Improved CD39 variant production

Abstract

The present invention relates to the use of miRNA technology for improving the recombinant production of CD39 or variants thereof in CHO cells. The miRNA is used for knocking down an endogenous protein CCL2 of CHO cells, and the protein is difficult to separate from CD39 or a variant thereof 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, wherein the level of host cell proteins is reduced, which interferes with the purification of CD39 and its variants. Artificial miRNAs targeting host cell proteins are used to reduce interference with the production of host cell proteins. Background Art

[0002] The generation of recombinant cell lines for the production of secreted proteins requires transfection of DNA vectors into host cells and the use of selection markers to enrich for stable transfectants. Secreted proteins may affect cellular parameters such as growth, viability and / or productivity, which often requires cell line engineering approaches to achieve high-expressing stable cell lines.

[0003] Similarly, the quality of secretory recombinant proteins is affected by cell-derived intrinsic factors. Typically, endogenously expressed proteins, such as cell surface receptors, enzymes or proteases (host cell proteins, i.e., HCPs), can be identified as the root cause of the undesirable effects that affect the quality of recombinant proteins (see, e.g., WO 2014 / 097113 A2). In addition, host cell proteins are well-known process-related impurities of biological products. When present in a drug product, these impurities may cause adverse effects (Vanderlaan et al., 2018, Biotechnology Progress [Biological Technology Progress] 34, 828–837) due to increased activity related to drug immunogenicity, inflammation and other specific residual host cell protein functions. The composition and abundance of HCPs present in multiple steps of the manufacturing process and in the final drug substance depend on many factors. These factors may be difficult to predict a priori and can usually only be understood through testing during process development.

[0004] The corresponding undesirable effect comprises the enzymatic cleavage of the polypeptide chain of recombinant protein, such as digestion or amino acid shearing of whole protein, i.e., removing one or several amino acids from the N or C-terminal of target protein. Other effects are unwanted post-translational modification or removal of required modification. In addition, the endogenous gene product of host cell may interact specifically with target protein. Such interaction may raise target protein from the supernatant of cell culture, making it difficult to remove endogenous protein in purification process, or even causing the activation of signal transduction pathway in host cell, thereby causing the growth, vigor and / or productivity of cell to reduce. In other cases, the endogenous gene product may only have chemical and physical properties similar to target protein height, which makes it difficult to develop purification method to effectively remove host cell protein without reducing the output of target protein. This is particularly relevant for therapeutic protein, wherein the host cell protein with high purity and low residual level in the final product is a prerequisite for obtaining and maintaining marketing authorization.

[0005] Therefore, the endogenous gene products of the host cell may significantly interfere with the recombinant production of the target protein. In view of the above situation, there is a need in the art to provide strategies to reduce the adverse effects of host cell proteins on the recombinant production of the target protein. Summary of the invention

[0006] The present inventors aim to provide a purification method for human CD39 and variants thereof produced in CHO cells, especially soluble variants of CD39 in which the membrane anchor sequence is deleted. During the development of the purification method, CCL2 was identified as a host cell protein with highly similar physicochemical properties to the CD39 variants, and therefore was not significantly removed during the purification step. Therefore, the present inventors established a production cell line of CD39 variants, in which CCL2 levels were reduced by using miRNA against this host cell protein. Therefore, the purity of the final CD39 variant can be significantly improved.

[0007] In view of this, in a first aspect, the present invention relates to a method for producing CD39 or a variant thereof, the method comprising the following steps:

[0008] (a) providing a CHO cell capable of producing CD39 or a variant thereof;

[0009] (b) culturing CHO cells in cell culture under conditions that allow production of CD39 or a variant thereof;

[0010] (c) obtaining CD39 or a variant thereof from a cell culture; and

[0011] (d) optionally processing CD39 or a variant thereof;

[0012] CHO cells were engineered to reduce the production of CCL2 in the CHO cells.

[0013] In a second aspect, the present invention provides a CHO cell capable of producing CD39 or a variant thereof, and the CHO cell is engineered to reduce the production of CCL2 in the CHO cell.

[0014] In certain embodiments of the above aspects, CHO cells produce miRNA targeting CCL2. In particular, CHO cells are engineered by introducing the vector nucleic acid according to the fifth aspect. In other embodiments of the above aspects, CHO cells are engineered by knocking out the CCL2 gene.

[0015] In a third aspect, the present invention provides a method for improving the production of CD39 or a variant thereof in CHO cells, the method comprising the steps of:

[0016] (ai) providing CHO cells capable of producing CD39 or a variant thereof;

[0017] (a-ii) CHO cells were engineered to reduce the production of CCL2 in CHO cells.

[0018] In certain embodiments, step (a-ii) of engineering CHO cells to reduce CCL2 production comprises introducing the vector nucleic acid according to the fifth aspect into CHO cells.

[0019] In a fourth aspect, the present invention provides an expression cassette for expressing miRNA in CHO cells, the expression cassette comprising a template sequence of a pri-miRNA, wherein the pri-miRNA is suitable for being processed in CHO cells to form a miRNA targeting CCL2.

[0020] In a fifth aspect, the present invention provides a vector nucleic acid for transfecting CHO cells, the vector nucleic acid comprising the expression cassette according to the fourth aspect.

[0021] In a sixth aspect, the present invention provides a method for producing a CHO cell capable of expressing CD39 or a variant thereof, the method comprising introducing the vector nucleic acid according to the fifth aspect into a CHO cell. The vector nucleic acid may additionally 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 a CHO cell or introduced into a CHO cell.

[0022] In a seventh aspect, the present invention provides a composition comprising CD39 or a variant thereof and CCL2, wherein the composition is obtained by using the CHO cell according to the second aspect, and wherein

[0023] (i) the amount of CCL2 in the composition is at least 10-fold lower than the same composition obtained by production using CHO cells that have not been engineered to reduce CCL2 production in CHO cells; and / or

[0024] (ii) The composition comprises CCL2 at a concentration of 100 ppm or less, preferably 25 ppm or less, more preferably 10 ppm or less.

[0025] In an eighth aspect, the present invention provides a CHO cell that is engineered to reduce the production of CCL2 in the CHO cell. In certain embodiments, the CHO cell produces a miRNA targeting CCL2. In particular, the CHO cell is engineered by introducing the vector nucleic acid according to the fifth aspect. In another embodiment of the above aspects, the CHO cell is engineered by knocking out the CCL2 gene.

