously into the tail vein (1 m (L / kg). The mice were obtained from WIGA and weighed approximately 22g. The experiments during their survival were conducted as follows: All work was carried out in accordance with Swiss animal welfare laws. Small volume was used with POCT Minivettes. Whole blood was collected in serum tubes at 0.25, 3, 8, 24, and 48 hours after administration (50 μg). (L / time point). Serum was separated and used for concentration determination.

[0249] Gyrolab technology works through centrifugal force and laser-induced fluorescence detection. This is an automated, nanoliter-scale immunoassay using a flow-through method. In rolab Bioaffy CD, streptavidin-coated beads are affinity The columns are pre-filled. Each CD contains 112 columns. The finity capture column contains 15 nL. The injected sample enters via capillary action. The tinification capture reagent binds to streptavidin-coated beads. Later, the sample solution is injected. It then binds to the capture molecule. Finally, the fluorophore-labeled detection reagent is applied. CD3 In case 9, depending on the availability of the APP tag, two different assay reads are used. Ta. 1) Anti-CD39 (40035) and anti-APP (27431) can be seen in Figure 10A. 2) Fab(40035) and anti-Fc / anti-CD39(40044) 1:1 premix (The complete EP28aa1-16 construct) can be seen in Figure 10B. Amine coupling When biotinylated by this method, antibody 40044 loses its activity, and therefore only antibody 40035 remains active. It was biotin-treated.

[0250] CD in Rexxip A containing 5% (v / v) mouse serum in a 1:2 dilution series All standard curves for 39 constructs were diluted. APP tagging constructs. The applicable concentration range for EP2 is 5000 ng / mL to 9.77 ng / mL. In the case of 8aa1-16, this was 10000 ng / mL to 9.77 ng / mL. 5 All mouse serum samples were mixed in a 1:1 ratio in Rexxip A containing %(v / v) mouse serum. Diluted to 00. CD39 contaminated in Rexxip A containing 5% (v / v) mouse serum. Diluted QC samples of the struct (50 and 50 for constructs with APP tags) (500 ng / mL and 1000 ng / mL for EP28aa1-16). Total The final concentration of the biotinylation capture reagent was 0.1 mg / mL, and the fluorescently labeled detection antibody It was diluted to 10 nM in Rexxip F.

[0251] (b) Results and interpretation The results are summarized in Table 34. As can be seen, all candidates show the same PK. Therefore, The selection of the replacement was not based on PK characteristics.

[0252] [Table 39]

[0253] 13. Example 12: In vivo active, AKI model (a) Materials and methods Before initiating I / R settings, a nephrectomy of the right kidney will be performed to alter the overall biological dynamics. To avoid compensatory mechanisms, the second kidney was removed. Anesthetized animals that were breathing spontaneously were kept at homeothermally. Place the device on the system's temperature-regulating blanket and cover it with sterile gauze to prevent hypothermia. Then, record body temperature via a rectal probe and adjust it within the range of 36.5-37.5°C. The area is anesthetized, shaved, and disinfected (betaseptic). After midline incision / abdominal surgery. The contents of the abdomen are moved to the left, and the right kidney is removed. The ureters and blood vessels are severed and ligated (9 (-0Ethicon), then the kidney is removed.

[0254] I / R injury induction: Immediately after nephrectomy of the right kidney, abdominal contents are moved to the right to induce renal ischemia. Next, the left renal artery is severed.

[0255] Micro-aneurysm clips are used to block blood flow to the kidneys and induce renal ischemia. Clamp the pedicle. The duration of renal ischemia begins from the time of clamping. The success of ischemia is confirmed by the color changing from red to dark purple in a few seconds. After ischemia, microarteriovenous tissue is used. Reperfusion is indicated by removing the aneurysm clip and the kidney changing color to red.

[0256] (b) Results and interpretation The results are shown in Figure 12. The candidates are those whose specific in vitro activity correlates with in vitro activity. The dose-response is shown in Figure 12A for the parent EP28, and Figure 12B for EP1x. Figure 12C shows the results for EP17 and the results for EP14. EP28 and EP1xEP17 showed similar dose-response behaviors, while in vitro activity was better. Higher EP14 levels are sufficient at lower doses.

