Improvement in the production of CD39 variants

Abstract

The present invention relates to the use of miRNA technology for improving the recombinant production of CD39 or its variants in CHO cells. The miRNA is used for knockdown of the endogenous protein CCL2 of CHO cells, which is difficult to separate from CD39 or its variants during purification.

Description

Technical Field

[0001] The present invention relates to the field of recombinant protein production. The present invention provides a method for producing CD39 and its variants, which reduces the level of host cell proteins that interfere with the purification of CD39 and its variants. The production of interfering host cell proteins is reduced using artificial miRNAs that target host cell proteins.

Background Art

[0002] The generation of recombinant cell lines for producing secreted proteins requires transfection of DNA vectors into host cells and enrichment of stable transfectants using selection markers. Secreted proteins can often affect cell parameters such as growth, viability, and / or productivity, which require cell line engineering methods to achieve stable cell lines with high expression.

[0003] Similarly, the quality of secreted recombinant proteins can be affected by endogenous cell-derived factors. In many cases, endogenously expressed proteins such as cell surface receptors, enzymes, or proteases (host cell proteins, HCPs) can be identified as the root cause of unwanted effects that affect the quality of recombinant proteins (see, for example, WO 2014 / 097113A2). Furthermore, host cell proteins are well known as process-related impurities in biopharmaceuticals. When present in drug products, these impurities can cause adverse effects due to drug immunogenicity, inflammation, and increased other activities associated with specific residual host cell protein functions (Vanderlaan et al., 2018, Biotechnology Progress 34, 828-837). The composition and abundance of HCPs present in various steps of the manufacturing process and in the final drug substance depend on many factors. These factors can be very difficult to predict in advance and are often learned only through testing during process development.

[0004] Among the undesirable effects are enzymatic cleavage of the polypeptide chain of the recombinant protein, such as digestion of the whole protein or amino acid clipping, i.e., removal of one or several amino acids from the N-terminus or C-terminus of the protein of interest. Other effects are undesirable post-translational modifications or removal of desired modifications. Further, the endogenous gene products of the host cell can specifically interact with the protein of interest. Such interactions can recruit the protein of interest from the supernatant of the cell culture, making it difficult to remove the endogenous protein during purification, or can lead to activation of signal transduction pathways within the host cell that result in a decrease in cell growth, viability and / or productivity. In other cases, the endogenous gene product may simply have chemical and physical properties very similar to the protein of interest, making it difficult to develop a purification process that efficiently removes the host cell protein without reducing the yield of the protein of interest. This is particularly relevant for therapeutic proteins, where high purity and low residual levels of host cell proteins in the final product are requirements for obtaining and maintaining marketing approval.

[0005] Thus, the endogenous gene products of the host cell can significantly interfere with the recombinant production of the protein of interest. In view of the above, there is a need in the art to provide strategies for reducing the undesirable effects of host cell proteins on the recombinant production of the protein of interest. SUMMARY OF THE INVENTION

[0006] The inventors aimed to provide a method for purifying human CD39 and its variants produced in CHO cells, particularly a soluble variant of CD39 lacking the membrane anchor sequence. During the development of the purification method, CCL2 was identified as a host cell protein that has physicochemical properties very similar to those of the CD39 variant and is thus not significantly removed during the purification process. Therefore, the inventors established a cell line producing a CD39 variant with reduced CCL2 levels by using miRNA against this host cell protein. As a result, the purity of the final CD39 variant could be significantly improved.

[0007] In view of this, in a first aspect, the present invention provides a method for producing CD39 or a variant thereof, comprising: (a) providing CHO cells capable of producing CD39 or a variant thereof; (b) culturing the CHO cells in a cell culture under conditions that allow the production of CD39 or a variant thereof; (c) obtaining CD39 or a variant thereof from the cell culture; (d) optionally treating CD39 or a variant thereof; and the CHO cells are engineered to reduce the production of CCL2 in the CHO cells. The present invention relates to a method for producing CD39 or a variant thereof.

[0008] In a second aspect, the present invention provides CHO cells capable of producing CD39 or a variant thereof and engineered to reduce the production of CCL2 in the CHO cells.

[0009] In certain embodiments of the above aspects, the CHO cells produce an miRNA targeting CCL2. In particular, the CHO cells are engineered by introduction of a vector nucleic acid according to a fifth aspect. In further embodiments of the above aspects, the CHO cells are engineered by knockout of the CCL2 gene.

[0010] In a third aspect, the present invention provides a method for improving the production of CD39 or a variant thereof in CHO cells, comprising: (a-i) providing a CHO cell capable of producing CD39 or a variant thereof; (a-ii) manipulating the CHO cell to reduce the production of CCL2 in the CHO cell; and thereby provides a method for improving the production of CD39 or a variant thereof in CHO cells.

[0011] In certain embodiments, the step (a-ii) of manipulating the CHO cell to reduce the production of CCL2 comprises introducing into the CHO cell a vector nucleic acid according to the fifth aspect.

[0012] In a fourth aspect, the present invention provides an expression cassette for the expression of miRNA in CHO cells, comprising a template sequence of pri-miRNA, wherein the pri-miRNA is suitable for being processed in CHO cells to form a miRNA targeting CCL2.

[0013] In a fifth aspect, the present invention provides a vector nucleic acid for the transfection of CHO cells, comprising the expression cassette according to the fourth aspect.

[0014] In a sixth aspect, the present invention provides a method for producing a CHO cell capable of expressing CD39 or a variant thereof, comprising introducing into the CHO cell the vector nucleic acid according to the fifth aspect. The vector nucleic acid may further comprise a coding sequence encoding CD39 or a variant thereof, or another nucleic acid comprising a coding sequence encoding CD39 or a variant thereof is present in or introduced into the CHO cell.

[0015] In a seventh aspect, the present invention provides a composition comprising CD39 or a variant thereof and CCL2, obtained by production using the CHO cell according to the second aspect. (i) The amount of CCL2 in the composition is at least 10-fold lower compared to the same composition obtained by production using CHO cells that were not engineered to reduce the production of CCL2 in CHO cells, and / or (ii) the composition contains CCL2 at a concentration of 100 ppm or less, preferably 25 ppm or less, more preferably 10 ppm or less, Provided is a composition comprising CD39 or a variant thereof and CCL2.

[0016] In an eighth aspect, the present invention provides CHO cells engineered to reduce the production of CCL2 in CHO cells. In certain embodiments, the CHO cells produce a miRNA that targets CCL2. In particular, the CHO cells are engineered by introduction of the vector nucleic acid according to the fifth aspect. In a further embodiment of the above aspect, the CHO cells are engineered by knockout of the CCL2 gene.

[0017] Other objects, features, advantages and aspects of the present invention will become apparent to those skilled in the art from the following description and the appended claims. However, it should be understood that the following description, the appended claims, and the specific examples, which illustrate preferred embodiments of the present application, are presented by way of illustration only. Various modifications and variations within the spirit and scope of the present invention will readily occur to those skilled in the art from reading the following.

[0018] Definitions As used herein, the following expressions are intended to have the meanings preferably indicated below, unless the context in which they are used indicates otherwise.

[0019] As used herein, the term "comprising" includes, in addition to its literal meaning, the terms "consisting essentially of" and "consisting of", and specifically refers to these. Thus, the term "comprising" refers to embodiments in which the "comprising" subject does not include further elements beyond the specifically recited elements, as well as embodiments in which the "comprising" subject may include and / or actually encompasses further elements. Similarly, the term "having" should be understood as the term "comprising", and also includes the terms "consisting essentially of" and "consisting of", and is specifically referred to. The term "consisting essentially of" refers, where possible, to embodiments in which the subject, in addition to the specifically recited elements of which it essentially consists, includes further elements of 20% or less, particularly 15% or less, 10% or less, or particularly 5% or less.

[0020] The term "nucleic acid" includes single-stranded and double-stranded nucleic acids, as well as ribonucleic acid and deoxyribonucleic acid. It may include naturally occurring nucleotides as well as synthetic nucleotides, and may be modified naturally or synthetically, for example, by methylation, 5'- and / or 3'-capping. In certain embodiments, the nucleic acid refers to double-stranded deoxyribonucleic acid.

[0021] The term "expression cassette" refers, in particular, to a nucleic acid construct that enables and can regulate the expression of a coding nucleic acid sequence and / or a template nucleic acid sequence introduced therein. An expression cassette may include a promoter, a ribosome binding site, an enhancer, and other control elements that regulate the transcription of a gene or the translation of mRNA. The exact structure of an expression cassette can vary depending on the species or cell type, but generally includes 5' non-transcribed sequences and 5' and 3' untranslated sequences that are involved in the initiation of transcription and translation, respectively, such as a TATA box, a capping sequence, a CAAT sequence, etc. More specifically, the 5' non-transcribed expression control sequence includes a promoter region that contains a promoter sequence for the transcriptional control of an operably linked nucleic acid. An expression cassette may also include an enhancer sequence or an upstream activator sequence. Some expression cassettes are used only for the transcription of a template nucleic acid sequence into an RNA product such as pri-miRNA. Such expression cassettes do not necessarily include regulatory elements for translation.

[0022] According to the present application, the template nucleic acid is understood as DNA that is transcribed into a functional RNA product or its precursor, particularly pri-miRNA. A functional RNA product has, in particular, alone or in combination with other RNA products and / or proteins, a biological activity, such as the activity of a miRNA that interferes (in combination with the proteins of RISC) with the expression of a target gene.

[0023] According to the present invention, the term "promoter" refers to a nucleic acid sequence that is located upstream (5') of the nucleic acid sequence to be expressed and controls the expression of the sequence by providing a recognition and binding site for RNA-polymerase. A "promoter" may include additional recognition and binding sites for additional factors involved in the regulation of gene transcription. A promoter can control the transcription of a prokaryotic gene or a eukaryotic gene. Furthermore, a promoter can be "inducible", i.e., capable of initiating transcription in response to an inducer, or "constitutive" if transcription is not controlled by an inducer. A gene under the control of an inducible promoter is not expressed or is expressed only to a low extent in the absence of the inducer. In the presence of the inducer, the gene is switched on or the transcription level increases. This is generally mediated by the binding of specific transcription factors.

[0024] The term "vector" is used herein in its most general sense and includes any intermediate vehicle for a nucleic acid that enables the nucleic acid to be introduced, for example, into prokaryotic and / or eukaryotic cells and, where appropriate, integrated into the genome. This type of vector is preferably replicated and / or expressed intracellularly. Vectors include plasmids, phagemids, bacteriophages or viral genomes. The term "plasmid" as used herein generally relates to an extrachromosomal genetic material that can replicate independently of chromosomal DNA, usually a circular DNA double-stranded construct. The vectors according to the present invention can exist in circular or linearized form. The "vector nucleic acid" as used herein is the nucleic acid that forms the vector or the nucleic acid part of the vector.

[0025] The terms "5'" and "3'" are conventions used to describe features of nucleic acid sequences related to either the position of genetic elements and / or the direction (5' to 3') of an event, such as transcription by RNA polymerase or translation by ribosomes proceeding in the 5' to 3' direction. Synonyms are upstream (5') and downstream (3'). Conventionally, DNA sequences, genetic maps, vector maps, and RNA sequences are drawn 5' to 3' from left to right, or the 5' to 3' direction is indicated by an arrow that points in the 3' direction. Thus, 5' (upstream) indicates a genetic element located towards the left-hand side when following this convention, and 3' (downstream) indicates a genetic element located towards the right-hand side.

[0026] "Polypeptide" or "polypeptide chain" refers to a molecule containing a polymer of amino acids linked together by peptide bonds. Polypeptides include polypeptides of any length, including proteins (e.g., having more than 50 amino acids) and peptides (e.g., having 2 - 49 amino acids). In particular, a polypeptide or polypeptide chain can be part of a protein consisting of two or more polypeptide chains. Polypeptides include any active or biologically active protein and / or peptide. A polypeptide can be a pharmaceutically or therapeutically active compound, or a research tool used in assays and the like.

[0027] When a target amino acid sequence shares identity with a reference amino acid sequence over its entire length of at least 75%, more preferably at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98% or at least 99%, the target amino acid sequence is "derived from" or "corresponds to" the reference amino acid sequence. In certain embodiments, a target amino acid sequence that is "derived from" or "corresponds to" a reference amino acid sequence is 100% identical to the reference amino acid sequence over its entire length. Similarly, when a target nucleotide sequence shares identity of at least 75%, more preferably at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98% or at least 99% with a reference nucleotide sequence over its entire length, the target nucleotide sequence is "derived from" or "corresponds to" the reference nucleotide sequence. In certain embodiments, a target nucleotide sequence that is "derived from" or "corresponds to" a reference nucleotide sequence is 100% identical to the reference nucleotide sequence over its entire length. "Identity" of an amino acid sequence or nucleotide sequence is preferably determined in accordance with the present invention over the entire length of the reference sequence.

[0028] As used herein, the abbreviation "microRNA" or "miRNA" refers to a single-stranded non-coding RNA molecule that plays a role in RNA silencing and post-transcriptional regulation of gene expression. miRNAs generally consist of 19 to 24 nucleotides, particularly about 22 nucleotides, especially 22 nucleotides. miRNA molecules can silence mRNAs containing complementary nucleotide sequences. Silencing of the target mRNA can occur by cleavage of the mRNA, destabilization of the mRNA, or interference with translation of the mRNA. Silencing of the target mRNA results in a decrease or loss of production of the protein encoded by the target mRNA. It is generally understood in the art that miRNAs associate with dicer and argonaute proteins to form an RNA-induced silencing complex (RISC) that binds to the target mRNA.