[0026] From the following description and the appended claims, other objects, features, advantages and aspects of the present invention will become apparent to those skilled in the art. However, it should be understood that the following description, the appended claims and the specific examples indicating the preferred embodiments of the present application are given by way of illustration only. By reading the following, various changes and modifications within the spirit and scope of the disclosed invention will become apparent to those skilled in the art.

[0027] definition

[0028] As used herein, the following expressions are generally intended to preferably have the meanings as described below, unless otherwise indicated in the context in which they are used.

[0029] In addition to its literal meaning, the expression "comprises" as used herein also includes and refers in particular to the expressions "consisting essentially of" and "consisting of". Thus, the expression "comprising" refers to embodiments in which the subject matter "comprising" the specifically listed elements does not comprise further elements, as well as embodiments in which the subject matter "comprising" the specifically listed elements may and / or does encompass further elements. Likewise, the expression "having" is to be understood as the expression "comprising", and also includes and refers in particular to the expressions "consisting essentially of" and "consisting of". Where possible, the term "consisting essentially of" refers in particular to embodiments in which the subject matter comprises 20% or less, in particular 15% or less, 10% or less, or especially 5% or less of further elements in addition to the specifically listed elements in which the subject matter consists essentially of.

[0030] The term "nucleic acid" includes single-stranded and double-stranded nucleic acids, ribonucleic acids and deoxyribonucleic acids. It may contain naturally occurring and synthetic nucleotides and may be modified naturally or synthetically (e.g., by methylation, 5'- and / or 3'-capping). In a specific embodiment, the nucleic acid refers to double-stranded deoxyribonucleic acid.

[0031] The term "expression cassette" refers in particular to a nucleic acid construct capable of initiating and regulating the expression of a coding nucleic acid sequence and / or a template nucleic acid sequence introduced therein. The expression cassette may include a promoter, a ribosome binding site, an enhancer, and other control elements regulating gene transcription or mRNA translation. The exact structure of the expression cassette may vary according to species or cell type, but generally includes 5'-untranscribed and 5'- and 3'-untranslated sequences that participate in transcription and translation initiation, such as TATA boxes, capping sequences, CAAT sequences, etc. More particularly, the 5'-untranscribed expression control sequence includes a promoter region that includes a promoter sequence for transcription control of an operably connected nucleic acid. The expression cassette may also include an enhancer sequence or an upstream activator sequence. Some expression cassettes are only used to transcribe the template nucleic acid sequence into an RNA product, such as pri-miRNA. Such an expression cassette may not necessarily include a regulatory element for translation.

[0032] According to the present application, template nucleic acid is understood to be a DNA that is transcribed into a functional RNA product or its precursor, in particular a pri-miRNA. The functional RNA product alone or in combination with other RNA products and / or proteins has a biological activity, for example, the activity of miRNA (in combination with RISC proteins) interfering with target gene expression.

[0033] According to the present invention, the term "promoter" refers to a nucleic acid sequence located upstream (5') of a nucleic acid sequence to be expressed, and controls the expression of the sequence by providing recognition and binding sites for RNA-polymerase."Promoter" may include other recognition and binding sites for other factors involved in gene transcription regulation. Promoters may control the transcription of prokaryotic or eukaryotic genes. In addition, promoters may be "inducible", i.e., initiate transcription in response to an inducing agent, or may be "constitutive" if transcription is not controlled by an inducing agent. If there is no inducing agent, the gene under the control of an inducible promoter is not expressed or is only expressed to a small extent. In the presence of an inducing agent, the gene is turned on or the transcription level increases. This is usually mediated by the combination of specific transcription factors.

[0034] As used herein, the term "vector" is used in its most general sense, and comprises any intermediate medium of nucleic acid, which can, for example, introduce the nucleic acid into prokaryotic and / or eukaryotic cells, and is integrated into the genome when appropriate. Such vectors are preferably replicated and / or expressed in cells. Vectors include plasmids, phagemids, phages or viral genomes. As used herein, the term "plasmid" generally relates to the construct of extrachromosomal genetic material, generally circular DNA duplexes, which can replicate independently of chromosomal DNA. According to the present invention, the vector can exist in a circular or linearized form. As used herein, "vector nucleic acid" is a nucleic acid forming a vector or a nucleic acid portion of a vector.

[0035] The term "5'" and "3'" convention is used to describe nucleic acid sequence features related to genetic element position and / or event direction (5' to 3'), such as being transcribed by RNA polymerase or translated by ribosomes in the 5' to 3' direction. Synonyms are upstream (5') and downstream (3'). By convention, DNA sequences, gene maps, vector cards and RNA sequences are drawn from left to right with 5' to 3', or indicated with arrows in the 5' to 3' direction, wherein the arrow points to the 3' direction. Therefore, when following this convention, 5' (upstream) indicates the genetic element positioned toward the left-hand side, and 3' (downstream) indicates the genetic element positioned toward the right-hand side.

[0036] "Polypeptide" or "polypeptide chain" refers to a molecule comprising 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 composed of two or more polypeptide chains. Polypeptides include proteins and / or peptides having any activity or biological activity. Polypeptides can be pharmaceutically or therapeutically active compounds, or research tools for use in assays, etc.

[0037] A target amino acid sequence is "derived" from or "corresponds to" a reference amino acid sequence if it is 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% identical to the reference amino acid sequence over its entire length. In a specific embodiment, a target amino acid sequence "derived" from or "corresponds to" a reference amino acid sequence is 100% identical to the reference amino acid sequence over its entire length. Similarly, a target nucleotide sequence is "derived" from or "corresponds to" a reference nucleotide sequence if it is 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% identical to the reference nucleotide sequence over its entire length. In a specific embodiment, a target nucleotide sequence "derived" from or "corresponds to" a reference nucleotide sequence is 100% identical to the reference nucleotide sequence over its entire length. Preferably, the "identity" of an amino acid sequence or a nucleotide sequence according to the invention is determined over the entire length of the reference sequence.