[0257] 14. Example 13: Titer, yield, and potential To produce selected candidates on a commercial scale, they must be expressible in relatively high yields. This is important. In the case of therapeutic proteins, this allows for the selection of high-productivity clones. In addition to the lack of shrinkage technology, the process is complex, making it less straightforward compared to therapeutic antibodies. There is a possibility.

[0258] Both candidates, EP14aa1-3 and EP28aa1-3, possess equivalent technical characteristics, This was extremely difficult. In particular, low-expression titers in early-expression batches (data not shown). This affects production costs, or because the control of host cell proteins is not robust, It could become even lower after the scale expands.

[0259] In order to improve protein expression through early clonal selection of both candidates, A purification process tailored to the specific needs of each individual was required. For this purpose, candidate EP28a was selected. A pool of cells expressing a1-3 and EP14aa1-3 was created.

[0260] As a host cell line for the production of EP28aa1-16 / EP14aa1-3 expressing cell lines The parent CHO cell line was used. The host cell line was, for example, a reference in both of them as a whole. Patent applications incorporated by, International Publication No. 2015092737 brochure and the same CHO, which is well known to those skilled in the art, as described in brochure No. 2015092735 -Derived from the K1 cell line. EP28aa1-16 / EP14aa1-3 recombinant cell line A single vial from the CHO strain was used for preparation.

[0261] Cells were grown in a chemically defined culture medium. Linearized positive cells were treated with 1 μg of SwAI. Mido DNA, an expression vector encoding EP28aa1-16 / EP14aa1-3 It was added for gene transfer. The gene transfer reaction was carried out in a chemically defined culture medium.

[0262] Following the manufacturer's instructions, use the AMAXA Gene Pulser to perform electro Gene transfer was performed by electroporation. The parental CHO cells used for gene transfer were: The cells were in the exponential growth phase, with a viability exceeding 95%. In total, there were 5 x 10 cells. 6Individual / gene transfer Three gene transfers were performed using [a specific method / library]. Immediately after gene transfer, a scientifically defined culture medium was used. The cells were transferred to a shaking flask containing the specified ingredients.

[0263] The cell pool was incubated at 36.5°C and 10% CO2 for 48 hours before the start of the selection process. The selection procedure was performed using the selection marker encoded in the current vector. Gene transfer and After 48 hours of growth under low folate conditions, 10 nM MTX was added to a chemically defined medium. The addition applied further selective pressure. 21 days after the start of MTX selection, primarily A pool of MTX-resistant cells emerged. After pooling, the retrieved cells were frozen. EP2 To determine the concentration of 8aa1-16 / EP14aa1-3, in a chemically defined medium... A standard fed-batch culture was established. Reverse-phase chromatography was used to determine the product concentration. -(RPC) was used. FACS single-cell sorting to obtain individualized clonal cell lines. / For cell printer procedures, CH28aa1-16 / EP14aa1-3 is produced. O-cell pooling was used.

[0264] 15. Example 14: Therapeutic use Extracellular ATP that activates P2X7R has several effects, including promoting graft-versus-host disease. It is clearly associated with the disease (Wilhelm et al. Graft-versu s-host disease is enhanced by extracellu lar ATP activating P2X7R.Nature Medicine 16:12, pages 1434-1439(2010)).

[0265] Furthermore, by adjusting the level of ADP in both in vitro and in vivo trials... CD39 is shown to be an important apirase in cardiovascular health. The enzyme is known to inhibit platelet aggregation by metabolizing extracellular ADP. ru.

[0266] Human apirase irreversibly binds clopidogrel (P) to ADP receptors on platelets. In contrast to other therapeutic agents such as LaVix (trademark), it does not covalently bind to platelets. This leads to a more rapid resolution of therapeutic inhibition, and therefore to patients with excessive platelet activation. A safer approach becomes possible. This is especially true for patients with excessive platelet activation. It provides a safer approach.