[0029] miRNAs are produced by transcription of the miRNA precursor (pri-miRNA) from the template DNA sequence. The pri-miRNA contains a hairpin stem-loop structure with a double-stranded stem having one side connected to a loop and the other side adjacent to single-stranded 5' and 3' extensions. The double-stranded stem contains a guide strand that forms the miRNA upon processing and a passenger strand that is essentially complementary to the guide strand. In particular, the passenger strand and the guide strand are complementary to each other except for the nucleotide pair at the end of the hairpin stem-loop structure, i.e., the nucleotide pair of the passenger and guide strands that is farthest from the loop structure. The guide strand and the passenger strand generally have lengths of about 19-24 nucleotides, particularly 22 nucleotides, respectively. The remaining portion of the pri-miRNA is referred to herein as the miRNA scaffold. Thus, from 5' to 3', the pri-miRNA includes: (i) a 5' miRNA scaffold stem consisting of the 5' portion of the stem structure up to the 5' single-stranded extension and the passenger strand; (ii) the passenger strand; (iii) the miRNA scaffold loop; (iv) the guide strand; (v) a 3' miRNA scaffold stem consisting of the 3' portion of the stem structure following the guide strand and the 3' single-stranded extension. Also, the positions of the passenger strand and the guide strand may be interchanged.

[0030] The pri-miRNA is processed by cleaving the 5' and 3' miRNA scaffold stems, resulting in a hairpin structure called pre-miRNA. From 5' to 3', the pre-miRNA consists of the passenger strand, the miRNA scaffold loop, and the guide strand, and the positions of the passenger strand and the guide strand may be interchanged. Then, the loop structure is cleaved and the resulting RNA duplex is separated into two single-stranded RNA molecules, the guide strand and the passenger strand. The guide strand complementary to the target mRNA molecule forms the RISC, where the passenger strand generally has no function.

[0031] The cells referred to in this specification are, in particular, host cells. According to the present invention, the term "host cell" relates to any cell that can be transformed or transfected with an exogenous nucleic acid. Mammalian cells, such as cells derived from human, mouse, hamster, pig, goat or primate, are particularly preferred. The cells can be derived from a number of tissue types and can include primary cells and cell lines. The nucleic acid can be present in the host cell in the form of a single copy or two or more copies, and in one embodiment, is expressed in the host cell. The host cell particularly refers to a cell present in a cell culture, in particular, a cell that does not exist in a living multicellular organism.

[0032] The term "CD39" as used herein refers to human protein CD39 (cluster of differentiation 39), also known as ectonucleoside triphosphate diphosphohydrolase-1 (NTPDase1 or Ecto-ATPDase1). CD39 is an ectonucleotidase present on the cell surface that has a catalytic site on the extracellular surface for catalyzing the hydrolysis of γ-phosphate residues and β-phosphate residues of triphosphonucleosides and diphosphonucleosides to monophosphonucleoside derivatives. In particular, human CD39 has the amino acid sequence of SEQ ID NO: 50 and / or is represented by UniProt entry P49961.

[0033] "Variant of CD39" refers to a protein derived from human CD39 and having an amino acid sequence that is at least 60% identical to human CD39 over the entire length of human CD39. In certain embodiments, the variant of CD39 consists of only the extracellular domain of human CD39 (amino acids 38 - 478 of SEQ ID NO: 50) or a part thereof. In particular, the variant of CD39 has an amino acid sequence that is at least 80% identical to the extracellular domain of human CD39 over the entire length of amino acid positions 38 - 478 of human CD39. In particular, the variant of CD39 is a soluble variant, i.e., a variant lacking the transmembrane domain of human CD39 and its membrane anchor. In certain embodiments, the variant has an N-terminal deletion of 30 - 50 amino acids, a C-terminal deletion of 20 - 40 amino acids, and / or a central deletion of 10 - 15 amino acids compared to human CD39. Furthermore, the variant may contain up to five point mutations compared to human CD39. Certain variants of CD39 are disclosed in WO 2020 / 016804 pamphlet. In particular, the variant of CD39 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 29 - 49.

[0034] As used herein, the term "CCL2" refers to Chinese hamster protein CCL2 (C-C motif chemokine receptor ligand 2), also known as monocyte chemoattractant protein 1 (MCP1). CCL2 particularly has the amino acid sequence of SEQ ID NO: 24. The amount and concentration of CCL2 in the compositions referred to herein are determined particularly by ELISA (enzyme-linked immunosorbent assay) or LC-MS (liquid chromatography - mass spectrometry, particularly by LC-MS). Each measurement is preferably carried out as described in Example 5, Sections 5 and 6 respectively.

[0035] The term "pharmaceutical composition" or "pharmaceutical preparation" particularly refers to a composition suitable for administration to humans or animals, i.e., a composition containing pharmaceutically acceptable ingredients. Preferably, the pharmaceutical composition contains an active compound or a salt or prodrug thereof together with a carrier, diluent or pharmaceutical excipient, such as a buffer, preservative and tonicity modifier.

[0036] The numbers presented in this specification may, in certain embodiments, be understood as approximate numbers. In particular, these numbers may preferably be up to 10% higher and / or lower, and in particular up to 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% higher and / or lower. In certain embodiments, the numbers presented in this specification are not approximate numbers and may only vary within the range of inaccuracies of technical measurements.

[0037] The numerical ranges described in this specification include the numbers that define the ranges. The headings provided herein do not limit the various aspects or embodiments of the invention that can be read by referring to the entire specification. According to one embodiment, the subject matter described herein, in the case of a method, includes specific steps, or in the case of a composition, includes specific components, and refers to the subject matter consisting of each step or component. It is preferable to select and combine the preferred aspects and embodiments described herein, and the specific subject matter resulting from each combination of the preferred embodiments also belongs to this disclosure.

Mode for Carrying Out the Invention

[0038] The present invention is based on the finding that the endogenous CHO protein CCL2 causes significant problems during the purification of recombinantly produced therapeutic CD39 variants. Typically, residual host proteins are removed from the drug by, for example, introducing new or optimized affinity purification steps, adapting the downstream purification process, or optimizing it. As observed during the purification process, the host cell protein CCL2 is not separated from the CD39 variant by established chromatographic steps, particularly anion exchange chromatography or hydrophobic interaction chromatography. This lack of separation is thought to be caused by the very similar physicochemical properties of CCL2 and the CD39 variant, particularly the similar charge distribution.

[0039] The inventors have solved this problem by using an miRNA approach to knockdown CCL2 in CHO host cells. Such an approach has the advantage that the presence of the problematic protein can be avoided from the very beginning of the manufacturing process and does not require further timely and potentially costly adaptations of downstream processes. When a vector that produces an miRNA targeting CCL2 mRNA is introduced into CHO host cells, a strong knockdown effect is shown and the level of CCL2 in the cell culture supernatant is successfully reduced significantly. Thereby, in the final composition of the purified CD39 variant, CCL2 was reduced to only trace amounts. Furthermore, the method for producing CD39 or its variant described herein enables the production of a pure and safe CD39 or its variant suitable for use as a drug.

[0040] 1. Method for producing CD39 and its variants In a first aspect, the present invention is a method for producing CD39 or its variant, comprising: (a) providing CHO cells capable of producing CD39 or its variant; (b) culturing the CHO cells in a cell culture under conditions that allow the production of CD39 or its variant; (c) obtaining CD39 or its variant from the cell culture; (d) optionally treating CD39 or its variant; comprising wherein the CHO cells are engineered to reduce the production of CCL2 in the CHO cells, relating to a method for producing CD39 or its variant.

[0041] The steps of the method are generally performed in the order shown.

[0042] In certain embodiments, the CHO cells are engineered to reduce the production of CCL2 in the CHO cells. The CHO cells can be engineered by any suitable method for reducing the production of CCL2. Reducing the production of CCL2 particularly refers to reducing the expression of CCL2. Generally, cell line engineering, particularly genetic engineering, is used. In certain embodiments, the production of CCL2 in the CHO cells is reduced by knockdown or knockout of CCL2 expression or degradation of the CCL2 protein.

[0043] Knockdown of CCL2 expression can be achieved by reducing the amount of functional mRNA encoding CCL2 in the cell and / or reducing the translation rate of the mRNA encoding CCL2 into the CCL2 protein. In certain embodiments, the CHO cells are engineered to produce a miRNA that targets the endogenous CCL2 of the CHO cells.

[0044] Knockout of CCL2 expression can be performed by deleting one or both alleles of the gene encoding CCL2 in the genome of the CHO cells or mutating one or both of these alleles so that they cannot be transcribed into functional mRNA. Knockout of CCL2 expression can be performed, for example, using homologous recombination, site-specific nucleases, CRISPR / Cas, TALEN or Cas-Clover.

[0045] Reducing the production of CCL2 particularly means that after engineering the CHO cells, the amount of CCL2 in the cells is 50% or less of the amount of CCL2 in the cells before engineering. In certain embodiments, the amount of CCL2 in the cells is 25% or less, particularly 10% or less or 5% or less of the amount of CCL2 in the cells before engineering.

[0046] In a further aspect, the invention is a method for producing CD39 or a variant thereof, comprising: (a) providing a host cell capable of producing CD39 or a variant thereof; (b) culturing the host cells in a cell culture under conditions that allow the production of CD39 or a variant thereof; (c) obtaining CD39 or a variant thereof from the cell culture; (d) optionally processing CD39 or a variant thereof; comprising wherein the host cell is engineered to reduce the production of CCL2 in the host cell provided is a method for producing CD39 or a variant thereof.

[0047] When the host cell is not engineered to reduce the production of CCL2, it can be any cell suitable for endogenously expressing CCL2 and producing CD39 or a variant thereof, particularly human CD39 or a variant thereof. In particular, the host cell is a mammalian cell. Mammalian cells can be selected from the group consisting of, but not limited to, cells derived from mice, such as COP, L, C127, Sp2 / 0, NS0, NS1, At20 and NIH3T3; cells derived from rats, such as PC12, PC12h, GH3, MtT, YB2 / 0 and Y0; cells derived from hamsters, such as BHK, CHO and DHFR gene-deficient CHO; cells derived from monkeys, such as COS1, COS3, COS7, CV1 and Vero; and cells derived from humans, such as Hela, HEK293, CAP, PER-C6 derived from the retina, cells derived from diploid fibroblasts, myeloma cells and HepG2. In certain embodiments, the host cell is a Chinese hamster ovary (CHO) cell. The host cell may be suitable for suspension culture and / or adherent culture, and can particularly be used in suspension culture. The features, embodiments and examples of the method for producing CD39 or a variant thereof according to the first aspect of the present invention are equally applicable to the method according to this further aspect of the present invention.

[0048] 1.1 miRNA targeting CCL2 In certain embodiments, the CHO cells are engineered to produce a miRNA that targets the endogenous CCL2 of the CHO cells. It is understood that the miRNA targeting CCL2 is at least partially complementary to the mRNA or pre-mRNA encoding CCL2, can bind to the mRNA or pre-mRNA encoding CCL2, and initiate its silencing. Silencing can occur by degradation of the targeted mRNA or by preventing the targeted mRNA from being translated. The miRNA can be complementary to a portion of the 5’UTR, a portion of the coding region, a portion of the intron, and / or a portion of the 3’UTR of the CCL2 mRNA. In particular, the miRNA targeting CCL2 is complementary to a portion of the 3’UTR of the CCL2 mRNA. Exemplary miRNAs targeting CCL2 have nucleotide sequences selected from the group consisting of SEQ ID NO: 26 and 28.

[0049] In certain embodiments, the CHO cells contain an expression cassette for the production of a miRNA targeting CCL2. The expression cassette may be present on a plasmid within the cell or may be integrated into the genome of the cell. The expression cassette contains the template sequence for the pri-miRNA, which is suitable for being processed in CHO cells to form a miRNA targeting CCL2. The expression cassette enables transcription of the template sequence into pri-RNA. The template sequence can be present anywhere within the transcription region of the expression cassette.

[0050] In certain embodiments, the template sequence is present within an intron sequence. When transcribed, the intron sequence is excised from the transcribed RNA, thereby forming the pri-miRNA.

[0051] Since the expression cassette contains the template sequence of pri-miRNA within the intron sequence, it may contain additional sequences for the expression of other products, such as a coding sequence for the production of a polypeptide of interest, a coding sequence for the production of a selectable marker, and a template sequence for other RNA products, particularly other pri-miRNAs. Alternatively, the expression cassette may be used only for the production of miRNA targeting CCL2.

[0052] The expression cassette may contain the template sequence of pri-miRNA within the intron sequence. When expressed, a pre-mRNA containing the intron sequence is formed. Then, the intron sequence is spliced from the pre-mRNA, thereby forming pri-miRNA, which is then further processed to finally provide miRNA. The formed pre-mRNA does not necessarily have to contain any sequence encoding a polypeptide.

[0053] In certain embodiments, the expression cassette further comprises a polymerase II promoter. This promoter is operably linked to the template sequence of pri-miRNA and controls its expression. The promoter can be any RNA polymerase II promoter suitable for gene expression in a host cell, particularly a CHO cell. For example, the promoter can be selected from the group consisting of the cytomegalovirus (CMV) promoter, simian virus 40 (SV40) promoter, ubiquitin C (UBC) promoter, elongation factor 1 alpha (EF1A) promoter, phosphoglycerate kinase (PGK) promoter, Rous sarcoma virus (RSV) promoter, BROAD3 promoter, mouse rosa26 promoter, pCEFL promoter, chicken beta-actin promoter (CBA), beta-actin promoter combined with CMV early enhancer (CAGG), alpha-1-antitrypsin promoter, and inducible promoters such as tetracycline-inducible promoter (e.g., pTRE), and vanillic acid-inducible promoter. In certain embodiments, the promoter is the CMV promoter or the SV40 promoter, particularly the CMV promoter.