[0038] As used herein, "microRNA", abbreviated as "miRNA", is a single-stranded non-coding RNA molecule that plays a role in RNA silencing and post-transcriptional regulation of gene expression. MiRNA is generally composed of 19 to 24 nucleotides, particularly about 22 nucleotides, especially 22 nucleotides. MiRNA molecules can silence mRNAs comprising complementary nucleotide sequences. The silencing of target mRNA may occur by cutting of mRNA, destabilization of mRNA, or blocking of mRNA translation. The silencing of target mRNA reduces or eliminates the production of proteins encoded by the target mRNA. It is generally understood in the art that miRNA associates with dicer and argonaute proteins to form an RNA-induced silencing complex (RISC) that binds to the target mRNA.

[0039] MiRNA is produced by transcribing a template DNA sequence into a miRNA precursor (pri-miRNA). Pri-miRNA comprises a hairpin stem-loop structure, wherein a double-stranded stem is connected to a loop on one side, and a single-stranded 5' and 3' extension is attached to the other side. The double-stranded stem comprises a guide strand (forming miRNA after processing) and a passenger strand (substantially complementary to the guide strand). In particular, except for the nucleotide pairs at the ends of the hairpin stem-loop structure, i.e., the nucleotide pairs of the passenger strand and the guide strand farthest from the loop structure, the passenger strand and the guide strand are complementary to each other. The guide strand and the passenger strand are generally each approximately 19 to 24 nucleotides in length, particularly 22 nucleotides. The remainder of pri-miRNA is referred to herein as miRNA scaffold. From 5' to 3', the pri-miRNA thus comprises (i) a 5'-miRNA scaffold stem, consisting of a 5' single-stranded extension and the 5' portion of the stem structure up to the passenger strand; (ii) the passenger strand; (iii) the miRNA scaffold loop; (iv) the guide strand; and (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. The positions of the passenger strand and the guide strand can also be switched.

[0040] The pri-miRNA is processed by cleaving the 5' and 3' miRNA scaffold stems, generating a hairpin structure called pre-miRNA. From 5' to 3', the pre-miRNA consists of a passenger strand, a miRNA scaffold loop, and a guide strand; the positions of the passenger and guide strands can also be switched. The loop structure is then cleaved, and the resulting RNA duplex is separated into two single-stranded RNA molecules, the guide strand and the passenger strand. The guide strand, which is complementary to the targeted mRNA molecule, forms the RISC, while the passenger strand usually has no function.

[0041] Cells mentioned herein are particularly host cells. According to the present invention, the term "host cell" refers to any cell that can be transformed or transfected with exogenous nucleic acids. Particularly preferred are mammalian cells, such as cells of humans, mice, hamsters, pigs, goats or primates. Cells can be derived from a variety of tissue types and include primary cells and cell lines. Nucleic acids can be present in host cells in the form of a single copy or two or more copies, and are expressed in host cells in one embodiment. Host cells particularly refer to cells present in cell culture, especially cells that are not present in living multicellular organisms.

[0042] As used herein, the term "CD39" refers to the human protein CD39 (cluster of differentiation 39), also known as ecto-nucleoside triphosphate diphosphohydrolase-1 (NTPDase1 or Ecto-ATPDase 1). CD39 is an ecto-nucleotidase present on the cell surface, having a catalytic site on the extracellular surface, which ecto-nucleotidase can catalyze the hydrolysis of the γ-phosphate residues and β-phosphate residues of triphosphate nucleosides and diphosphate nucleosides to monophosphate nucleoside derivatives. In particular, human CD39 has an amino acid sequence of SEQ ID NO: 50 and / or is represented by UniProt entry P49961.

[0043] "CD39 variant" refers to a protein derived from human CD39, which has an amino acid sequence at least 60% identical to human CD39 over the full length of human CD39. In certain embodiments, the variant of CD39 consists only of the extracellular domain of human CD39 or a portion thereof (SEQ ID NO:50 amino acids 38 to 478). In particular, the variant of CD39 has an amino acid sequence at least 80% identical to the extracellular domain of human CD39 over the full length of amino acid positions 38 to 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, compared to human CD39, the variant has an N-terminal deletion of 30 to 50 amino acids, a C-terminal deletion of 20 to 40 amino acids, and / or a central deletion of 10 to 15 amino acids. In addition, compared to human CD39, the variant may include up to 5 point mutations. Certain variants of CD39 are disclosed in WO 2020 / 016804. In particular, the variants of CD39 have an amino acid sequence selected from the group consisting of SEQ ID NO: 29 to 49.

[0044] As used herein, the term "CCL2" refers to the Chinese hamster protein CCL2 (CC motif chemokine receptor ligand 2), also known as monocyte chemoattractant protein 1 (MCP1). In particular, CCL2 has the amino acid sequence of SEQ ID NO: 24. As mentioned herein, the amount and concentration of CCL2 in the composition are determined in particular by ELISA (enzyme-linked immunosorbent assay) or LC-MS (liquid chromatography-mass spectrometry), in particular by LC-MS. Preferably, the corresponding measurements are performed as described in Example 5, Section 5 and 6, respectively.

[0045] The term "pharmaceutical composition" or "pharmaceutical formulation" refers in particular to a composition suitable for administration to humans or animals, i.e., a composition containing pharmaceutically acceptable components. Preferably, the pharmaceutical composition comprises an active compound or a salt or prodrug thereof, and a carrier, diluent or pharmaceutical excipient, such as a buffer, a preservative and a tonicity regulator.

[0046] The numbers given herein may be understood in certain embodiments as approximate numbers. In particular, the numbers may preferably be up to 10% higher and / or lower, in particular up to 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% higher and / or lower. In particular embodiments, the numbers given herein are not approximate numbers and may only vary within the inaccuracy of technical measurements.

[0047] Numerical ranges described herein include numbers that define ranges. The titles provided herein are not limitations on the various aspects or embodiments of the present invention, which can be read with reference to the entire specification. According to one embodiment, the subject matter described herein as comprising certain steps in the case of a method or as comprising certain components in the case of a composition refers to the subject matter consisting of the corresponding steps or components. Preferably, the preferred aspects and embodiments described herein are selected and combined, and the specific subject matter produced by the corresponding combinations of the preferred embodiments also belongs to the present disclosure. DETAILED DESCRIPTION

[0048] The present invention is based on the discovery 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 adjusting or by optimizing the downstream purification process (e.g., by introducing a novel or optimized affinity purification step). As observed during the purification process, the host cell protein CCL2 was not separated from the CD39 variants by the established chromatographic steps (especially by anion exchange chromatography or hydrophobic interaction chromatography). It is assumed that this lack of separation is caused by the highly similar physicochemical properties of CCL2 and the CD39 variants, in particular by the similar charge distribution.