[0267] Therefore, the therapeutic use of compounds that reduce the level of extracellular ATP, such as the compound according to the present invention There is clear justification for this.

[0268] Specific non-limiting examples of therapeutic uses of the compounds according to the present invention include acute injuries caused by trauma and / or hypoxia. Organ damage, such as acute respiratory distress syndrome (ARDS), lung injury, renal failure, and acute kidney injury. Acute kidney injury (AKI) (including acute kidney injury following coronary artery bypass grafting), kidney or other solid organ Post-transplant organ dysfunction or vascular disease following organ transplantation (including xenotransplantation), such as occlusive hematopoiesis. Treatment of individuals suffering from vascular diseases, transplants and xenotransplants, and the following conditions: stroke, coronary artery disease. Disease or injury resulting from myocardial infarction, atherosclerosis, arteriosclerosis, embolism, eclampsia Pre-symptoms, angioplasty, vascular injury, transplantation, neonatal hypoxic-ischemic encephalopathy, platelet-related ischemic injury ( (including pulmonary ischemia, coronary artery ischemia and cerebral ischemia), ischemia-reperfusion injury (IRI), thrombotic disorders (coronary artery blood Thrombosis, including cerebral artery thrombosis, intracardiac thrombosis, peripheral artery thrombosis and venous thrombosis), kidney or This refers to organ dysfunction following the transplantation of other solid organs (including xenotransplantation). Other non-limiting examples of the therapeutic uses of the compound include burns or radiation injuries, treatment of sepsis, To improve wound healing, reduce bleeding or the risk of bleeding, prevent organ damage, prevent graft-versus-host disease, or This is to prevent transplant rejection.

[0269] Particularly preferred therapeutic uses of the compounds according to the present invention include acute kidney injury (AKI), such as coronary artery disease. This is acute kidney injury, sepsis, or rhabdomyolysis following a vascular bypass transplant. It increases patient mortality and there is no standard of care (SoC). The main cause of AKI in the intensive care unit. The main causes are sepsis (47.5%), major surgery (34%), cardiogenic shock (27%), and hematologic disorders. The causes are fluid volume reduction (26%) and nephrotoxic compounds (19%). Furthermore, AKI is a chronic kidney disease ( It is an independent and strong risk factor for the development of CKD. 20-30% of patients undergoing major cardiac surgery. 0% will result in acute kidney injury. Another preferred embodiment is acute kidney injury associated with cardiac surgery. This invention relates to the use of isolated apirase for the treatment of [condition].

[0270] In another embodiment, this disclosure relates to post-transplant organ dysfunction (DGF), acute respiratory distress syndrome. Group (ARDS), acute myocardial infarction (AMI), traumatic brain injury (TBI) / acute ischemic attack ( Treatment of a combination of these conditions, often referred to as AIS (arterial insufficiency) or multiple organ failure (MOF). The present invention relates to an isolated apirase for use.

[0271] 16. Acute kidney injury (AKI) is a common complication of sepsis. It affects 28% of patients with sepsis. This leads to AKI. In a further preferred embodiment, the present disclosure relates to acute kidney injury associated with sepsis. This invention relates to the use of isolated apirase for the treatment of injury.

[0272] Example 15: Therapeutic composition Therapeutic proteins are generally available in an immediate-administration aqueous form or in a suitable diluent before administration. It is formulated as either a freeze-dried product for composition. The protein is, for example, prefilled sylvestre. It can be formulated in a syringe either as a lyophilized product or as an aqueous composition.

[0273] Appropriate formulations include aqueous pharmaceutical compositions or therapeutic protein-based formulations for delivery to the patient. It can be reconstituted to give a solution with a high concentration of components and a low level of protein aggregation. It may be possible to provide freeze-dried products. High concentrations of protein are substances that must be delivered to the patient. It is useful because it reduces the amount (dose) of the drug. By reducing the volume of administration, it is possible to reduce the amount of the drug in the patient. The time required to deliver a fixed dose is reduced. The aqueous solution of the present invention with high protein concentration. The composition is particularly suitable for subcutaneous administration.