[0054] In certain embodiments, the expression cassette further comprises a terminator. The terminator is operably linked to the template sequence of the pri-miRNA and controls its expression. As used herein, the term "terminator" particularly refers to a transcription terminator that terminates the transcription of DNA into RNA by RNA polymerase II.

[0055] The template sequence of the pri-miRNA is particularly located between the promoter and the terminator of the expression cassette.

[0056] In certain embodiments, the expression cassette comprises, for example, a coding sequence encoding a polypeptide of interest or a selectable marker. In these embodiments, the expression cassette may further comprise a 5' untranslated region (5'UTR) and a 3' untranslated region (3'UTR). The intron sequence containing the template sequence of the pri-miRNA may be present within the 5'UTR, 3'UTR or the coding sequence. In particular, the intron sequence is present within the 5'UTR or 3'UTR, particularly within the 5'UTR. In alternative embodiments, the expression cassette does not comprise a coding sequence encoding a polypeptide.

[0057] The intron sequence containing the template sequence of the pri-miRNA particularly comprises a splice donor site upstream of the pri-miRNA and a corresponding splice acceptor site downstream of the pri-miRNA. By these splice donor and acceptor sites, the pri-miRNA is spliced from the pre-mRNA after transcription.

[0058] In certain embodiments, the intron sequence comprises two or more template sequences for the pri-miRNA. In these embodiments, the intron sequence comprises a splice donor site upstream of the first template sequence of the pri-miRNA, i.e., the most 5'-side template sequence, and a corresponding splice acceptor site downstream of the last template sequence of the pri-miRNA, i.e., the most 3'-side template sequence. Adjacent template sequences within the intron sequence may be separated from each other by a spacer sequence. Such a spacer sequence particularly forms an RNA stem-loop structure such as the sequence of SEQ ID NO: 22.

[0059] In certain embodiments, the expression cassette comprises only one intron sequence having one or more template sequences for the pri-miRNA. In alternative embodiments, the expression cassette comprises two or more intron sequences having one or more template sequences for the pri-miRNA.

[0060] In particular, the pri-miRNAs of two or more template sequences present within the same or different intron sequences are different from each other. In certain embodiments, all of the miRNAs produced from the pri-miRNA target CCL2, but bind to different portions of the CCL2 mRNA or pre-mRNA.

[0061] The expression cassette may comprise a coding sequence encoding a polypeptide. The coding sequence may in particular encode a selectable marker. The coding sequence is preferably operably linked to a polymerase II promoter and a terminator of the expression cassette. In embodiments where the expression cassette comprises a template sequence for the pri-miRNA and a coding sequence for a selectable marker, the expression of the miRNA is linked to the selectable marker expression. Thus, by increasing the selection pressure during clone selection, the miRNA level also increases.

[0062] The selectable marker may be selected from the group consisting of folate receptor (FAR), dihydrofolate reductase (DHFR), glutamine synthetase, puromycin, hygromycin, neomycin, zeocin and blasticidin. In certain embodiments, the selectable marker is folate receptor (FAR).

[0063] The expression cassette comprises a template sequence for the pri-miRNA. The pri-miRNA produced from the expression cassette may have any structure suitable for processing by the host cell to obtain a functional miRNA that targets CCL2. The functional miRNA induces a decrease in the level of CCL2 in the host cell.

[0064] In certain embodiments, the pri-miRNA comprises a passenger strand and a guide strand. The guide strand specifically comprises or consists of the miRNA formed after processing of the pri-miRNA by the host cell. Further, the pri-miRNA can comprise miRNA scaffold loops and / or miRNA scaffold stems, particularly the 5'miRNA scaffold stem and the 3'miRNA scaffold stem. In certain embodiments, the pri-miRNA comprises, from 5' to 3', a 5'miRNA scaffold stem, a passenger strand, a miRNA scaffold loop, a guide strand, and a 3'miRNA scaffold stem. In alternative embodiments, the pri-miRNA comprises, from 5' to 3', a 5'miRNA scaffold stem, a guide strand, a miRNA scaffold loop, a passenger strand, and a 3'miRNA scaffold stem. Embodiments in which the passenger strand is located upstream of the guide strand are preferred.

[0065] In particular, the passenger strand and the guide strand of the pri-miRNA have artificial sequences. The artificial sequences in this regard refer to sequences that do not exist as passenger or guide strands in naturally occurring miRNAs. In particular, the sequences of the passenger strand and the guide strand are not found in naturally occurring miRNAs.

[0066] In certain embodiments, the guide strand has the nucleotide sequence of SEQ ID NO: 26 and the passenger strand has the nucleotide sequence of SEQ ID NO: 25. In other embodiments, the guide strand has the nucleotide sequence of SEQ ID NO: 28 and the passenger strand has the nucleotide sequence of SEQ ID NO: 27.

[0067] In certain embodiments, one or more of the scaffold sequences of pri-miRNA or pre-miRNA are derived from naturally occurring pri-miRNAs, particularly those that are naturally occurring in mammals, particularly humans. In certain embodiments, all of the scaffold sequences of the pri-miRNA are derived from naturally occurring pri-miRNAs, particularly those that are naturally occurring in mammals, particularly humans. In particular, all of the scaffold sequences of the pri-miRNA are derived from the same naturally occurring pri-miRNA. The scaffold sequences of the pri-miRNA particularly include a 5'miRNA scaffold stem, a miRNA scaffold loop, and a 3'miRNA scaffold stem. Suitable naturally occurring pri-miRNAs from which the scaffold sequences can be derived include miR-30A, miR-E, SIBR, eSIBR, miR-1, miR-155, miR-16, miR-16-1, miR-16-2, miR-3G, miRGE, miR100, miR125b, miR-130a, miR-190a, miR-193a, miR-211, miR-26a, miR-340, miR-7-2, miR-96, and miR-44. Thus, in one embodiment, the 5'miRNA scaffold stem, the miRNA scaffold loop, and the 3'miRNA scaffold stem are derived from one or more pre-miRNAs selected from the group consisting of miR-30A, miR-E, SIBR, eSIBR, miR-1, miR-155, miR-16, miR-16-1, miR-16-2, miR-3G, miRGE, miR100, miR125b, miR-130a, miR-190a, miR-193a, miR-211, miR-26a, miR-340, miR-7-2, miR-96, and miR-44. In certain embodiments, the naturally occurring pri-miRNA from which the scaffold sequence is derived is miR-30A.

[0068] In certain embodiments, all of the pri-miRNA scaffold sequences share at least 80%, particularly at least 90%, and particularly at least 95% nucleotide sequence identity over their entire length with the corresponding scaffold sequences of naturally occurring pri-miRNAs. In certain embodiments, the 5'miRNA scaffold stem of the pri-miRNA shares at least 80%, particularly at least 85%, and particularly at least 90% nucleotide sequence identity over its entire length with the corresponding scaffold sequence of a naturally occurring pri-miRNA. In certain embodiments, the 3'miRNA scaffold stem of the pri-miRNA shares at least 80%, particularly at least 90%, and particularly at least 95% nucleotide sequence identity over its entire length with the corresponding scaffold sequence of a naturally occurring pri-miRNA. In certain embodiments, the miRNA scaffold loop of the pri-miRNA shares at least 60%, particularly at least 70%, and particularly at least 75% nucleotide sequence identity over its entire length with the corresponding scaffold sequence of a naturally occurring pri-miRNA. In these embodiments, the naturally occurring pri-miRNA can be particularly miR-30A.

[0069] In certain embodiments, the 5'miRNA scaffold stem of the pri-miRNA comprises the nucleotide sequence of any one of SEQ ID NOs: 1-7 or a sequence derived therefrom. In particular, the 5'miRNA scaffold stem of the pri-miRNA comprises the nucleotide sequence of any one of SEQ ID NOs: 1-7, or a sequence sharing at least 90%, preferably at least 95%, more preferably at least 98%, and most preferably 100% nucleotide sequence identity therewith. In particular, the 5'miRNA scaffold stem of the pri-miRNA consists of the nucleotide sequence of any one of SEQ ID NOs: 1-4, particularly SEQ ID NO: 1.

[0070] In certain embodiments, the miRNA scaffold loop of pri-miRNA comprises the nucleotide sequence of any one of SEQ ID NOs: 8-10 or a sequence derived therefrom. In particular, the miRNA scaffold loop of pri-miRNA comprises the nucleotide sequence of any one of SEQ ID NOs: 8-10, or a sequence sharing at least 75%, preferably at least 85%, more preferably at least 90%, and most preferably 100% nucleotide sequence identity therewith. In particular, the miRNA scaffold loop of pri-miRNA consists of the nucleotide sequence of any one of SEQ ID NOs: 8-10, particularly the nucleotide sequence of SEQ ID NO: 8.

[0071] In certain embodiments, the 3'miRNA scaffold stem of pri-miRNA comprises the nucleotide sequence of any one of SEQ ID NOs: 11-17 or a sequence derived therefrom. In particular, the 3'miRNA scaffold stem of pri-miRNA comprises the nucleotide sequence of any one of SEQ ID NOs: 11-17, or a sequence sharing at least 90%, preferably at least 95%, more preferably at least 98%, and most preferably 100% nucleotide sequence identity therewith. In particular, the 3'miRNA scaffold stem of pri-miRNA consists of the nucleotide sequence of any one of SEQ ID NOs: 11-14, particularly the nucleotide sequence of SEQ ID NO: 14.

[0072] In certain embodiments, the template sequence of the pri-miRNA comprises at least one recognition site, particularly two recognition sites, for a DNA restriction enzyme. In particular, the two recognition sites are for different DNA restriction enzymes and generate different overhangs upon cleavage. The two recognition sites are preferably adjacent, on both sides, to the pre-miRNA portion of the pri-miRNA comprising the guide strand, the passenger strand, and the miRNA scaffold loop. In particular, one of the recognition sites is located within the sequence transcribed into the 5'miRNA scaffold stem and the other recognition site is located within the sequence transcribed into the 3'miRNA scaffold stem. In certain embodiments, the recognition sites are located within the sequences transcribed into the single-stranded portions of the 5' and 3'miRNA scaffold stems. The recognition sites are particularly unique recognition sites within the expression cassette, particularly within the entire vector having the expression cassette.

[0073] 1.2 Knockout of the CCL2 gene In certain embodiments, CHO cells are engineered by knockout of the CCL2 gene. Knockout of the CCL2 gene refers to deletion or inactivation of the CCL2 gene. In certain embodiments, both alleles of the CCL2 gene in the CHO cells are knocked out. The CCL2 gene can be completely removed, or its transcription can be impaired, or translation of the complete CCL2 protein can be inhibited. In particular, mutations can be introduced into the gene that result in a frameshift of the coding sequence of the CCL2 mRNA.

[0074] Knockout of CCL2 expression can be performed, for example, using homologous recombination, site-specific nucleases, CRISPR / Cas, TALENs, or Cas-Clover. In particular, knockout using Cas-Clover can be performed using a gRNA pair of a gRNA comprising the nucleotide sequence of SEQ ID NO: 53 and another gRNA comprising the nucleotide sequence of SEQ ID NO: 54.

[0075] 1.3 Production and purification process In certain embodiments, the method comprises, between step (a) and step (b), (a1) inoculating CHO cells into a cell culture medium to provide a cell culture; (a2) culturing the CHO cells in the cell culture under conditions that allow the number of CHO cells in the cell culture to increase; further comprising.

[0076] Suitable conditions for culturing host cells and increasing their cell numbers to express CD39 or its variant can be readily determined by those skilled in the art and are already known in the art. In certain embodiments, the vector nucleic acid encoding CD39 or its variant in CHO cells comprises one or more selectable marker genes. In these embodiments, the culture conditions of step (a2) and / or (b) may include the presence of the corresponding selectable agent(s) in the cell culture medium.

[0077] Obtaining CD39 or its variant from the cell culture in step (c) particularly includes isolating CD39 or its variant from the cell culture. Isolation of CD39 or its variant particularly refers to the separation of CD39 or its variant from the remaining components of the cell culture. As used herein, the term "cell culture" particularly includes the cell culture medium and CHO cells. In certain embodiments, CD39 or its variant is secreted by CHO cells. In these embodiments, CD39 or its variant is isolated from the cell culture medium. Separation of CD39 or its variant from the cell culture medium can be performed, for example, by chromatography and / or filtration methods.

[0078] Suitable methods and means for isolating CD39 or its variants are known in the art and can be readily applied by those skilled in the art. Exemplary isolation methods include, for example, filtration steps such as tangential flow filtration, alternating flow filtration, depth filtration, ultrafiltration and diafiltration, and / or chromatography steps such as affinity chromatography, anion and / or cation exchange chromatography, hydrophilic interaction chromatography, size exclusion chromatography and reverse phase chromatography. The step of obtaining CD39 or its variant from a cell culture may particularly include a filtration step, a capture chromatography step and one or more polishing chromatography steps. Further, step (c) may further include one or more virus inactivation steps.

[0079] In certain embodiments, step (c) includes performing anion exchange chromatography and / or hydrophobic interaction chromatography. In particular, step (c) (c1) separating CHO cells from a cell culture supernatant containing CD39 or its variant; (c2) separating CD39 or its variant from the cell culture supernatant using anion exchange chromatography; (c3) further purifying CD39 or its variant using hydrophobic interaction chromatography; (c4) further purifying CD39 or its variant using anion exchange chromatography; and includes.

[0080] Step (c1) can be carried out, in particular, using a filtration method such as tangential flow filtration or cross-flow filtration, where the CHO cells are retained in the retentate and the cell culture supernatant containing CD39 or a variant thereof passes through the filter to become the permeate. Step (c1) may also be carried out using a centrifugation method. In addition to steps (c1) to (c4), step (c) may further include one or more ultrafiltration and / or diafiltration steps for buffer exchange between or after chromatographic steps, as well as a virus inactivation and / or virus retention step. Virus inactivation can be achieved, in particular, using incubation at low pH for a time sufficient to inactivate substantially all viruses, and virus retention can be achieved using sterile filtration.