[0049] The present inventors have solved this problem by knocking down CCL2 in CHO host cells using a miRNA method. Such a method has the following advantages: the presence of problematic proteins can be avoided from the beginning of the production process, and there is no need to further adjust the downstream process in a timely and potentially costly manner. Transfecting CHO host cells with a vector that produces miRNA targeting CCL2 mRNA has shown a strong knockdown effect and successfully significantly reduced the CCL2 level in the cell culture supernatant. Therefore, in the final composition of the purified CD39 variant, CCL2 is reduced to a negligible residual amount. In addition, the method for producing CD39 or its variants as described herein allows the production of pure and safe CD39 or its variants, which are suitable for use as drugs.

[0050] 1. Methods for producing CD39 and its variants

[0051] In a first aspect, the present invention provides a method for producing CD39 or a variant thereof, the method comprising the following steps:

[0052] (a) providing a CHO cell capable of producing CD39 or a variant thereof;

[0053] (b) cultivating CHO cells in cell culture under conditions that allow production of CD39 or a variant thereof;

[0054] (c) obtaining CD39 or a variant thereof from a cell culture; and

[0055] (d) optionally processing CD39 or a variant thereof;

[0056] CHO cells were engineered to reduce the production of CCL2 in the CHO cells.

[0057] The steps of the method are generally performed in the order indicated.

[0058] In a particular embodiment, CHO cells are engineered to reduce the production of CCL2 in CHO cells. CHO cells can be engineered in any suitable manner to reduce the production of CCL2. Reducing the production of CCL2 particularly refers to reducing the expression of CCL2. Cell line engineering, especially genetic engineering, is often used. In certain embodiments, the production of CCL2 in CHO cells is reduced by knocking down or knocking out CCL2 expression or degrading CCL2 protein.

[0059] Knockdown of CCL2 expression can be performed by reducing the amount of functional mRNA encoding CCL2 in the cell and / or reducing the rate at which mRNA encoding CCL2 is translated into CCL2 protein. In a specific embodiment, CHO cells are engineered to produce miRNA that targets endogenous CCL2 of CHO cells.

[0060] Knockout of CCL2 expression can be performed by deleting one or both alleles of the gene encoding CCL2 in the CHO cell genome, or mutating one or both of these alleles so that they cannot be transcribed into functional mRNA. Knockout of CCL2 expression can be accomplished using, for example, homologous recombination, site-specific nucleases, CRISPR / Cas, TALEN or Cas-Clover.

[0061] Reducing the production of CCL2 specifically 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, in particular 10% or less, or 5% or less of the amount of CCL2 in the cells before engineering.

[0062] In another aspect, the present invention provides a method for producing CD39 or a variant thereof, the method comprising the steps of:

[0063] (a) providing a host cell capable of producing CD39 or a variant thereof;

[0064] (b) cultivating the host cell in cell culture under conditions that allow production of CD39 or a variant thereof;

[0065] (c) obtaining CD39 or a variant thereof from a cell culture; and

[0066] (d) optionally processing CD39 or a variant thereof;

[0067] Wherein the host cell is engineered to reduce the production of CCL2 in the host cell.

[0068] The host cell can be any cell suitable for producing CD39 or its variant (especially human CD39 or its variant) and endogenously expressing CCL2 when not engineered to reduce CCL2 production. In particular, the host cell is a mammalian cell. The mammalian cell can be selected from, but not limited to, a group consisting of cells derived from the following animals: mice, such as COP, L, C127, Sp2 / 0, NS0, NS1, At20, and NIH3T3; rats, such as PC12, PC12h, GH3, MtT, YB2 / 0, and Y0; hamsters, such as BHK, CHO, and DHFR gene-deficient CHO; monkeys, such as COS1, COS3, COS7, CV1, and Vero; and humans, such as Hela, HEK293, CAP, retina-derived PER-C6, cells derived from diploid fibroblasts, myeloma cells, and HepG2. In a specific 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 is particularly useful for suspension culture.The features, embodiments and examples of the method for producing CD39 or variants thereof according to the first aspect of the invention are also applicable to the method according to the other aspects of the invention.

[0069] 1.1 miRNAs targeting CCL2

[0070] In certain embodiments, CHO cells are engineered to produce miRNAs targeting endogenous CCL2 of CHO cells. It should be understood that the miRNA targeting CCL2 is at least partially complementary to the mRNA or precursor mRNA encoding CCL2 and is capable of binding to the mRNA or precursor mRNA encoding CCL2 and initiating the silencing of the mRNA or precursor mRNA encoding CCL2. Silencing may occur via degradation of the targeted mRNA or preventing the targeted mRNA from being translated. The miRNA may 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. An exemplary miRNA targeting CCL2 has a nucleotide sequence selected from the group consisting of SEQ ID NO: 26 and 28.

[0071] In a specific embodiment, a CHO cell comprises an expression cassette for producing a miRNA targeting CCL2. The expression cassette may be present on a plasmid in the cell, or may be integrated into the genome of the cell. The expression cassette comprises a template sequence for a pri-miRNA, wherein the pri-miRNA is suitable for processing in a CHO cell to form a miRNA targeting CCL2. The expression cassette enables the template sequence to be transcribed into a pri-RNA. The template sequence may be present anywhere within the transcription region of the expression cassette.

[0072] In certain embodiments, the template sequence is present within an intron sequence. After transcription, the intron sequence is excised from the transcribed RNA, thereby forming a pri-miRNA.

[0073] Since the expression cassette contains the template sequence of the pri-miRNA in the intron sequence, it may contain further sequences for expressing other products, such as coding sequences for producing a polypeptide of interest, coding sequences for producing a selectable marker, and template sequences for other RNA products (especially other pri-miRNAs). Alternatively, the expression cassette can be specifically used to produce a miRNA targeting CCL2.