[0274] Accordingly, the present invention relates to the administration of a therapeutic protein to a target, for example, for subcutaneous administration. To provide an aqueous pharmaceutical composition suitable for [the purpose].

[0275] When combined with a pharmaceutically acceptable carrier, therapeutic proteins can be used as a pharmaceutical composition. Such compositions can be used, in addition to therapeutic proteins, include carriers, various diluents, and fillers. It may contain agents, salts, buffers, stabilizers, solubilizers, and other substances well known in the art. Physical characteristics depend on the route of administration. Pharmaceutical compositions for use in the disclosed manner are specific It may also contain additional therapeutic agents for treating target damage.

[0276] 17. Example 16: Route of administration Generally, the protein according to the present invention is administered by injection, for example, intravenously, intraperitoneally, or subcutaneously. It is administered by one of the following methods. The method for carrying out this administration is known to those skilled in the art. To obtain a composition that can be administered orally orally, or that can pass through mucous membranes. It may also be possible. As recognized by those skilled in the art, appropriate for a specific selected route of administration. Thus, some appropriate means for administration can be used.

[0277] Possible routes of administration include parenteral (e.g., intravenous (IV or IV), intramuscular ( IM), intradermal, subcutaneous (SC or SC) or intravenous infusion), oral and pulmonary (e.g., inhalation), nasal cavity These include percutaneous (local), transmucosal, intra-arterial, continuous intravenous infusion, and rectal administration. Parenteral, intradermal or The solution or suspension used for subcutaneous application consists of the following components: sterile diluent, e.g., Water for spraying, saline solution, fixing oil, polyethylene glycol, glycerin, propylene glycol Coal or other synthetic solvents; antimicrobial agents, such as benzyl alcohol or methylparaben. Antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, for example For example, ethylenediaminetetraacetic acid; buffer solutions, such as acetic acid, citric acid or phosphoric acid, and It may also contain agents for adjusting isotonicity, such as sodium chloride or dextrose. The pH can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral formulations are , in glass or plastic ampoules, disposable syringes or multi-dose vials It can be enclosed in.

[0278] By administering a "loading dose" of the protein according to the present invention to a subject requiring treatment, Then, apirase therapy can be initiated. The "loading dose" is the amount of the present invention administered to the subject. The initial dose of protein is intended, and here the dose of the protein according to the present invention administered is more It falls within the high dose range. "Loading dose" refers to a single dose, for example, an intravenous administration of protein. In some cases, it is administered as a single intravenous infusion, or the complete "loading dose" is administered over a period of approximately 24 hours (or the disease). Depending on the severity, if multiple intravenous administrations are necessary, multiple doses will be administered (within the first month). For example, if protein is administered intravenously, it may be administered as multiple intravenous infusions. After administering the "loading dose," one or more further therapeutic doses of the protein according to the present invention are administered. The effective dose is administered. For example, a schedule of weekly administration or once every two weeks for three weeks. Subsequent therapeutically effective doses may be administered according to the dosage schedule, either once every interval or once every four weeks. In such embodiments, the subsequent therapeutically effective dose generally falls within a lower dose range. ru.

[0279] Alternatively, in some embodiments, the post-treatment of the protein according to the present invention after the "loading dose" The therapeutically effective dose is administered according to the "maintenance schedule" and is used for the treatment of proteins according to the present invention. The effective dose is once a month, once every 6 weeks, once every 2 months, and once every 10 weeks. Once every 3 months, once every 14 weeks, once every 4 months, once every 18 weeks. Once every 5 months, once every 22 weeks, once every 6 months, once every 7 months, and every 8 months Once every 10 months, once every 9 months, once every 11 months, or every 12 months It is administered once per instance. In such embodiments, the therapeutic effect of the protein according to the present invention The dosage is administered at more frequent intervals, for example, once every two weeks to once a month, with subsequent doses being given. If administered, it should fall within a lower dose range, or especially when subsequent doses are administered less frequently. When administered at intervals, for example, if subsequent doses are administered 1 to 12 months apart, the effect is greater. It falls within the recommended dosage range.