[0081] The obtained CD39 or a variant thereof can optionally be subjected to further processing step (d), for example a modification and / or formulation step, in order to produce CD39 or a variant thereof with the desired quality and composition. Such further processing steps and methods are generally known in the art. The formulation step may include buffer exchange, addition of formulation components, pH adjustment and concentration adjustment. Any combination of these and further steps can be used.

[0082] In certain embodiments, the method for producing CD39 or a variant thereof further includes, as step (d) or part of step (d), the step of providing a pharmaceutical formulation comprising CD39 or a variant thereof. Providing a pharmaceutical formulation comprising CD39 or a variant thereof, or formulating CD39 or a variant thereof as a pharmaceutical composition, in particular includes exchanging the buffer solution or buffer solution components of the composition comprising CD39 or a variant thereof. Further, this step may include lyophilization of CD39 or a variant thereof. In particular, CD39 or a variant thereof is transferred to a composition containing only pharmaceutically acceptable components.

[0083] Methods for producing CD39 or variants thereof provide CD39 or variants thereof with higher purity, particularly as compared to the same methods using CHO cells that have not been engineered to reduce the production of CCL2 in CHO cells. In certain embodiments, the CD39 or variant thereof obtained after step (c) is present in a composition containing CCL2 at a concentration that is at least 10-fold lower, preferably 25-fold lower, more preferably 100-fold lower, as compared to a composition obtained by the same method after step (c) using CHO cells that have not been engineered to reduce the production of CCL2 in CHO cells. In particular, the CD39 or variant thereof obtained after step (c) is present in a composition containing CCL2 at a concentration of 100 ppm or less, preferably 25 ppm or less, more preferably 10 ppm or less.

[0084] In certain embodiments, the CD39 or variant thereof obtained after step (c1) is present in a composition containing CCL2 at a concentration that is at least 10-fold lower, preferably 25-fold lower, more preferably 100-fold lower, as compared to a composition obtained by the same method after step (c1) using CHO cells that have not been engineered to reduce the production of CCL2 in CHO cells. In particular, the CD39 or variant thereof obtained after step (c1) is present in a composition containing CCL2 at a concentration of 100 ppm or less, preferably 25 ppm or less, more preferably 10 ppm or less.

[0085] In certain embodiments, the CD39 or variant thereof obtained after step (c4) is present in a composition containing CCL2 at a concentration that is at least 10-fold lower, preferably 25-fold lower, more preferably 100-fold lower, as compared to a composition obtained by the same method after step (c4) using CHO cells that have not been engineered to reduce the production of CCL2 in CHO cells. In particular, the CD39 or variant thereof obtained after step (c4) is present in a composition containing CCL2 at a concentration of 25 ppm or less, preferably 10 ppm or less, more preferably 5 ppm or less.

[0086] In certain embodiments, the CD39 or its variant obtained after step (d) is in a composition containing CCL2 at a concentration that is at least 10-fold lower, preferably 25-fold lower, more preferably 100-fold lower, compared to the composition obtained in the same manner after step (d) using CHO cells that have not been engineered to reduce the production of CCL2 in CHO cells. In particular, the CD39 or its variant obtained after step (d) is in a composition containing CCL2 at a concentration of 25 ppm or less, preferably 10 ppm or less, more preferably 5 ppm or less.

[0087] 1.4 CD39 and its variants The CD39 or its variant specifically produced by the method is a human CD39 or a variant of human CD39. In certain embodiments, the CD39 or its variant does not contain a membrane anchor. In this embodiment, it is in particular a soluble variant of CD39. In certain embodiments, the variant has N-terminal, C-terminal and / or central deletions, in particular N-terminal, C-terminal and central deletions, compared to human CD39. The N-terminal deletion can in particular contain 30 to 50 amino acids. The C-terminal deletion can in particular contain 20 to 40 amino acids. The central deletion can in particular contain 10 to 15 amino acids. Furthermore, the variant in certain embodiments contains up to 5 point mutations compared to human CD39.

[0088] In certain embodiments, the CD39 variant has an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-49. In particular, the CD39 variant contains the amino acid sequence of SEQ ID NO: 49 and in particular consists of the amino acid sequence of SEQ ID NO: 49.

[0089] 2. CHO host cells In a second aspect, the present invention provides a CHO cell that can produce CD39 or its variant and has been engineered to reduce the production of CCL2 in CHO cells. The CHO cell can be engineered as described above herein with respect to the method for producing CD39 or its variant.

[0090] In a further aspect, the present invention provides a host cell that is capable of producing CD39 or a variant thereof and is engineered to reduce the production of CCL2 in the host cell. The host cell is, in particular, a host cell as described above herein with respect to a method of producing CD39 or a variant thereof and can be engineered as described above herein with respect to a method of producing CD39 or a variant thereof. The features and embodiments described herein for CHO cells generally apply equally to the host cell.

[0091] The host cell can be any cell that is suitable for endogenously expressing CCL2 and produces CD39 or a variant thereof, particularly human CD39 or a variant thereof, when not engineered to reduce the production of CCL2. In particular, the host cell is a mammalian cell. The mammalian cell can be selected from the group consisting of, but not limited to, cells derived from mouse, such as COP, L, C127, Sp2 / 0, NS0, NS1, At20 and NIH3T3; cells derived from rat, such as PC12, PC12h, GH3, MtT, YB2 / 0 and Y0; cells derived from hamster, such as BHK, CHO and DHFR gene-deficient CHO; cells derived from monkey, such as COS1, COS3, COS7, CV1 and Vero; and cells derived from human, such as Hela, HEK293, CAP, PER-C6 derived from retina, cells derived from diploid fibroblasts, myeloma cells and HepG2. In a particular embodiment, the host cell is a Chinese hamster ovary (CHO) cell. The host cell may be suitable for suspension culture and / or adherent culture and can be used particularly in suspension culture.

[0092] The CHO cells or host cells may contain additional exogenous nucleic acids in addition to the expression cassette according to the fourth aspect of the present invention or the vector nucleic acid according to the fifth aspect. In particular, the CHO cells or host cells may contain an expression cassette for the expression of CD39 or a variant thereof that is not present on the vector nucleic acid according to the fifth aspect but rather on the expression cassette according to the fourth aspect. The expression cassette for the expression of CD39 or a variant thereof may be present on an additional vector nucleic acid or may be integrated into the genome of the CHO cells or host cells.

[0093] In certain embodiments, the coding sequence of CD39 or a variant thereof is present in the CHO cells or host cells: (i) within an expression cassette that expresses an miRNA targeting CCL2, (ii) within an additional expression cassette on the same vector nucleic acid as the expression cassette that expresses an miRNA targeting CCL2, or (iii) on an additional vector nucleic acid or within the genome of the CHO cells or host cells and is present.

[0094] CD39 or a variant thereof is, in particular, the CD39 or a variant thereof described herein.

[0095] In a further aspect, the present invention provides mammalian cells, particularly CHO cells, engineered to reduce the production of CCL2 in mammalian cells. The mammalian cells can be engineered as described above herein with respect to the method of producing CD39 or a variant thereof.

[0096] In an eighth aspect, the present invention provides CHO cells engineered to reduce the production of CCL2 in CHO cells. In certain embodiments, the CHO cells produce an miRNA targeting CCL2. In particular, the CHO cells are engineered by introduction of the vector nucleic acid according to the fifth aspect. In a further embodiment of the above aspect, the CHO cells are engineered by knockout of the CCL2 gene.

[0097] The embodiments, features, and examples of CHO cells according to the second aspect are similarly applicable to CHO cells according to the eighth aspect, except that the CHO cells according to the eighth aspect do not necessarily have to be able to produce CD39 or its variant.

[0098] 3. Methods for improving the production of CD39 and its variants In a third aspect, the present invention provides a method for improving the production of CD39 or its variant in CHO cells, comprising: (a-i) providing a CHO cell capable of producing CD39 or its variant; (a-ii) manipulating the CHO cell to reduce the production of CCL2 in the CHO cell; and provides a method for improving the production of CD39 or its variant in CHO cells.

[0099] The manipulation of the CHO cell can be performed as described above herein, particularly with respect to methods of producing CD39 or its variant. In certain embodiments, the CHO cell is manipulated in step (a-ii) by knockdown or knockout of CCL2 expression in the CHO cell or induction of degradation of the CCL2 protein. In particular, the CHO cell is manipulated in step (a-ii) to produce a miRNA that targets the endogenous CCL2 of the CHO cell. In certain embodiments, the step (a-ii) of manipulating the CHO cell to reduce the production of CCL2 comprises introducing into the CHO cell a vector nucleic acid encoding a miRNA targeting CCL2, particularly a vector nucleic acid comprising an expression cassette for the expression of a miRNA targeting CCL2. In particular, the vector nucleic acids described herein are used.

[0100] Embodiments, features, and examples of the method for producing CD39 or its variant described herein are equally applicable to the method for improving the production of CD39 or its variant in CHO cells. In particular, the CHO cells, CD39 or its variant, miRNA targeting CCL2, and / or the expression cassette can be as defined above herein with respect to the method for producing CD39 or its variant.

[0101] In certain embodiments, the step (a-ii) of manipulating the CHO cells results in manipulated CHO cells in which the level of CCL2 mRNA is reduced by at least 5-fold, preferably at least 10-fold, more preferably at least 25-fold, compared to the same CHO cells prior to step (a-ii). In certain embodiments, the step (a-ii) of manipulating the CHO cells results in manipulated CHO cells in which the level of CCL2 protein is reduced by at least 5-fold, preferably at least 10-fold, more preferably at least 25-fold, compared to the same CHO cells prior to step (a-ii).

[0102] A method for improving the production of CD39 or its variant in CHO cells comprises (b) culturing the CHO cells obtained in step (a-ii) in a cell culture under conditions that allow for the growth of the CHO cells and the simultaneous and / or subsequent production of CD39 or its variant; (c) obtaining the CD39 or its variant from the cell culture; (d) optionally processing the CD39 or its variant; and further comprises.

[0103] Embodiments, features, and examples of steps (a), (b), (c), and (d) of the method for producing CD39 or its variant described herein are equally applicable to steps (a-i), (b), (c), and (d) of the method for improving the production of CD39 or its variant in CHO cells, respectively.

[0104] 4. Expression Cassette and Vector Nucleic Acid In a fourth aspect, the present invention provides an expression cassette for the expression of miRNA in CHO cells, comprising a template sequence of pri-miRNA, wherein the pri-miRNA is suitable for being processed in CHO cells to form a miRNA targeting CCL2.

[0105] The features, embodiments and examples of the expression cassette described above herein with respect to a method of producing CD39 or a variant thereof are equally applicable to the expression cassette according to the fourth aspect of the present invention.

[0106] In a fifth aspect, the present invention provides a vector nucleic acid for transfection of CHO cells, comprising an expression cassette according to the fourth aspect. The vector nucleic acid can be any vector nucleic acid suitable for transfection of CHO cells. In certain embodiments, the vector nucleic acid is a plasmid. In other embodiments, the vector nucleic acid is a viral vector.

[0107] The vector nucleic acid may contain additional elements in addition to the expression cassette. For example, the vector nucleic acid may contain an origin of replication (ORI), a coding sequence encoding a polypeptide of interest, a selectable marker gene, and / or an antibiotic resistance gene. The polypeptide of interest can in particular be CD39 or a variant thereof, in particular a CD39 or a variant thereof as described herein.

[0108] In certain embodiments, the vector nucleic acid further comprises a coding sequence encoding CD39 or a variant thereof, in particular a CD39 or a variant thereof as described herein. The coding sequence encoding CD39 or a variant thereof may be present in the same expression cassette as the template sequence of pri-miRNA, or may be present in an additional expression cassette present in the vector nucleic acid.

[0109] In certain embodiments, the vector nucleic acid does not contain a coding sequence encoding a polypeptide of interest. In specific embodiments where the vector nucleic acid does not contain a coding sequence encoding a polypeptide of interest, the expression cassette according to the fourth aspect of the present invention does not contain a coding sequence of the polypeptide. In these embodiments, the vector nucleic acid particularly contains a further expression cassette containing a selectable marker gene. In alternative embodiments where the vector nucleic acid does not contain a coding sequence encoding a polypeptide of interest, the expression cassette according to the fourth aspect of the present invention contains a coding sequence encoding a selectable marker.

[0110] In certain embodiments, the vector nucleic acid contains a coding sequence encoding a polypeptide of interest. The coding sequence encoding the polypeptide of interest may be present within the expression cassette according to the fourth aspect of the present invention or may be present within a further expression cassette. In specific embodiments, the vector nucleic acid contains at least two expression cassettes, a first expression cassette for expressing the polypeptide of interest and a second expression cassette for expressing a selectable marker, and either the first or the second expression cassette is an expression cassette according to the fourth aspect of the present invention. In alternative embodiments, the vector nucleic acid contains at least three expression cassettes, the first expression cassette is an expression cassette according to the fourth aspect of the present invention, the second expression cassette is for expressing the polypeptide of interest, and the third expression cassette is for expressing a selectable marker. The polypeptide of interest may particularly be CD39 or a variant thereof, particularly CD39 or a variant thereof as described herein.

[0111] In certain embodiments, two or more expression cassettes of the vector nucleic acid are expression cassettes according to the fourth aspect of the present invention. Each of these expression cassettes may contain the template sequences of the same or different pri-miRNAs, particularly different pri-miRNAs. The miRNAs produced from the pri-miRNAs may particularly all target CCL2, but bind to different portions of the mRNA or pre-mRNA of CCL2.