[0074] The expression cassette may contain a template sequence for a pri-miRNA within an intron sequence. After expression, a pre-mRNA containing the intron sequence is formed. The intron sequence is then spliced ​​from the pre-mRNA to form the pri-miRNA, which is then further processed to ultimately provide the miRNA. The pre-mRNA formed need not contain any sequence encoding a polypeptide.

[0075] In certain embodiments, the expression cassette further comprises a polymerase II promoter. The promoter is functionally connected to the template sequence of the pri-miRNA and controls the expression of the pri-miRNA. The promoter can be any RNA polymerase II promoter suitable for expressing genes in a host cell (especially a CHO cell). For example, the promoter can be selected from the group consisting of: cytomegalovirus (CMV) promoter, simian virus 40 (SV40) promoter, ubiquitin C (UBC) promoter, elongation factor 1α (EF1A) promoter, phosphoglycerate kinase (PGK) promoter, Rous sarcoma virus (Rous sarcoma virus (RSV)) promoter, BROAD3 promoter, mouse rosa 26 promoter, pCEFL promoter, chicken β-actin promoter (CBA), β-actin promoter (CAGG) coupled to CMV early enhancer, α-1-antitrypsin promoter, and inducible promoters, such as tetracycline inducible promoters (e.g., pTRE) and vanillic acid inducible promoters. In a particular embodiment, the promoter is a CMV promoter or a SV40 promoter, in particular a CMV promoter.

[0076] In certain embodiments, the expression cassette further comprises a terminator. The terminator is functionally connected to the template sequence of the pri-miRNA and controls the expression of the pri-miRNA. The term "terminator" as used herein refers to a transcription terminator that terminates transcription of DNA into RNA, especially by RNA polymerase II.

[0077] The template sequence of the pri-miRNA is particularly located between the promoter and the terminator of the expression cassette.

[0078] In certain embodiments, the expression cassette comprises a coding sequence encoding, for example, a polypeptide of interest or a selective 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 of the template sequence comprising the pri-miRNA may be present in the 5'UTR, 3'UTR or the coding sequence. In particular, the intron sequence is present in the 5'UTR or 3'UTR, especially in the 5'UTR. In an alternative embodiment, the expression cassette does not comprise a coding sequence encoding a polypeptide.

[0079] The intron sequence containing the template sequence of the pri-miRNA specifically contains a splice donor site upstream of the pri-miRNA and a corresponding splice acceptor site downstream of the pri-miRNA. Using these splice donor and acceptor sites, the pri-miRNA is spliced ​​out from the pre-mRNA after transcription.

[0080] In certain embodiments, the intron sequence comprises two or more template sequences of pri-miRNA. In these embodiments, the intron sequence comprises a splicing donor site upstream of the template sequence of the first pri-miRNA (i.e., the most 5' template sequence), and a corresponding splicing acceptor site downstream of the template sequence of the last pri-miRNA (i.e., the most 3' template sequence). Adjacent template sequences in the intron sequence can 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.

[0081] In certain embodiments, the expression cassette comprises only one intron sequence with one or more template sequences of a pri-miRNA. In alternative embodiments, the expression cassette comprises two or more intron sequences with one or more template sequences of a pri-miRNA.

[0082] The pri-miRNAs of two or more template sequences present in the same or different intronic sequences are particularly different from each other. In a specific embodiment, the miRNAs generated by the pri-miRNAs all target CCL2, but bind to different parts of the mRNA or pre-mRNA of CCL2.

[0083] The expression cassette can comprise the coding sequence of the coded polypeptide. The coding sequence can encode the selective marker in particular. The coding sequence is preferably functionally connected with the polymerase II promoter and terminator of the expression cassette. In the embodiment in which the expression cassette comprises the template sequence of the pri-miRNA and the coding sequence of the selective marker, the expression of the miRNA is related to the expression of the selective marker. Therefore, by increasing the selection pressure in the cloning selection process, the miRNA level also can increase.

[0084] The selectable marker may be selected from the group consisting of folate receptor (FAR), dihydrofolate reductase (DHFR), glutamine synthetase, puromycin, hygromycin, neomycin, bleomycin and blasticidin. In certain embodiments, the selectable marker is folate receptor (FAR).

[0085] The expression cassette comprises a template sequence of the pri-miRNA. The pri-miRNA produced from the expression cassette may have any structure suitable for host cell processing to obtain a functional miRNA targeting CCL2. The functional miRNA induces a decrease in the level of CCL2 in the host cell.

[0086] In certain embodiments, the pri-miRNA comprises a passenger strand and a guide strand. The guide strand particularly comprises or is composed of a miRNA formed after the host cell processes the pri-miRNA. In addition, the pri-miRNA may comprise a miRNA scaffold loop and / or a miRNA scaffold stem, especially a 5'miRNA scaffold stem and a 3'miRNA scaffold stem. In a specific embodiment, from 5' to 3', the pri-miRNA comprises a 5'miRNA scaffold stem, a passenger strand, a miRNA scaffold loop, a guide strand, and a 3'miRNA scaffold stem. In an alternative embodiment, from 5' to 3', the pri-miRNA comprises a 5'miRNA scaffold stem, a guide strand, a miRNA scaffold loop, a passenger strand, and a 3'miRNA scaffold stem. An embodiment in which the passenger strand is located upstream of the guide strand is preferred.

[0087] In particular, the passenger strand and guide strand of the pri-miRNA have artificial sequences. In this regard, artificial sequences refer to sequences that do not exist as passenger strands or guide strands in naturally occurring miRNAs. In particular, the sequences of passenger strands and guide strands are not found in naturally occurring miRNAs.

[0088] 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.

[0089] In certain embodiments, one or more scaffold sequences of a pri-miRNA or pre-miRNA are derived from naturally occurring pri-miRNAs, in particular pri-miRNAs naturally occurring in mammals, in particular humans. In a specific embodiment, all scaffold sequences of a pri-miRNA are derived from naturally occurring pri-miRNAs, in particular pri-miRNAs naturally occurring in mammals, in particular humans. In particular, all scaffold sequences of a pri-miRNA are derived from the same naturally occurring pri-miRNA. The scaffold sequence of a pri-miRNA particularly comprises a 5' miRNA scaffold stem, a miRNA scaffold loop, and a 3' miRNA scaffold stem. Suitable naturally occurring pri-miRNAs from which 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, miRNA scaffold loop, and 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 a specific embodiment, the naturally occurring pri-miRNA from which the scaffold sequence is derived is miR-30A.