[0280] The timing of administration is generally measured from the first day of administration of the active compound, and this is the "baseline." Also known as "line," however, different healthcare providers use different naming conventions.

[0281] It is worth noting that Week 0 may be referred to as Week 1 by some healthcare providers, while Day 0 may be called Day 1 by some healthcare providers. Therefore, depending on the doctor, for example... For example, on the 21st day of the 3rd week, on the 22nd day of the 3rd week, on the 21st day of the 4th week, It could be called a dose given on the 22nd day, while on the other hand, it could refer to the same dosing schedule. For consistency, the first week of administration will be referred to as Week 0 in this specification, while the following week of administration... The first day is called Day 1. However, this naming convention is used simply for consistency and not limited to It should not be interpreted as meaning that weekly administration is done by a doctor designating a specific week as "Week 1" or " Those skilled in the art will understand that this is the provision of a weekly dose of protein, regardless of whether it is called "week 2" or not. This will be understood. Examples of dosage regimens as described herein can be seen in Figures 1 and 2. The dosage does not need to be provided at a precise time point, but rather, for example, as long as it is provided at the appropriate week. The dose that would normally be given on day 29 could be provided, for example, from day 24 to day 34, or for example, on day 30. It will be understood that...

[0282] As used herein, "[Tampa] to enable delivery of [specified dose]" The phrase "a container having a sufficient amount of substance" refers to certain types of containers (e.g., vials, pens, syringes). (i) therein, a protein (e.g., a pharmaceutical composition) that can be used to provide the desired dose. It is used to mean that the volume of (as part of) is being arranged. For example, desired If the dose is 500 mg, the clinician may use a protein prescription with a concentration of 250 mg / mL. From a container containing [the substance], a container containing 2 mL of a protein formulation with a concentration of 500 mg / mL. From 1 mL, 0.5 mL from a container containing a protein formulation with a concentration of 1000 mg / mL L, etc., may be used. In each such case, these containers are for the desired 500 mg. It has a sufficient amount of protein to enable the delivery of a certain quantity.

[0283] As used herein, "to enable the delivery of [specified dose] via [route of administration]" The phrase "prescribed in the specified dose" means that it is administered via a designated route of administration (e.g., via sc or IV). Certain pharmaceutical compositions may be used to provide a desired dose of protein. Used to mean. For example, if the desired subcutaneous dose is 500 mg, clinicians This includes a 2 mL protein formulation with a concentration of 250 mg / mL, and a 500 mg / mL concentration... 1 mL of protein formulation with a certain degree, 0.5 mL of a solution with a concentration of 1000 mg / mL Protein formulations may be used. In each of these cases, these proteins The protein formulation is at a sufficiently high concentration to enable subcutaneous delivery of this protein. Delivery generally requires the delivery of a volume of less than approximately 2 mL, preferably less than approximately 1 mL. However, larger volumes can be delivered over time, for example, using a patch / pump mechanism. .

[0284] The use of proteins for the manufacture of drugs for treating tissue damage in patients is described herein. It is indicated that this medication is prescribed to include a container, and each container contains at least approximately 75 mg, 1 A device to enable delivery of 50 mg, 300 mg, or 600 mg protein / unit dose. It contains a sufficient amount of protein.

[0285] The use of proteins for the manufacture of drugs for treating tissue damage in patients is described herein. It was shown that this drug contains 75 mg, 150 mg, and 300 mg of protein in a 600 mg dose. / To enable systemic delivery (e.g., IV or SC delivery) at a single dose. It is prescribed based on the dosage.

[0286] 18. Example 17: Kit This disclosure relates to a kit for treating patients with (as applicable) protein-induced tissue damage. This also includes. Such kits include proteins (e.g., in liquid or lyophilized form) or this The kit contains a pharmaceutical composition containing the protein (mentioned above). Means for administering the substance (e.g., syringes and vials, pre-filled syringes, pre-filled syringes) This may include a filtrate pen, patch / pump, and instructions for use. These instructions may include specific dosage instructions. It may be disclosed that the patient is provided with protein as part of the treatment plan. These kits are, for example, For example, for delivery in combination with the disclosed protein, further treatment for psoriasis It may also contain therapeutic agents (listed above).