[0112] 5. Method for producing CHO cells In a sixth aspect, the present invention provides a method for producing CHO cells capable of expressing CD39 or a variant thereof, comprising introducing the vector nucleic acid according to the fifth aspect into CHO cells. The vector nucleic acid may further comprise a coding sequence encoding CD39 or a variant thereof, or another nucleic acid comprising a coding sequence encoding CD39 or a variant thereof is present in or introduced into the CHO cells.

[0113] Thus, in certain embodiments, a method for generating CHO cells comprises introducing the vector nucleic acid according to the fifth aspect into CHO cells, wherein the vector nucleic acid comprises a coding sequence of CD39 or a variant thereof, either within an expression cassette that expresses an miRNA targeting CCL2 or within a further expression cassette. In an alternative embodiment, a method for producing CHO cells comprises introducing the vector nucleic acid according to the fifth aspect into CHO cells, the step wherein the vector nucleic acid does not comprise a coding sequence of CD39 or a variant thereof, and introducing into the CHO cells a further vector nucleic acid suitable for recombinant expression of CD39 or a variant thereof, wherein the different vector nucleic acids can be introduced into the CHO cells simultaneously or sequentially in any order. In still further embodiments, a method for producing CHO cells comprises (a) providing CHO cells capable of expressing CD39 or a variant thereof, and (b) introducing the vector nucleic acid according to the fifth aspect into the CHO cells. In these embodiments, the vector nucleic acid according to the fifth aspect preferably does not comprise a coding sequence of CD39 or a variant thereof.

[0114] CD39 or a variant thereof is in particular the CD39 or a variant thereof described herein.

[0115] The vector nucleic acid is artificially introduced into CHO cells. In particular, the vector nucleic acid is introduced by transfection. The transfection in this regard can be transient or stable, and in particular, stable transfection is used. Thus, in certain embodiments, the produced CHO cells contain an expression cassette according to the fifth aspect stably integrated into their genome.

[0116] In a further aspect, the present invention provides the use of an expression cassette according to the fourth aspect or a vector nucleic acid according to the fifth aspect or a CHO cell according to the sixth aspect for the production of CD39 or a variant thereof. The features, embodiments and examples of the methods for producing CD39 or a variant thereof described herein apply equally to this use.

[0117] The present invention further provides the use of an expression cassette according to the fourth aspect or a vector nucleic acid according to the fifth aspect for improving the production of CD39 or a variant thereof by CHO cells, which comprises introducing an expression cassette or a vector nucleic acid into CHO cells capable of producing CD39 or a variant thereof. In certain embodiments, the vector nucleic acid introduced into the CHO cells does not contain the coding sequence of CD39 or a variant thereof. Improving the production of CD39 or a variant thereof particularly includes increasing the purity of CD39 or a variant thereof. The features, embodiments and examples of the methods for improving the production of CD39 or a variant thereof in CHO cells described herein apply equally to this use.

[0118] 6. Composition comprising CD39 or a variant thereof In a seventh aspect, the present invention is a composition comprising CD39 or a variant thereof and CCL2, obtained by production using CHO cells according to the second aspect, (i) the amount of CCL2 in the composition is at least 10-fold lower compared to the same composition obtained by production using CHO cells whose production of CCL2 in the CHO cells has not been engineered to be reduced, and / or (ii) The composition contains CCL2 at a concentration of 100 ppm or less, preferably 25 ppm or less, more preferably 10 ppm or less. Provided is a composition comprising CD39 or a variant thereof and CCL2.

[0119] In certain embodiments, the composition is obtained by step (c1) of the method for producing CD39 or a variant thereof according to the first aspect of the present invention. In these embodiments, the composition can in particular be a cell culture supernatant or a cell-free bulk harvest of a cell culture. The composition according to these embodiments can in particular contain CCL2 at a concentration of 100 ppm or less, preferably 25 ppm or less, more preferably 10 ppm or less.

[0120] In further embodiments, the composition is obtained by step (c4) of the method for producing CD39 or a variant thereof according to the first aspect of the present invention. In these embodiments, the composition can in particular be a purified composition essentially free of proteins other than CD39 or a variant thereof. A composition essentially free of proteins other than CD39 or a variant thereof contains, in particular, 5% or less, preferably 2% or less, more preferably 1% or less of proteins other than CD39 or a variant thereof, based on the total amount of proteins in the composition. The composition according to these embodiments can in particular contain CCL2 at a concentration of 25 ppm or less, preferably 10 ppm or less, more preferably 5 ppm or less.

[0121] In further embodiments, the composition is obtained by step (d) of the method for producing CD39 or a variant thereof according to the first aspect of the present invention. In these embodiments, the composition can in particular be a pharmaceutical composition suitable for use in the treatment of a patient, in particular a human patient. The composition according to these embodiments can in particular contain CCL2 at a concentration of 25 ppm or less, preferably 10 ppm or less, more preferably 5 ppm or less.

[0122] 7. Specific Embodiments Hereinafter, specific embodiments of the present invention will be described. These embodiments can be combined with further embodiments, features, and examples described herein.

[0123] Embodiment 1. A method for producing CD39 or a variant thereof, comprising: (a) providing CHO cells capable of producing CD39 or a variant thereof; (b) culturing the CHO cells in a cell culture under conditions that allow the production of CD39 or a variant thereof; (c) obtaining CD39 or a variant thereof from the cell culture; (d) optionally treating CD39 or a variant thereof; wherein the CHO cells are engineered to reduce the production of CCL2 in the CHO cells. A method for producing CD39 or a variant thereof.

[0124] Embodiment 2. The method according to Embodiment 1, wherein the production of CCL2 in the CHO cells is reduced by knockdown or knockout of CCL2 expression or degradation of the CCL2 protein.

[0125] Embodiment 3. The method according to Embodiment 1 or 2, wherein the CHO cells are engineered to produce an miRNA that targets the endogenous CCL2 of the CHO cells.

[0126] Embodiment 4. The method according to Embodiment 3, wherein the miRNA targeting CCL2 has a nucleotide sequence selected from the group consisting of SEQ ID NO: 26 and 28.

[0127] Embodiment 5. The method according to Embodiment 3 or 4, wherein the CHO cells contain an expression cassette for the production of an miRNA that targets CCL2.

[0128] Embodiment 6. The method according to embodiment 5, wherein the expression cassette comprises an intron sequence containing the template sequence of pri-miRNA, and the pri-miRNA is suitable for being processed in CHO cells to form a miRNA targeting CCL2.

[0129] Embodiment 7. The pri-miRNA comprises, from 5' to 3', (i) a 5'miRNA scaffold stem optionally containing the nucleotide sequence of SEQ ID NOs: 1 to 4, (ii) a passenger strand having a nucleotide sequence complementary to the sequence of the miRNA and optionally containing one or two mismatches, (iii) a miRNA scaffold loop optionally containing the nucleotide sequence of SEQ ID NO: 8, (iv) a guide strand having the nucleotide sequence of the miRNA, (v) a 3'miRNA scaffold stem optionally containing the nucleotide sequences of SEQ ID NOs: 11 to 14, and the positions of the passenger strand and the guide strand may be interchanged. Optionally, the passenger strand has the nucleotide sequence of SEQ ID NO: 25 and the guide strand has the nucleotide sequence of SEQ ID NO: 26, or the passenger strand has the nucleotide sequence of SEQ ID NO: 27 and the guide strand has the nucleotide sequence of SEQ ID NO: 28. The method according to embodiment 6.

[0130] Embodiment 8. The method according to any one of embodiments 5 to 7, wherein the expression cassette comprises two or more template sequences for pri-miRNA, the pri-miRNA is suitable for being processed in CHO cells to form a miRNA targeting CCL2, and the miRNA processed from the pri-miRNA binds to different parts of the RNA of CCL2, particularly mRNA or pre-mRNA.

[0131] Embodiment 9. The method according to embodiment 8, wherein different miRNAs targeting CCL2 have nucleotide sequences selected from the group consisting of SEQ ID NOs: 26 and 28.

[0132] Embodiment 10. A method for producing CD39 or a variant thereof, comprising: (a) providing CHO cells capable of producing CD39 or a variant thereof; (b) culturing the CHO cells in a cell culture under conditions that allow the production of CD39 or a variant thereof; (c) obtaining CD39 or a variant thereof from the cell culture; (d) optionally processing CD39 or a variant thereof. A method for producing CD39 or a variant thereof, comprising the above steps.

[0133] Embodiment 11. The method according to any one of Embodiments 1 to 10, wherein step (c) comprises performing anion exchange chromatography and / or hydrophobic interaction chromatography.

[0134] Embodiment 12. In step (c): (c1) separating CHO cells from the cell culture supernatant containing CD39 or a variant thereof; (c2) separating CD39 or a variant thereof from the cell culture supernatant using anion exchange chromatography; (c3) further purifying CD39 or a variant thereof using hydrophobic interaction chromatography; (c4) further purifying CD39 or a variant thereof using anion exchange chromatography. The method according to any one of Embodiments 1 to 11, comprising the above steps.

[0135] Embodiment 13. The method according to any one of Embodiments 1 to 12, wherein step (d) comprises providing a pharmaceutical formulation comprising CD39 or a variant thereof.

[0136] Embodiment 14. The following features: (i) a soluble variant lacking the membrane anchor of human CD39 (ii) having an N-terminal, C-terminal, and central deletion as compared to human CD39, (iii) having an N-terminal deletion of 30-50 amino acids, a C-terminal deletion of 20-40 amino acids, and a central deletion of 10-15 amino acids as compared to human CD39, (iv) containing up to five point mutations as compared to human CD39, (v) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-49, A method according to any one of Embodiments 1-13, for producing a variant of CD39 having one or more of the above.

[0137] Embodiment 15. A method according to any one of Embodiments 1-14, for producing a variant of CD39 comprising the amino acid sequence of SEQ ID NO: 49.

[0138] Embodiment 16. A method according to any one of Embodiments 1-15, for producing a variant of CD39 consisting of the amino acid sequence of SEQ ID NO: 49.

[0139] Embodiment 17. A method according to any one of Embodiments 1-16, wherein the CD39 or its variant obtained after step (c) is in a composition containing CCL2 at a concentration of 100 ppm or less, preferably 25 ppm or less, more preferably 10 ppm or less.

[0140] Embodiment 18. A method according to any one of Embodiments 12, wherein the CD39 or its variant obtained after step (c1) is in a composition containing CCL2 at a concentration of 100 ppm or less, preferably 25 ppm or less, more preferably 10 ppm or less.

[0141] Embodiment 19. A method according to any one of Embodiments 12, wherein the CD39 or its variant obtained after step (c4) is in a composition containing CCL2 at a concentration of 25 ppm or less, preferably 10 ppm or less, more preferably 5 ppm or less.

[0142] The method according to any one of Embodiments 1 to 19, wherein the CD39 or its variant obtained after step (d) is in a composition containing CCL2 at a concentration of 25 ppm or less, preferably 10 ppm or less, more preferably 5 ppm or less.

[0143] Embodiment 21. The CD39 or its variant obtained after step (c) is in a composition containing CCL2 at a concentration at least 10-fold lower, preferably 25-fold lower, more preferably 100-fold lower, as compared to the composition obtained in the same manner after step (c) using CHO cells that have not been engineered to reduce the production of CCL2 in CHO cells. The method according to any one of Embodiments 1 to 20.

[0144] Embodiment 22. The CD39 or its variant obtained after step (c1) is in a composition containing CCL2 at a concentration at least 10-fold lower, preferably 25-fold lower, more preferably 100-fold lower, as compared to the composition obtained in the same manner after step (c1) using CHO cells that have not been engineered to reduce the production of CCL2 in CHO cells. The method according to any one of Embodiments 1 to 20.

[0145] Embodiment 23. The CD39 or its variant obtained after step (c4) is in a composition containing CCL2 at a concentration at least 10-fold lower, preferably 25-fold lower, more preferably 100-fold lower, as compared to the composition obtained in the same manner after step (c4) using CHO cells that have not been engineered to reduce the production of CCL2 in CHO cells. The method according to any one of Embodiments 1 to 20.

[0146] Embodiment 24. The CD39 or its variant obtained after step (d) is in a composition containing CCL2 at a concentration at least 10-fold lower, preferably 25-fold lower, more preferably 100-fold lower, as compared to the composition obtained in the same manner after step (d) using CHO cells that have not been engineered to reduce the production of CCL2 in CHO cells. The method according to any one of Embodiments 1 to 20.

[0147] Embodiment 25. A CHO cell capable of producing CD39 or a variant thereof and engineered to reduce the production of CCL2 in CHO cells.

[0148] Embodiment 26. The cell according to Embodiment 25, wherein the production of CCL2 is reduced by knockdown or knockout of CCL2 expression or degradation of the CCL2 protein.

[0149] Embodiment 27. The cell according to Embodiment 25 or 26, which is engineered to produce an miRNA targeting endogenous CCL2 in CHO cells.

[0150] Embodiment 28. The cell according to Embodiment 27, wherein the miRNA targeting CCL2 has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 26 and 28.

[0151] Embodiment 29. The cell according to Embodiment 27 or 28, comprising an expression cassette for producing an miRNA targeting CCL2.

[0152] Embodiment 30. The cell according to Embodiment 29, wherein the expression cassette comprises an intron sequence containing the template sequence of pri-miRNA, and the pri-miRNA is suitable for being processed in CHO cells to form an miRNA targeting CCL2.