[0090] In a particular embodiment, all scaffold sequences of the pri-miRNA have 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 the naturally occurring pri-miRNA. In certain embodiments, the 5'miRNA scaffold stem of the pri-miRNA has at least 80%, particularly at least 85%, and particularly at least 90% nucleotide sequence identity over its entire length with the corresponding scaffold sequences of the naturally occurring pri-miRNA. In certain embodiments, the 3'miRNA scaffold stem of the pri-miRNA has at least 80%, particularly at least 90%, and particularly at least 95% nucleotide sequence identity over its entire length with the corresponding scaffold sequences of the naturally occurring pri-miRNA. In certain embodiments, the miRNA scaffold loop of the pri-miRNA has at least 60%, particularly at least 70%, and particularly at least 75% nucleotide sequence identity over its entire length with the corresponding scaffold sequences of the naturally occurring pri-miRNA. In these embodiments, the naturally occurring pri-miRNA may be particularly miR-30A.

[0091] In certain embodiments, the 5'miRNA scaffold stem of the pri-miRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 1-7 or a derivative thereof. In particular, the 5'miRNA scaffold stem of the pri-miRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 1-7 or a sequence having 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 a nucleotide sequence of any one of SEQ ID NOs: 1-4 (particularly SEQ ID NO: 1).

[0092] In certain embodiments, the miRNA scaffold loop of the pri-miRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 8-10 or a derivative thereof. In particular, the miRNA scaffold loop of the pri-miRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 8-10 or a sequence having at least 75%, preferably at least 85%, more preferably at least 90%, and most preferably 100% nucleotide sequence identity thereto. In particular, the miRNA scaffold loop of the pri-miRNA consists of a nucleotide sequence of any one of SEQ ID NOs: 8-10, in particular SEQ ID NO: 8.

[0093] In certain embodiments, the 3'miRNA scaffold stem of the pri-miRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 11-17 or a derivative thereof. In particular, the 3'miRNA scaffold stem of the pri-miRNA comprises a nucleotide sequence of any one of SEQ ID NOs: 11-17 or a sequence having at least 90%, preferably at least 95%, more preferably at least 98%, and most preferably 100% nucleotide sequence identity thereto. In particular, the 3'miRNA scaffold stem of the pri-miRNA consists of a nucleotide sequence of any one of SEQ ID NOs: 11-14 (in particular SEQ ID NO: 14).

[0094] In certain embodiments, the template sequence of the pri-miRNA comprises at least one recognition site, especially two recognition sites, of a DNA restriction enzyme. In particular, the two recognition sites are used for different DNA restriction enzymes and produce different overhangs after cutting. The two recognition sites are preferably flanked on both sides by the pre-miRNA portion of the pri-miRNA - it comprises a guide strand, a passenger strand and a miRNA scaffold loop. In particular, one of the recognition sites is located within a sequence that is transcribed into a 5' miRNA scaffold stem, and the other recognition site is located within a sequence that is transcribed into a 3' miRNA scaffold stem. In a particular embodiment, the recognition site is located within a sequence that is transcribed into a single-stranded portion of a 5' and 3' miRNA scaffold stem. The recognition site is particularly a unique recognition site within an expression cassette, especially within the entire vector comprising the expression cassette.

[0095] 1.2 Knockout of CCL2 gene

[0096] In certain embodiments, CHO cells are engineered by knocking out the CCL2 gene. Knockout of the CCL2 gene refers to the deletion or inactivation of the CCL2 gene. In a specific embodiment, both alleles of the CCL2 gene in the CHO cell are knocked out. The CCL2 gene can be completely removed, or its transcription may be impaired, or the translation of the complete CCL2 protein may be inhibited. In particular, mutations can be introduced into genes that cause frameshifts in the coding sequence of CCL2 mRNA.

[0097] Knockout of CCL2 expression can be accomplished using, for example, homologous recombination, site-specific nucleases, CRISPR / Cas, TALEN, or Cas-Clover. In particular, knockout using Cas-Clover can be performed using a gRNA pair (including a gRNA comprising a nucleotide sequence of SEQ ID NO: 53 and another gRNA comprising a nucleotide sequence of SEQ ID NO: 54).

[0098] 1.3 Production and purification steps

[0099] In some embodiments, the method further comprises the following steps between steps (a) and (b):

[0100] (a1) inoculating a cell culture medium with CHO cells to provide a cell culture, and (a2) culturing the CHO cells in the cell culture under conditions that allow the number of the CHO cells in the cell culture to increase.

[0101] Technicians can easily determine suitable conditions for culturing host cells, increasing their cell number and expressing CD39 or its variants, which are also known in the art. In certain embodiments, the vector nucleic acid encoding CD39 or its variants in CHO cells comprises one or more selective marker genes. In these embodiments, the culture conditions in steps (a2) and / or (b) may include the presence of a corresponding selection agent in the cell culture medium.

[0102] Obtaining CD39 or its variant from the cell culture in step (c) particularly comprises separating CD39 or its variant from the cell culture. The separation 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 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 separated from the cell culture medium. Separating CD39 or its variant from the cell culture medium can be performed, for example, by chromatography and / or filtration.

[0103] Suitable methods and means for isolating CD39 or its variants are known in the art and can be easily applied by those skilled in the art. Exemplary separation methods include, for example, filtration steps (e.g., tangential flow filtration, alternating flow filtration, depth filtration, ultrafiltration and diafiltration) and / or chromatography steps (e.g., affinity chromatography, anion and / or cation exchange chromatography, hydrophilic interaction chromatography, size exclusion chromatography and reverse phase chromatography). In particular, the step of obtaining CD39 or its variants from cell culture may include a filtration step, a capture chromatography step and one or more polishing chromatography steps. In addition, step (c) may further include one or more viral inactivation steps.