[0287] The phrase "means for administration" refers to pre-filled syringes, vials and syringes. Includes injection pens, automatic infusion devices, IV infusion and bags, pumps, patches / pumps, etc. To show any available equipment for systemic administration of drugs to a patient, without limitation. It is used. Using such items, the patient administers the drug himself (i.e., by himself / herself) Drugs may be administered (for this purpose), or a caregiver or physician may administer drugs.

[0288] a) A pharmaceutical composition containing a therapeutically effective amount of protein; b) Administering the protein to a patient means for; and a) providing subcutaneous administration of protein to patients who need it. A kit for the treatment of a patient with tissue damage, including instructions, is disclosed herein.

[0289] Sequence List Table 35 discloses useful amino acid and nucleotide sequences for carrying out the present invention.

[0290] [Table 40]

[0291] [Table 41]

[0292] [Table 42]

[0293] [Table 43]

[0294] [Table 44]

[0295] [Table 45]

[0296] Table 46

[0297] Table 47

[0298] Table 48

[0299] Table 49

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[0301] Table 51

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[0303] Table 53

[0304] Table 54

[0305] Table 55

[0306] Table 56

[0307] Table 57

[0308] Table 58

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[0345] Table 95

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[0350] Table 100

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[0373]

Table 123

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[0375] Table 125

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[0380] Table 130

[0381] Table 131

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[0384] Table 134

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[0386] Table 136

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[0388] Table 138

[0389] Table 139

[0390] Table 140

Claims

1. At least two selected from the list consisting of N-terminal deletion, C-terminal deletion, and central modification Solubilized human apirase having the following modification.

2. Solubilized human apyramid according to claim 1, having an N-terminal deletion, a C-terminal deletion, and a central modification. -ze.

3. a) The N-terminal deletion is 30 to 50 amino acids long, b) The C-terminal deletion is 20 to 40 amino acids long, c) The central modification includes deletions and / or point mutations, The solubilized human apirase according to claim 1 or 2.

4. The central modification comprises the deletion of 10 to 15 consecutive amino acids, according to any one of claims 1 to 3. Solubilized human apirase as described in item 1.

5. The deletion in the central region corresponds to amino acid number 19 in the wild-type CD39 sequence described in Sequence ID No.

1. The solubilized human apirase according to claim 4, wherein deletions 3 to 204 are present.

6. The aforementioned central modification is K71E, N73Q, V95A, G102D, Y104S, T10 6S, R113M, L149M, V151A, E173D, T229A, L254M, K 258R, W263R, E276D, N292Q, R304G, I319T, N327Q , A362N, F365S, N371Q, K405N, Y412F, L424Q, H43 6D, I437N, F439S, G441D, N457Q, P463S and S469R Selected from the following groups, 1, 2, 3, relative to the wild-type CD39 sequence described in Sequence ID No. 1 A possible method according to any one of claims 1 to 5, comprising a point mutation comprising four or five point mutations. Lysozyme human apirase.

7. Sequence ID 4, Sequence ID 6, Sequence ID 32, Sequence ID 54, Sequence ID 56, Sequence ID 70 The sequence comprising, selected from the group consisting of sequence number 76 and sequence number 78, according to claims 1 to 6 Solubilized human apirase as described in any one of the items.

8. Sequence ID 131, Sequence ID 133, Sequence ID 135, Sequence ID 137, Sequence ID 139 The present invention relates to any one of claims 1 to 7, which includes an array selected from the group consisting of and sequence number 141. The solubilized human apirase described.

9. Sequence IDs 213, 227, 219, 225, and 217 , SEQ ID NO: 209, SEQ ID NO: 221, SEQ ID NO: 72, SEQ ID NO: 215, SEQ ID NO: 223, Includes a sequence selected from the group consisting of SEQ ID NOs: 211, 58, and 229. The solubilized human apirase according to claim 8, comprising either or the same.