[0153] Embodiment 31. The pri-miRNA, from 5' to 3', (i) a 5'miRNA scaffold stem optionally containing the nucleotide sequence of SEQ ID NOs: 1-4, and (ii) a passenger strand having a nucleotide sequence complementary to the sequence of the miRNA and optionally containing one or two mismatches, and (iii) an miRNA scaffold loop optionally containing the nucleotide sequence of SEQ ID NO: 8, and (iv) a guide strand having the nucleotide sequence of the miRNA. (v) A 3'miRNA scaffold stem optionally containing the nucleotide sequences of SEQ ID NOs: 11 to 14, comprising The positions of the passenger strand and the guide strand may be interchanged. Optionally, the passenger strand has the nucleotide sequence of SEQ ID NO: 25, the guide strand has the nucleotide sequence of SEQ ID NO: 26, or the passenger strand has the nucleotide sequence of SEQ ID NO: 27 and the guide strand has the nucleotide sequence of SEQ ID NO: 28. The cell according to Embodiment 30.

[0154] Embodiment 32. The expression cassette contains two or more template sequences for pri-miRNA, the pri-miRNA is suitable for being processed in CHO cells to form a miRNA targeting CCL2, and the miRNA processed from the pri-miRNA binds to different parts of the RNA of CCL2, particularly mRNA or pre-mRNA. The cell according to any one of Embodiments 29 to 31.

[0155] Embodiment 33. Different miRNAs targeting CCL2 have nucleotide sequences selected from the group consisting of SEQ ID NOs: 26 and 28. The cell according to Embodiment 32.

[0156] Embodiment 34. The following features: (i) A soluble variant lacking the membrane anchor of human CD39 (ii) Having N-terminal, C-terminal and central deletions compared to human CD39 (iii) Having an N-terminal deletion of 30 to 50 amino acids, a C-terminal deletion of 20 to 40 amino acids, and a central deletion of 10 to 15 amino acids compared to human CD39 (iv) Containing a maximum of five point mutations compared to human CD39 (v) Having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29 to 49 The cell according to any one of Embodiments 25 to 33, which can produce a variant of CD39 having one or more of the above.

[0157] The cell according to any one of Embodiments 25 to 34, which is capable of producing a variant of CD39 containing the amino acid sequence of SEQ ID NO: 49.

[0158] The cell according to any one of Embodiments 25 to 35, which is capable of producing a variant of CD39 consisting of the amino acid sequence of SEQ ID NO: 49.

[0159] Embodiment 37. A method for improving the production of CD39 or its variant in CHO cells, comprising: (a-i) providing a CHO cell capable of producing CD39 or its variant; (a-ii) manipulating the CHO cell to reduce the production of CCL2 in the CHO cell; A method for improving the production of CD39 or its variant in CHO cells, comprising the above steps.

[0160] Embodiment 38. The method according to Embodiment 37, wherein in step (a-ii), the CHO cell is manipulated by knockdown or knockout of CCL2 expression in the CHO cell or induction of degradation of the CCL2 protein.

[0161] Embodiment 39. The method according to Embodiment 37 or 38, wherein in step (a-ii), the CHO cell is manipulated to produce an miRNA targeting endogenous CCL2 of the CHO cell.

[0162] Embodiment 40. The method according to Embodiment 39, wherein the miRNA targeting CCL2 has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 26 and 28.

[0163] Embodiment 41. The method according to Embodiment 39 or 40, wherein in step (a-ii), an expression cassette for producing an miRNA targeting CCL2 is introduced into the CHO cell.

[0164] Embodiment 42. The method according to embodiment 41, wherein the expression cassette comprises an intron sequence containing the template sequence of pri-miRNA, and the pri-miRNA is suitable for being processed in CHO cells to form a miRNA targeting CCL2.

[0165] Embodiment 43. The pri-miRNA, from 5' to 3', (i) a 5'miRNA scaffold stem optionally containing the nucleotide sequence of SEQ ID NOs: 1-4, (ii) a passenger strand having a nucleotide sequence complementary to the sequence of the miRNA and optionally containing one or two mismatches, (iii) a miRNA scaffold loop optionally containing the nucleotide sequence of SEQ ID NO: 8, (iv) a guide strand having the nucleotide sequence of the miRNA, (v) a 3'miRNA scaffold stem optionally containing the nucleotide sequences of SEQ ID NOs: 11-14, and the positions of the passenger strand and the guide strand may be interchanged, and optionally, the passenger strand has the nucleotide sequence of SEQ ID NO: 25 and the guide strand has the nucleotide sequence of SEQ ID NO: 26, or the passenger strand has the nucleotide sequence of SEQ ID NO: 27 and the guide strand has the nucleotide sequence of SEQ ID NO: 28, The method according to embodiment 42.

[0166] Embodiment 44. The expression cassette comprises two or more template sequences for pri-miRNA, the pri-miRNA is suitable for being processed in CHO cells to form a miRNA targeting CCL2, and the miRNA processed from the pri-miRNA binds to different parts of the RNA of CCL2, particularly mRNA or pre-mRNA. The method according to any one of embodiments 41-43.

[0167] Embodiment 45. The method according to embodiment 44, wherein different miRNAs targeting CCL2 have nucleotide sequences selected from the group consisting of SEQ ID NOs: 26 and 28.

[0168] Embodiment 46. The method according to any one of Embodiments 41 to 45, wherein step (a-ii) includes introducing a vector nucleic acid into CHO cells, and the vector nucleic acid contains an expression cassette for the expression of miRNA targeting CCL2.

[0169] Embodiment 47. The method according to any one of Embodiments 37 to 46, wherein by manipulating CHO cells, the CCL2 mRNA level in the CHO cells is reduced by at least 5-fold, preferably at least 10-fold, more preferably at least 25-fold, compared to the same CHO cells before step (a-ii).

[0170] Embodiment 48. The method according to any one of Embodiments 37 to 47, wherein by manipulating CHO cells, the CCL2 protein level in the CHO cells is reduced by at least 5-fold, preferably at least 10-fold, more preferably at least 25-fold, compared to the same CHO cells before step (a-ii).

[0171] Embodiment 49. The method (b) culturing the CHO cells obtained in step (a-ii) in a cell culture under conditions that allow the growth of the CHO cells and the simultaneous and / or subsequent production of CD39 or its variant; (c) obtaining the CD39 or its variant from the cell culture; (d) optionally processing the CD39 or its variant; and further includes the method according to any one of Embodiments 37 to 48.

[0172] Embodiment 50. The method according to Embodiment 49, wherein step (c) includes performing anion exchange chromatography and / or hydrophobic interaction chromatography.

[0173] Embodiment 51. Step (c) (c1) separating the CHO cells from the cell culture supernatant containing CD39 or its variant; (c2) a step of separating CD39 or its variant from a cell culture supernatant using anion exchange chromatography; (c3) a step of further purifying CD39 or its variant using hydrophobic interaction chromatography; (c4) a step of further purifying CD39 or its variant using anion exchange chromatography; The method according to embodiment 49 or 50, comprising:

[0174] Embodiment 52. The method according to any one of embodiments 49 to 51, wherein step (d) comprises providing a pharmaceutical preparation comprising CD39 or its variant.

[0175] Embodiment 53. The method according to any one of embodiments 49 to 52, wherein the CD39 or its variant obtained after step (c) is in a composition containing CCL2 at a concentration of 100 ppm or less, preferably 25 ppm or less, more preferably 10 ppm or less.

[0176] Embodiment 54. The method according to any one of embodiments 51 to 53, wherein the CD39 or its variant obtained after step (c1) is in a composition containing CCL2 at a concentration of 100 ppm or less, preferably 25 ppm or less, more preferably 10 ppm or less.

[0177] Embodiment 55. The method according to any one of embodiments 51 to 54, wherein the CD39 or its variant obtained after step (c4) is in a composition containing CCL2 at a concentration of 25 ppm or less, preferably 10 ppm or less, more preferably 5 ppm or less.

[0178] Embodiment 56. The method according to any one of embodiments 49 to 55, wherein the CD39 or its variant obtained after step (d) is in a composition containing CCL2 at a concentration of 25 ppm or less, preferably 10 ppm or less, more preferably 5 ppm or less.

[0179] The method according to any one of Embodiments 49 to 56, wherein the CD39 or its variant obtained after step (c) is in a composition containing CCL2 at a concentration that is at least 10-fold lower, preferably 25-fold lower, more preferably 100-fold lower, compared to the composition obtained in the same manner after step (c) using CHO cells that have not been engineered to reduce the production of CCL2 in CHO cells.

[0180] The method according to any one of Embodiments 51 to 57, wherein the CD39 or its variant obtained after step (c1) is in a composition containing CCL2 at a concentration that is at least 10-fold lower, preferably 25-fold lower, more preferably 100-fold lower, compared to the composition obtained in the same manner after step (c1) using CHO cells that have not been engineered to reduce the production of CCL2 in CHO cells.

[0181] The method according to any one of Embodiments 51 to 58, wherein the CD39 or its variant obtained after step (c4) is in a composition containing CCL2 at a concentration that is at least 10-fold lower, preferably 25-fold lower, more preferably 100-fold lower, compared to the composition obtained in the same manner after step (c4) using CHO cells that have not been engineered to reduce the production of CCL2 in CHO cells.

[0182] The method according to any one of Embodiments 49 to 59, wherein the CD39 or its variant obtained after step (d) is in a composition containing CCL2 at a concentration that is at least 10-fold lower, preferably 25-fold lower, more preferably 100-fold lower, compared to the composition obtained in the same manner after step (d) using CHO cells that have not been engineered to reduce the production of CCL2 in CHO cells.

[0183] Embodiment 61. The following features: (i) A soluble variant lacking the membrane anchor of human CD39, (ii) Having N-terminal, C-terminal, and central deletions compared to human CD39, (iii) having an N-terminal deletion of 30 to 50 amino acids, a C-terminal deletion of 20 to 40 amino acids, and a central deletion of 10 to 15 amino acids as compared to human CD39, (iv) containing up to five point mutations as compared to human CD39, (v) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29 to 49, The method according to any one of Embodiments 37 to 60, which is for improving the production of a variant of CD39 having one or more of the above.

[0184] Embodiment 62. The method according to any one of Embodiments 37 to 61, which is for improving the production of a variant of CD39 containing the amino acid sequence of SEQ ID NO: 49.

[0185] Embodiment 63. The method according to any one of Embodiments 37 to 62, which is for improving the production of a variant of CD39 consisting of the amino acid sequence of SEQ ID NO: 49.

[0186] Embodiment 64. An expression cassette for miRNA expression in CHO cells, containing a template sequence of pri-miRNA, wherein the pri-miRNA is suitable for being processed in CHO cells to form a miRNA targeting CCL2.

[0187] Embodiment 65. The expression cassette according to Embodiment 64, wherein the miRNA targeting CCL2 has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 26 and 28.

[0188] Embodiment 66. The expression cassette according to Embodiment 64 or 65, wherein the template sequence of pri-miRNA is present within an intron sequence.

[0189] Embodiment 67. The pri-miRNA, from 5' to 3', (i) Optionally containing a 5'miRNA scaffold stem with the nucleotide sequences of SEQ ID NOs: 1 to 4, (ii) a passenger strand having a nucleotide sequence complementary to the miRNA sequence and optionally containing one or two mismatches, (iii) an miRNA scaffold loop optionally containing the nucleotide sequence of SEQ ID NO: 8, (iv) a guide strand having the nucleotide sequence of the miRNA, (v) a 3'miRNA scaffold stem optionally containing the nucleotide sequences of SEQ ID NOs: 11 to 14, comprising the positions of the passenger strand and the guide strand may be interchanged, and optionally, the passenger strand has the nucleotide sequence of SEQ ID NO: 25 and the guide strand has the nucleotide sequence of SEQ ID NO: 26, or the passenger strand has the nucleotide sequence of SEQ ID NO: 27 and the guide strand has the nucleotide sequence of SEQ ID NO: 28, The method according to any one of Embodiments 64 to 66.

[0190] An expression cassette according to any one of Embodiments 64 to 67, comprising two or more template sequences for pri-miRNA, wherein the pri-miRNA is processed in CHO cells and is suitable for forming an miRNA targeting CCL2, and the miRNA processed from the pri-miRNA binds to different parts of the RNA, particularly the mRNA or pre-mRNA of CCL2.

[0191] An expression cassette according to Embodiment 68, wherein different miRNAs targeting CCL2 have nucleotide sequences selected from the group consisting of SEQ ID NOs: 26 and 28.

[0192] A vector nucleic acid for transfection of CHO cells, comprising the expression cassette according to any one of Embodiments 64 to 69.

[0193] A vector nucleic acid according to Embodiment 70, further comprising a coding sequence encoding CD39 or a variant thereof.

[0194] The vector nucleic acid according to Embodiment 71, wherein the coding sequence encoding CD39 or a variant thereof is present in the same expression cassette as the template sequence of pri-miRNA.

[0195] The vector nucleic acid according to Embodiment 71, wherein the coding sequence encoding CD39 or a variant thereof is present in an additional expression cassette present within the vector nucleic acid.

[0196] Embodiment 74. The coding sequence has the following characteristics: (i) A soluble variant lacking the membrane anchor of human CD39, (ii) Having N-terminal, C-terminal, and central deletions compared to human CD39, (iii) Having an N-terminal deletion of 30 to 50 amino acids, a C-terminal deletion of 20 to 40 amino acids, and a central deletion of 10 to 15 amino acids compared to human CD39, (iv) Containing a maximum of five point mutations compared to human CD39, (v) Having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29 to 49, The vector nucleic acid according to any one of Embodiments 71 to 73, encoding a variant of CD39 having one or more of the above.

[0197] Embodiment 75. The vector nucleic acid according to any one of Embodiments 71 to 74, wherein the coding sequence encodes a variant of CD39 containing the amino acid sequence of SEQ ID NO: 49.