[0104] In certain embodiments, step (c) comprises performing anion exchange chromatography and / or hydrophobic interaction chromatography. In particular, step (c) comprises:

[0105] (c1) isolating the CHO cells from the cell culture supernatant containing CD39 or its variants;

[0106] (c2) isolating CD39 or its variant from the cell culture supernatant using anion exchange chromatography;

[0107] (c3) further purifying CD39 or its variant using hydrophobic interaction chromatography; and

[0108] (c4) further purifying CD39 or its variant using anion exchange chromatography.

[0109] In particular, step (c1) can be carried out using a filtration method (e.g., tangential flow filtration or alternating flow filtration), wherein CHO cells are retained in the retentate, and the cell culture supernatant containing CD39 or its variants passes through the filter to become a permeate. In addition, step (c1) can also be carried out using a centrifugation method. In addition to steps (c1) to (c4), step (c) can also further include one or more ultrafiltration and / or diafiltration steps, which are used to perform buffer exchange between or after the chromatography step and the virus inactivation and / or virus retention step. In particular, virus inactivation can be achieved using a time sufficient to inactivate substantially all viruses at low pH, and virus retention can be achieved using sterile filtration.

[0110] The obtained CD39 or its variant can optionally be subjected to additional processing steps (d), such as, for example, modification and / or formulation steps, to produce CD39 or its variant with desired quality and composition. Such additional 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 steps and additional steps can be used.

[0111] In certain embodiments, the method for producing CD39 or a variant thereof further comprises (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 into a pharmaceutical composition particularly comprises exchanging a buffer solution or a buffer solution component of a composition comprising CD39 or a variant thereof. Furthermore, this step may comprise lyophilization of CD39 or a variant thereof. In particular, CD39 or a variant thereof is transferred to a composition comprising only pharmaceutically acceptable ingredients.

[0112] Compared to the same method using CHO cells that have not been engineered to reduce the production of CCL2 in CHO cells, the method of producing CD39 or its variants particularly provides CD39 or its variants with higher purity. In certain embodiments, the CD39 or its variants obtained after step (c) are present in a composition, which is compared to the 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, and contains a concentration of at least 10 times lower, preferably 25 times lower, and more preferably 100 times lower CCL2. In particular, the CD39 or its variants obtained after step (c) are present in a composition, which contains a concentration of 100ppm or less, preferably 25ppm or less, more preferably 10ppm or less CCL2.

[0113] In certain embodiments, the CD39 or variant thereof obtained after step (c1) is present in a composition comprising a concentration of CCL2 at least 10 times lower, preferably 25 times lower, more preferably 100 times lower, compared to a composition obtained by the same method after step (c1) using CHO cells that have not been engineered to reduce CCL2 production in CHO cells. In particular, the CD39 or variant thereof obtained after step (c1) is present in a composition comprising a concentration of CCL2 of 100 ppm or less, preferably 25 ppm or less, more preferably 10 ppm or less.

[0114] In certain embodiments, the CD39 or variant thereof obtained after step (c4) is present in a composition comprising a concentration of CCL2 at least 10 times lower, preferably 25 times lower, more preferably 100 times lower, compared to a composition obtained by the same method after step (c4) using CHO cells that have not been engineered to reduce CCL2 production in CHO cells. In particular, the CD39 or variant thereof obtained after step (c4) is present in a composition comprising a concentration of CCL2 of 25 ppm or less, preferably 10 ppm or less, more preferably 5 ppm or less.

[0115] In certain embodiments, the CD39 or variant thereof obtained after step (d) is present in a composition comprising a concentration of CCL2 at least 10-fold lower, preferably 25-fold lower, more preferably 100-fold lower, compared to a composition obtained by the same method after step (d) using CHO cells that have not been engineered to reduce CCL2 production in CHO cells. In particular, the CD39 or variant thereof obtained after step (d) is present in a composition comprising a concentration of CCL2 of 25 ppm or less, preferably 10 ppm or less, more preferably 5 ppm or less.

[0116] 1.4CD39 and its variants

[0117] In particular, the CD39 or its variant produced by the method is a variant of human CD39 or human CD39. In certain embodiments, CD39 or its variant does not contain any membrane anchor. In this embodiment, the variant is particularly a soluble variant of CD39. In a specific embodiment, compared with human CD39, the variant has an N-terminal deletion, a C-terminal deletion and / or a central deletion, especially an N-terminal deletion, a C-terminal deletion and a central deletion. In particular, the N-terminal deletion may contain 30 to 50 amino acids. In particular, the C-terminal deletion may contain 20 to 40 amino acids. In particular, the central deletion may contain 10 to 15 amino acids. In addition, compared with human CD39, the variant in some embodiments contains up to 5 point mutations.

[0118] In certain embodiments, the CD39 variant has an amino acid sequence selected from the group consisting of SEQ ID NO: 29-49. In particular, the CD39 variant comprises the amino acid sequence of SEQ ID NO: 49, in particular consisting of the amino acid sequence of SEQ ID NO: 49.

[0119] 2. CHO host cells

[0120] In a second aspect, the present invention provides a CHO cell capable of producing CD39 or a variant thereof, and the CHO cell is engineered to reduce the production of CCL2 in the CHO cell. In particular, the CHO cell can be engineered as described above for the method of producing CD39 or a variant thereof.

[0121] In another aspect, the present invention provides a host cell capable of producing CD39 or a variant thereof, and the host cell is engineered to reduce the production of CCL2 in the host cell. In particular, the host cell is a host cell as described above for the method of producing CD39 or a variant thereof, and can be engineered as described above for the method of producing CD39 or a variant thereof. The features and embodiments described herein for CHO cells are also generally applicable to host cells.

[0122] The host cell can be any cell suitable for producing CD39 or its variant (especially human CD39 or its variant) and endogenously expressing CCL2 when not engineered to reduce CCL2 production. In particular, the host cell is a mammalian cell. The mammalian cell can be selected from, but not limited to, a group consisting of cells derived from the following animals: mice, such as COP, L, C127, Sp2 / 0, NS0, NS1, At20, and NIH3T3; rats, such as PC12, PC12h, GH3, MtT, YB2 / 0, and Y0; hamsters, such as BHK, CHO, and DHFR gene-deficient CHO; monkeys, such as COS1, COS3, COS7, CV1, and Vero; and humans, such as Hela, HEK293, CAP, retina-derived PER-C6, cells derived from diploid fibroblasts, myeloma cells, and HepG2. In a specific 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 is particularly useful for suspension culture.