10. A sequence selected from the group consisting of sequence numbers 58, 72, and 229. The solubilized human apirase according to claim 9.

11. A therapeutically effective dose of apirase according to any one of claims 1 to 10, and one or more pharmaceuticals A pharmaceutical composition comprising a suitably acceptable carrier.

12. The pharmaceutical composition according to claim 11, further comprising one or more further active ingredients.

13. An isolated apirase according to any one of claims 1 to 10 for use as a pharmaceutical agent.

14. Isolation apparatus according to any one of claims 1 to 10 for use in the treatment of tissue injury -ze.

15. The aforementioned tissue damage is acute brain injury (stroke); acute multiple organ failure; kidney or other solid organ damage. Post-transplant organ dysfunction; burn damage; radiation damage; trauma and / or Acute injury due to hypoxia, such as acute respiratory distress syndrome (ARDS) or lung injury; acute kidney injury For example, thoracic surgery (e.g., aortic valve replacement, coronary artery bypass surgery) or sepsis or rhabdomyolysis. Acute kidney injury as a result of the alleviation of symptoms or the toxic effects of antibiotics or other drugs; acute Myocardial injury, an isolated apirase for use according to claim 14.

16. Any one of claims 1 to 10 for use in the treatment of acute kidney injury associated with cardiac surgery Listed isolated apirase.

17. Post-transplant organ dysfunction (DGF), acute respiratory distress syndrome (ARDS), acute myocardial infarction (AMI), Traumatic Brain Injury (TBI) / Acute Ischemic Attack (AIS), Ischemia-Reperfusion Injury (I) In the treatment of combinations of these conditions, often referred to as RI (reactive oxygen species) or multiple organ failure (MOF), An isolated apirase according to any one of claims 1 to 10 for use.

18. Any one of claims 1 to 10 for use in the treatment of acute kidney injury associated with sepsis. The isolated apirase described above.

19. This includes administering a therapeutically effective dose of solubilized human apirase to a subject. A method for treating tissue damage in a medical setting.

20. The solubilized human apirase is the apirase described in any one of claims 1 to 10. The method according to claim 19.

21. The aforementioned tissue damage may manifest as acute brain injury (stroke); acute multiple organ failure; or kidney or other solid organ failure. Post-plant organ dysfunction; burn damage; radiation damage; trauma and / or acute Acute injuries due to hypoxia, such as respiratory distress syndrome (ARDS) or lung injury; acute kidney injury, For example, thoracic surgery (e.g., aortic valve replacement, coronary artery bypass surgery) or sepsis or rhabdomyolysis Secondary acute kidney injury due to the toxic effects of antibiotics or other drugs; acute cardiac The method according to claim 19 or 20, which is a muscle injury.

22. A method for treating acute kidney injury associated with cardiac surgery, and for which such treatment is necessary. A method comprising administering a therapeutically effective dose of solubilized human apirase to a subject.

23. Post-transplant organ dysfunction (DGF), acute respiratory distress syndrome (ARDS), acute myocardial infarction (AMI), Traumatic Brain Injury (TBI) / Acute Ischemic Attack (AIS), Ischemia-Reperfusion Injury (IR) I) Methods for treating combinations of these conditions, often referred to as multiple organ failure (MOF). Therefore, to subjects requiring such treatment, a therapeutically effective dose of solubilized human apirase is administered. A method including administration.

24. A method for treating acute kidney injury associated with sepsis, and for which such treatment is necessary. A method comprising administering a therapeutically effective dose of solubilized human apirase to a subject.

25. An isolated nucleic acid molecule encoding any apirase according to any one of claims 1 to 10.

26. Suitable for recombinant production of isolated apirase according to any one of claims 1 to 10. A cloning or expression vector containing one or more nucleic acid sequences as described in item 25.

27. A host cell comprising one or more cloning or expression vectors as described in claim 26.

28. The method includes culturing the host cells described in claim 27 and purifying and recovering the apirase. A process for producing apirase as described in any one of the requests 1 to 10.

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