[0198] Embodiment 76. The vector nucleic acid according to any one of Embodiments 71 to 74, wherein the coding sequence encodes a variant of CD39 consisting of the amino acid sequence of SEQ ID NO: 49.

[0199] Embodiment 77. A method for producing CHO cells capable of expressing CD39 or a variant thereof, comprising introducing the vector nucleic acid according to any one of Embodiments 70 to 76 into CHO cells.

[0200] Method according to embodiment 77, wherein the CHO cells do not contain a coding sequence encoding CD39 or a variant thereof before introduction of the vector nucleic acid, and the vector nucleic acid according to any one of embodiments 71 to 76 is introduced into the CHO cells.

[0201] Method according to embodiment 77, wherein the vector nucleic acid according to embodiment 70 that does not contain a coding sequence encoding CD39 or a variant thereof is introduced into CHO cells, and the CHO cells contain a coding sequence encoding CD39 or a variant thereof before introduction of the vector nucleic acid.

[0202] Method according to embodiment 77, wherein the vector nucleic acid according to embodiment 70 that does not contain a coding sequence encoding CD39 or a variant thereof is introduced into CHO cells, and a further vector nucleic acid containing a coding sequence encoding CD39 or a variant thereof is introduced into the CHO cells, wherein the introduction of the two different vector nucleic acids is performed either simultaneously or subsequently in any order.

[0203] Embodiment 81. The CHO cells have the following characteristics: (i) A soluble variant lacking the membrane anchor of human CD39 (ii) Having N-terminal, C-terminal, and central deletions compared to human CD39 (iii) Having an N-terminal deletion of 30 to 50 amino acids, a C-terminal deletion of 20 to 40 amino acids, and a central deletion of 10 to 15 amino acids compared to human CD39 (iv) Containing up to five point mutations compared to human CD39 (v) Having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29 to 49 Method according to any one of embodiments 77 to 80, which can express a variant of CD39 having one or more of the above.

[0204] Method according to any one of embodiments 77 to 81, wherein the CHO cells can express a variant of CD39 containing the amino acid sequence of SEQ ID NO: 49.

[0205] The method according to any one of Embodiments 77 to 82, wherein the CHO cell can express a variant of CD39 consisting of the amino acid sequence of SEQ ID NO: 49.

[0206] A composition comprising CD39 or a variant thereof and CCL2, wherein the composition is obtained by production using the CHO cell according to any one of Embodiments 25 to 36, and the amount of CCL2 in the composition is at least 10-fold lower, preferably 25-fold lower, more preferably 100-fold lower, compared to the same composition obtained by production using a CHO cell that has not been engineered to reduce the production of CCL2 in the CHO cell.

[0207] A composition comprising CD39 or a variant thereof and CCL2, wherein the composition is obtained by production using a CHO cell, and the composition contains CCL2 at a concentration of 100 ppm or less, preferably 25 ppm or less, more preferably 10 ppm or less.

[0208] The composition according to Embodiment 84, which is the composition according to Embodiment 85.

[0209] The composition according to any one of Embodiments 84 to 86, which is obtained by steps (a), (b) and (c1) of the method according to Embodiment 12.

[0210] The composition according to Embodiment 87, which is a cell culture supernatant or a cell-free bulk harvest of a cell culture.

[0211] The composition according to Embodiment 87 or 88, wherein the composition contains CCL2 at a concentration of 100 ppm or less, preferably 25 ppm or less, more preferably 10 ppm or less.

[0212] Embodiment 90. A composition according to any one of Embodiments 84 to 86, obtained by steps (a), (b) and (c) of the method according to any one of Embodiments 1 to 24.

[0213] Embodiment 91. A composition according to any one of Embodiments 84 to 86, obtained by steps (a), (b), (c1), (c2), (c3) and (c4) of the method according to Embodiment 12.

[0214] Embodiment 92. The composition according to Embodiment 90 or 91, which is a purified composition essentially free of proteins other than CD39 or its variants.

[0215] Embodiment 93. A composition according to any one of Embodiments 84 to 86, obtained by steps (a), (b), (c) and (d) of the method according to any one of Embodiments 1 to 24.

[0216] Embodiment 94. The composition according to any one of Embodiments 90 to 93, which is a purified composition essentially free of proteins other than CD39 or its variants.

[0217] Embodiment 95. The composition according to any one of Embodiments 90 to 94, wherein the composition contains 5% or less, preferably 2% or less, more preferably 1% or less of proteins other than CD39 or its variants based on the total amount of proteins in the composition.

[0218] Embodiment 96. The composition according to any one of Embodiments 90 to 95, wherein the composition contains CCL2 at a concentration of 25 ppm or less, preferably 10 ppm or less, more preferably 5 ppm or less.

[0219] Embodiment 97. CHO cells engineered to reduce the production of CCL2 in CHO cells.

[0220] Embodiment 98. The cell according to Embodiment 97, wherein the production of CCL2 is reduced by knockdown or knockout of CCL2 expression or degradation of the CCL2 protein.

[0221] The cell according to embodiment 97 or 98, which has been engineered to produce an miRNA that targets the endogenous CCL2 of CHO cells.

[0222] The cell according to embodiment 99, wherein the miRNA targeting CCL2 has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 26 and 28.

[0223] The cell according to embodiment 99 or 100, comprising an expression cassette for producing an miRNA that targets CCL2.

[0224] The cell according to embodiment 101, wherein the expression cassette comprises an intron sequence containing the template sequence of pri-miRNA, and the pri-miRNA is suitable for being processed in CHO cells to form an miRNA that targets CCL2.

[0225] Embodiment 103. The pri-miRNA, from 5' to 3', (i) Optionally includes a 5'miRNA scaffold stem with the nucleotide sequences of SEQ ID NOs: 1-4, (ii) A passenger strand having a nucleotide sequence complementary to the sequence of the miRNA and optionally containing one or two mismatches, (iii) Optionally includes an miRNA scaffold loop with the nucleotide sequence of SEQ ID NO: 8, (iv) A guide strand having the nucleotide sequence of the miRNA, (v) Optionally includes a 3'miRNA scaffold stem with the nucleotide sequences of SEQ ID NOs: 11-14, and The positions of the passenger strand and the guide strand may be interchanged. Optionally, the passenger strand has the nucleotide sequence of SEQ ID NO: 25 and the guide strand has the nucleotide sequence of SEQ ID NO: 26, or the passenger strand has the nucleotide sequence of SEQ ID NO: 27 and the guide strand has the nucleotide sequence of SEQ ID NO: 28. The cell according to Embodiment 102.

[0226] Embodiment 104. The expression cassette contains two or more template sequences for pri-miRNA, the pri-miRNA is suitable for being processed in CHO cells to form a miRNA targeting CCL2, and the miRNA processed from the pri-miRNA binds to different parts of the RNA of CCL2, particularly mRNA or pre-mRNA. The cell according to any one of Embodiments 101 to 103.

[0227] Embodiment 105. The different miRNAs targeting CCL2 have a nucleotide sequence selected from the group consisting of SEQ ID NOs: 26 and 28. The cell according to Embodiment 104.

Brief Description of the Drawings

[0228]

Figure 1

Figure 2

Figure 3

Figure 4

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Figure 11-1

Figure 11-2

Example

[0229] Example 1: Identification of CCL2 as an important impurity of a human CD39 variant recombinantly produced in CHO cells. A variant of human CD39 having the amino acid sequence of SEQ ID NO: 49 (hereinafter "CD39*") was produced by a CHO production cell line ("CD39* cell line") and isolated from the cell culture using a standard purification process. The CD39* drug product purification process followed a 3-chromatography step process including anion exchange, hydrophobic interaction and multimodal anion exchange chromatography, low pH virus inactivation, virus removal filtration and two ultrafiltration / diafiltration steps (Figure 1).

[0230] Application of the CD39* purification process showed depletion of host cell proteins (HCP) using a plurality of ELISA assays specific for HCP. Analysis of individual HCPs using an orthogonal LC-MS method revealed one prominent protein: CCL2 (CC-chemokine ligand 2; also known as MCP-1 (monocyte chemoattractant protein 1)), which was not detected by ELISA.

[0231]

Table 1

[0232] CCL2 was further observed not to be removed during the purification process since its concentration in the product remained almost constant after each purification step.

[0233] Further analysis of the upstream process (USP) and downstream process (DSP) of CD39* production showed that the formation of CCL2 in the cell culture increased with increasing culture time (Figure 2), and that the elution profile of CCL2 in the AEC capture step significantly overlapped with the elution profile of CD39* (Figure 3).

[0234] Example 2: miRNA vector design. The vectors encoding intron-miRNA (pCMV04_A and pCMV04_B) were based on the standard vector (pCMV). The vectors were modified by inserting the intron-miRNA sequence into the cloning site of the CMV promoter-driven expression cassette. The sequence environment selected for the miRNA scaffold is similar to that of human miR-30A and miR-E molecules (e.g., Fellmann et al., 2011, Molecular Cell 41, 733-746) and is expected to result in optimal processing of the resulting miRNA sequence. However, the sequence was further modified by: i) replacing the EcoRI restriction site with a BglII restriction site, ii) replacing the sequence downstream of the XhoI restriction site with a CHO-derived sequence, and iii) adding additional miR-30A scaffolds upstream and downstream of the published sequence. Two different miRNA sequences targeting endogenous CCL2 in CHO cells: CCL2_A AIM, which contains a guide strand having the nucleotide sequence of SEQ ID NO: 26 and a passenger strand having the nucleotide sequence of SEQ ID NO: 25, and CCL2_B AIM, which contains a guide strand having the nucleotide sequence of SEQ ID NO: 28 and a passenger strand having the nucleotide sequence of SEQ ID NO: 27, were tested. Additionally, a vector with an AIM targeting an irrelevant endogenous protein in CHO cells was used as a control.

[0235] Example 3: Generation of CCL2 knockdown cell lines. The strategy for generating CCL2 KD cell lines is shown in Figure 4. CHO parental cell lines were transfected with the vector pCMV03 (hereinafter "CD39*") encoding a variant of human CD39, and pool selection was performed using MTX in low folate medium. The pool was subjected to single cell cloning, and monoclonal cell lines expressing CD39* (referred to as CD39* cell lines) were selected. Subsequently, using the primary seed lot (PSL) of the CD39* cell line, the cells were transfected with a vector encoding an artificial intron miRNA targeting endogenous CCL2, and a pool was generated using puromycin.

[0236] Two different miRNAs targeting CHO CCL2 mRNA, called CCL2_A and CCL2_B, were generated, both targeting the 3’UTR of the transcript. For pool generation, miRNAs targeting different genes (control AIM), the parental CD39* cell line, and the empty parental cell line (CHO) as a control were included. All samples (triplicate pool generation for the knockdown approach) were inoculated into an optimized fed-batch run, and cell growth, gene expression, and CD39* titers were evaluated on different days (Figures 5 - 7). Also, CCL2 protein levels at the harvest level were evaluated using CCL2 ELISA (Figure 8).

[0237] After confirmation of efficient CCL2 knockdown at the pool generation and mRNA levels, the CCL2_A pool was selected for single-cell cloning, and 96 growing clones were inoculated into a 24dwp standard fed-batch to evaluate CCL2 knockdown efficiency and CD39* productivity (Figures 9 and 10).

[0238] The top 30 clones were further characterized. Based on multiple parameters (USP, DSP, CCL2 data, CD39* protein characteristics), the top 3 clones were selected and inoculated into a 7L bioreactor. CCL2 expression was significantly decreased in the CCL2 knockdown clones, resulting in an increase in CD39* titer (Figure 11).

[0239]

Table 2

[0240] After collecting the culture supernatant containing CD39*, the concentration of CCL2 in the cell-free supernatant was determined.

[0241]

Table 3

[0242] Subsequent application of the downstream process, including anion exchange chromatography, HIC, and MAC, resulted in a CD39* composition (drug substance) with a CLL2 content below the limit of quantification (4 ppm).

[0243]

Table 4

[0244] Example 4: Preparation of a CCL2 knockout cell line. Gene editing technology represents an alternative approach to the vectorized RNAi approach using artificial intron miRNAs. Gene knockout at the DNA level encoding the CCL2 gene results in complete functional inactivation due to the loss of essential gene information. The inventors prepared a single gRNA pair for the targeting of Cas-Clover to the CCL2 gene on exons 1 and 2 (guide CCL2-01: SEQ ID NO: 51; guide CCL2-02: SEQ ID NO: 52; guide CCL2-03: SEQ ID NO: 53; guide CCL2-04: SEQ ID NO: 54). Cas-Clover, a dCas9 protein fused to the nuclease Clo51, can homodimerize at the CCL2 locus, resulting in the formation of insertions and deletions (indels), and ultimately a frameshift mutation in the CCL2 gene. The mRNA encoding Cas-Clover was transfected into MaKo cells seeded in 24-well plates one day before transfection. Six hours later, the sgRNA pair targeting the CCL2 gene was co-transfected into Cas-Clover-expressing cells. One day after transfection, the live cell population was sorted into single cells using fluorescence-activated cell sorting (FACS). After a 16-day growth period in 96-well plates, the genomic DNA of the clonal cells was isolated for NGS genotyping. PCR amplicons prepared using primers adjacent to the predicted gRNA editing sites were sequenced as Nextera XT libraries on Illumina's MiSeq. The sequencing reads were aligned to the CCL2 gene sequence to identify indels. Clone 048 transfected with the sgRNA pair guide_SA03 and guide_SA04 revealed two deletions (40 bp and 15 bp - 13 bp) without detection of the wild-type sequence, confirming a homozygous CCL2 gene knockout in the CHO MaKo cell line.

[0245] Example 5: Materials and methods. In Examples 1 to 4, the following materials and methods were used.

[0246] 1. Expression vector construction The vectors used in the examples consist of the following elements: the hCMV promoter / enhancer driving the expression of individual genes, the polyadenylation signal (polyA), the folate receptor, DHFR, the puromycin and hygromycin resistance genes as selection markers, the origin of replication of E. coli (CoIEori) and the beta-lactamase gene for ampicillin (amp) resistance enabling amplification in bacteria. Different plasmid settings were evaluated and are shown in more detail in the figures.

[0247] 2. Cell Lines, Cultures, Transfections and Selections CHO cell lines were cultured by suspending them in their own chemically defined culture medium at 300 rpm (24 dwp) or 150 rpm (shaking flask), 10% CO2, 36.5 °C in a non-humidified shaking incubator cabinet in 24 deep well plates or shaking flasks. Cell viability and growth rate were monitored by an automated system (ViCell, Beckman Coulter) or using analytical flow cytometry (CytoFlex, Beckman Coulter). Cells were passaged 2 - 3 times a week into fresh medium and maintained in the logarithmic growth phase.

[0248] The linearized expression vector was transfected by electroporation (Amaxa Nucleofection system, Lonza, Germany). The transfection reaction was carried out in a chemically defined culture medium according to the manufacturer's instructions. The parental CHO cells used for transfection were in the exponential growth phase with a cell viability of over 95%. Transfection was carried out with 5×10 6 cells per transfection. Immediately after transfection, the cells were transferred to a shaking flask containing chemically defined culture medium. The cell pool was incubated at 36.5 °C and 10% CO2 for 48 hours before starting the selection process.

[0249] As described above, a selection procedure was performed using the selectable markers encoded by the individual expression vectors. Proteins FoIR and DHFR are involved in the same molecular pathway, where FolR transports folic acid and the folic acid analogue MTX into the cell, and DHFR converts it into important precursors for purine and methionine synthesis. Combining these as selection principles enables the adoption of a specific, powerful selection regime to enrich recombinant cells expressing both recombinant proteins. Puromycin selection is driven by inhibition of protein synthesis, and a vector encoding a puromycin resistance marker enables cells to survive in the presence of puromycin.

[0250] Forty-eight hours after transfection and growth under low folate conditions, an additional selection pressure was applied by adding 10 nM MTX to the chemically defined culture medium. Alternatively, puromycin was used as the selection agent. Forty-eight hours after transfection, 0.003 mg / mL puromycin was added to the chemically defined culture medium. After pool recovery, the cells were frozen in culture medium supplemented with 7.5% DMSO and cell pellets were prepared.

[0251] 3. Gene expression analysis by quantitative real-time PCR RNA extraction was performed using the Qiagen RNeasy Mini Kit according to the manufacturer's instructions. For real-time qPCR, cDNA was synthesized from 200 ng / μl diluted RNA using the High Capacity RNA-to-cDNA Master Mix (Applied Biosystems), and 10-fold diluted cDNA was analyzed in triplicate using the QuantiFast SYBR Green PCR Kit (Qiagen) or the TaqMan Primer / Probe system and TaqMan Mastermix (Applied Biosystems). GAPDH was amplified as an endogenous control for normalization. Amplification and analysis were performed using the ABI PRISM® 7900HT Sequence Detection System. For the calculation of the relative quantity (RQ) of gene expression for sample comparison, the comparative 2 -ΔΔCt method was used to normalize the data.

[0252] 4. Upstream processing Following selection, the material was generated in either a shake flask fed-batch, 24 deep well plate culture, or ambr15 bioreactor. The fed-batch cultures were inoculated at a cell seeding density of 4E5 vc / ml (starting from day 3, a proprietary feed solution was added, and the culture temperature was shifted to 33 °C on day 5). During the culture, in-process controls were performed to monitor the concentration of CD39*. Cell culture samples for RNA isolation were taken on day 10 of the process. The individual cultures were incubated for 14 days. At the end of the culture process, the cells were separated from the culture supernatant by centrifugation and / or depth filtration, followed by sterile filtration, and then further downstream processing was carried out.

[0253] 5. CCL2 protein analysis by ELISA The amount of the Chinese hamster (CHO) host cell protein "CC-chemokine ligand 2" (CCL2) was determined using a sandwich ELISA. Samples were added to microtiter plates coated with an anti-CCL2 antibody (capture antibody). The bound CCL2 was then quantified by incubation with a biotinylated anti-CCL2 antibody (detection antibody), followed by streptavidin-peroxidase as the substrate and tetramethylbenzidine (TMB), and measurement of the absorbance at 450 nm. The CCL2 levels in the samples were calculated based on a rat or CHO CCL2 standard, preferably a CHO CCL2 standard.

[0254] 6. Determination of CCL2 by LC-MS The amount of the Chinese hamster (CHO) host cell protein "CC-chemokine ligand 2" (CCL2) was determined using LC-MS absolute quantification. A CCL2 heavy-labeled peptide derived from the CCL2 sequence was spiked into the tryptic digest of the drug substance. The endogenous CCL2 protein was then quantified by comparing the LC-MS intensity of the endogenous CCL2 unlabeled peptide with the LC-MS intensity of the spiked CCL2 heavy-labeled peptide.

[0255] The method demonstrated linear detection of the CCL2 heavy-labeled peptide over the range of 33 ng / mg DS to 8100 ng / mg DS (R2 > 0.99). The CCL2 quantification in CD39* was not affected by the concentration of the heavy-labeled peptide used in the experiment (99% agreement), which was confirmed using alternative data analysis (peak area vs MS intensity). This method showed an accuracy of 9% CV. As a conclusion, this method is considered suitable for quantifying CCL2 in the CD39* drug substance.

[0256]

Table 5

[0257]

Table 6

[0258]

Table 7

[0259]

Table 8

[0260]

Table 9

[0261]

Table 10

[0262]

Table 11

Claims

1. A method for producing CD39 or its variants, (a) A step of providing CHO cells capable of producing CD39 or a variant thereof, (b) A step of culturing the CHO cells in a cell culture under conditions that enable the production of CD39 or its variants, (c) A step of obtaining CD39 or its variant from the cell culture, (d) A step of optionally processing CD39 or its variant, Includes, The CHO cells are manipulated to reduce the production of CCL2 in the CHO cells. A method for producing CD39 or its variants.

2. The method according to claim 1, wherein the production of CCL2 in the CHO cells is reduced by knockdown or knockout of CCL2 expression or degradation of the CCL2 protein.

3. The method according to claim 1, wherein the CHO cells are manipulated to produce miRNAs that target endogenous CCL2 of the CHO cells, and the miRNAs that target CCL2 preferably have a nucleotide sequence selected from the group consisting of SEQ ID NOs: 26 and 28.

4. The method according to claim 3, wherein the CHO cells include an expression cassette for producing miRNA targeting CCL2, the expression cassette includes an intron sequence containing a template sequence for prim-miRNA, and the prim-miRNA is suitable for being processed in the CHO cells to form miRNA targeting CCL2.

5. The aforementioned prim-miRNA is divided from 5' to 3'. (i) A 5' miRNA scaffold stem which optionally includes the nucleotide sequences of SEQ ID NOs: 1 to 4, (ii) A passenger strand having a nucleotide sequence complementary to the sequence of the miRNA, and optionally containing one or two mismatches, (iii) A miRNA scaffold loop which optionally includes the nucleotide sequence of Sequence ID No. 8, (iv) A guide strand having the nucleotide sequence of the miRNA, (v) A 3' miRNA scaffold stem which optionally includes the nucleotide sequences of SEQ ID NOs. 11 to 14, Includes, The positions of the passenger strand and the guide strand may be swapped, and in some cases, the passenger strand may have the nucleotide sequence of SEQ ID NO: 25 and the guide strand may have the nucleotide sequence of SEQ ID NO: 26, or the passenger strand may have the nucleotide sequence of SEQ ID NO: 27 and the guide strand may have the nucleotide sequence of SEQ ID NO:

28. The method according to claim 4.

6. The following features: (i) A soluble variant of human CD39 lacking a membrane anchor, (ii) Compared to human CD39, it has N-terminal, C-terminal and central deletions. (iii) Compared to human CD39, it has an N-terminal deletion of 30-50 amino acids, a C-terminal deletion of 20-40 amino acids, and a central deletion of 10-15 amino acids. (iv) Contains up to 5 point mutations compared to human CD39, (v) Having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29 to 49, The method according to claim 1, for producing a variant of CD39 having one or more of the following.

7. The method according to claim 1, wherein the CD39 or its variant obtained after step (c) is in a composition containing CCL2 at a concentration of 100 ppm or less, preferably 25 ppm or less, more preferably 10 ppm or less, and / or in a composition containing CCL2 at a concentration at least 10 times lower, preferably 25 times lower, more preferably 100 times lower, compared to a composition obtained in the same way after step (c) using CHO cells that have not been manipulated to reduce the production of CCL2 in the CHO cells, and / or the CD39 or its variant obtained after step (d) is in a composition containing CCL2 at a concentration of 25 ppm or less, preferably 10 ppm or less, more preferably 5 ppm or less, and / or in a composition containing CCL2 at a concentration at least 10 times lower, preferably 25 times lower, more preferably 100 times lower, compared to a composition obtained in the same way after step (d) using CHO cells that have not been manipulated to reduce the production of CCL2 in the CHO cells.

8. Step (c) is, (c1) A step of separating the CHO cells from the cell culture supernatant containing CD39 or the variant thereof, (c2) A step of separating CD39 or its variant from the cell culture supernatant using anion exchange chromatography, (c3) A step of further purifying CD39 or its variant using hydrophobic interaction chromatography, (c4) A step of further purifying CD39 or its variant using anion exchange chromatography, The method according to claim 1, including the method described in claim 1.

9. The method according to claim 8, wherein the CD39 or its variant obtained after step (c1) is in a composition containing CCL2 at a concentration of 100 ppm or less, preferably 25 ppm or less, more preferably 10 ppm or less, and / or in a composition containing CCL2 at a concentration at least 10 times lower, preferably 25 times lower, more preferably 100 times lower, compared to a composition obtained in the same way after step (c1) using CHO cells that have not been manipulated to reduce the production of CCL2 in the CHO cells, and / or the CD39 or its variant obtained after step (c4) is in a composition containing CCL2 at a concentration of 25 ppm or less, preferably 10 ppm or less, more preferably 5 ppm or less, and / or in a composition containing CCL2 at a concentration at least 10 times lower, preferably 25 times lower, more preferably 100 times lower, compared to a composition obtained in the same way after step (c4) using CHO cells that have not been manipulated to reduce the production of CCL2 in the CHO cells.

10. The method according to any one of claims 1 to 9, wherein step (d) is to provide a pharmaceutical formulation comprising CD39 or a variant thereof.

11. CHO cells capable of producing CD39 or a variant thereof, and engineered to reduce the production of CCL2 in the CHO cells.

12. The CHO cell according to claim 11, wherein the CHO cell is manipulated to reduce the production of CCL2 in the CHO cell according to any one of claims 2 to 7.

13. A method for improving the production of CD39 or its variants in CHO cells, (a-i) A step of providing CHO cells capable of producing CD39 or its variants, (a-ii) A step of manipulating the CHO cells to reduce the production of CCL2 in the CHO cells, A method for improving the production of CD39 or its variants in CHO cells, including the following.

14. The method according to claim 13, wherein the CHO cells are operated in step (a-ii) of any one of claims 2 to 7.

15. The method according to claim 13, wherein step (a-ii) comprises introducing a vector nucleic acid into the CHO cells, the vector nucleic acid comprising an expression cassette for the expression of a miRNA targeting CCL2.

16. The method according to claim 13, wherein by manipulating the CHO cells, the CCL2 mRNA level and / or CCL2 protein level in the CHO cells is reduced by at least 5 times, preferably at least 10 times, and more preferably at least 25 times, compared to the same CHO cells prior to step (a-ii).

17. (b) A step of culturing the CHO cells obtained in step (a-ii) in a cell culture under conditions that enable proliferation of the CHO cells and simultaneous and / or subsequent production of CD39 or its variants, (c) A step of obtaining the CD39 or its variant from the cell culture, (d) A step of optionally processing CD39 or its variant, The method according to claim 13, further comprising:

18. Step (c) is, (c1) A step of separating the CHO cells from the cell culture supernatant containing CD39 or the variant thereof, (c2) A step of separating CD39 or its variant from the cell culture supernatant using anion exchange chromatography, (c3) A step of further purifying CD39 or its variant using hydrophobic interaction chromatography, (c4) A step of further purifying CD39 or its variant using anion exchange chromatography, Includes, and / or Step (d) includes providing a pharmaceutical formulation containing CD39 or its variant, The method according to claim 17.

19. An expression cassette for the expression of miRNA in CHO cells, comprising a template sequence of prim-miRNA, wherein the prim-miRNA is suitable for processing in CHO cells to form a miRNA that targets CCL2.

20. An expression cassette according to claim 19, having the features described in any one of claims 3 to 5.

21. A vector nucleic acid for transfection of CHO cells, comprising the expression cassette according to claim 19.

22. A method for producing CHO cells capable of expressing CD39 or a variant thereof, comprising introducing the vector nucleic acid described in claim 21 into CHO cells.

23. A composition comprising CD39 or a variant thereof and CCL2, obtained by production using the CHO cells described in claim 11, (i) The amount of CCL2 in the composition is at least 10 times lower than that of the same composition obtained by production using CHO cells that have not been manipulated to reduce the production of CCL2 in the CHO cells, and / or (ii) The composition contains CCL2 at a concentration of 100 ppm or less, preferably 25 ppm or less, more preferably 10 ppm or less. A composition comprising CD39 or a variant thereof and CCL2.

24. CHO cells manipulated to reduce the production of CCL2 in the CHO cells, particularly as defined in any one of claims 2 to 7.

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