[0123] In addition to the expression cassette according to the fourth aspect of the invention or the vector nucleic acid according to the fifth aspect of the invention, the CHO cell or host cell may also contain other exogenous nucleic acids. In particular, the CHO cell or host cell may contain an expression cassette for expressing CD39 or a variant thereof, which is not an expression cassette according to the fourth aspect and is not present on the vector nucleic acid according to the fifth aspect. The expression cassette for expressing CD39 or a variant thereof may be present on another vector nucleic acid or integrated into the genome of the CHO cell or host cell.

[0124] In certain embodiments, the coding sequence of CD39 or its variant is present in a CHO cell or a host cell:

[0125] (i) within an expression cassette expressing a miRNA targeting CCL2,

[0126] (ii) within another expression cassette on the same vector nucleic acid as the expression cassette for expressing the miRNA targeting CCL2, or

[0127] (iii) on another vector nucleic acid or in the genome of a CHO cell or host cell.

[0128] In particular, the CD39 or a variant thereof is CD39 or a variant thereof as described herein.

[0129] In a further aspect, the present invention provides a mammalian cell (particularly a CHO cell) engineered to reduce the production of CCL2 in the mammalian cell. The mammalian cell may be engineered as described above for the method of producing CD39 or a variant thereof.

[0130] In an eighth aspect, the present invention provides a CHO cell that is engineered to reduce the production of CCL2 in the CHO cell. In certain embodiments, the CHO cell produces a miRNA targeting CCL2. In particular, the CHO cell is engineered by introducing the vector nucleic acid according to the fifth aspect. In another embodiment of the above aspects, the CHO cell is engineered by knocking out the CCL2 gene.

[0131] Except that the CHO cell according to the eighth aspect does not necessarily have to be able to produce CD39 or its variants, the embodiments, features and examples of the CHO cell according to the second aspect are also applicable to the CHO cell according to the eighth aspect.

[0132] 3. Methods for improving the production of CD39 and its variants

[0133] In a third aspect, the present invention provides a method for improving the production of CD39 or a variant thereof in CHO cells, the method comprising the steps of:

[0134] (ai) providing CHO cells capable of producing CD39 or a variant thereof;

[0135] (a-ii) CHO cells were engineered to reduce the production of CCL2 in CHO cells.

[0136] In particular, the engineering of CHO cells can be carried out as described above for the method for producing CD39 or its variants. In certain embodiments, CHO cells are engineered by knocking down or knocking out CCL2 expression in CHO cells or inducing the degradation of CCL2 protein in CHO cells in step (a-ii). In particular, the CHO cells in step (a-ii) are engineered to produce the miRNA targeting the endogenous CCL2 of CHO cells. In a specific embodiment, the step (a-ii) of engineering CHO cells to reduce the production of CCL2 includes introducing the vector nucleic acid (especially the vector nucleic acid comprising the expression cassette for expressing the miRNA targeting CCL2) encoding the miRNA targeting CCL2 into the CHO cells. In particular, a vector nucleic acid as described herein is used.

[0137] The embodiments, features and examples of the method for producing CD39 or its variants as described herein are also applicable to improving the method for producing CD39 or its variants in CHO cells. In particular, the CHO cells, CD39 or its variants, the miRNA targeting CCL2 and / or the expression cassette can be as defined above for the method for producing CD39 or its variants.

[0138] In certain embodiments, step (a-ii) of engineering CHO cells produces engineered 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 cell before step (a-ii). In specific embodiments, step (a-ii) of engineering CHO cells produces engineered 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 cell before step (a-ii).

[0139] A method for improving the production of CD39 or a variant thereof in CHO cells, the method may further comprise the following steps:

[0140] (b) culturing the CHO cells obtained in step (a-ii) in a cell culture under conditions that allow the CHO cells to proliferate and simultaneously and / or subsequently produce CD39 or a variant thereof;

[0141] (c) obtaining the CD39 or its variant from a cell culture; and

[0142] (d) optionally processing CD39 or a variant thereof.

[0143] The embodiments, features and examples of steps (a), (b), (c) and (d) of the method for producing CD39 or its variants as described herein are also equally applicable to steps (ai), (b), (c) and (d), respectively, of the method for improving the production of CD39 or its variants in CHO cells.

[0144] 4. Expression Cassette and Vector Nucleic Acid

[0145] In a fourth aspect, the present invention provides an expression cassette for expressing miRNA in CHO cells, the expression cassette comprising a template sequence of a pri-miRNA, wherein the pri-miRNA is suitable for being processed in CHO cells to form a miRNA targeting CCL2.

[0146] The features, embodiments and examples of the expression cassette described above in relation to the method for producing CD39 or variants thereof are also applicable to the expression cassette according to the fourth aspect of the present invention.

[0147] In a fifth aspect, the present invention provides a vector nucleic acid for transfecting CHO cells, the vector nucleic acid comprising the expression cassette according to the fourth aspect. The vector nucleic acid can be any vector nucleic acid suitable for transfecting CHO cells. In certain embodiments, the vector nucleic acid is a plasmid. In other embodiments, the vector nucleic acid is a viral vector.

[0148] In addition to the expression cassette, the vector nucleic acid may also include other elements. For example, the vector nucleic acid may include an origin of replication (ORI), a coding sequence encoding a target polypeptide, a selective marker gene and / or an antibiotic resistance gene. In particular, the target polypeptide may be CD39 or a variant thereof, especially CD39 or a variant thereof as described herein.

[0149] In certain embodiments, the vector nucleic acid further comprises a coding sequence encoding CD39 or a variant thereof, particularly 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 the pri-miRNA, or may be present in another expression cassette present in the vector nucleic acid.

[0150] In certain embodiments, the vector nucleic acid does not comprise a coding sequence encoding a polypeptide of interest. In specific embodiments in which the vector nucleic acid does not comprise a coding sequence encoding a polypeptide of interest, the expression cassette according to the fourth aspect of the invention does not comprise a coding sequence for a polypeptide of interest. In these embodiments, the vector nucleic acid particularly comprises another expression cassette containing a selective marker gene. In alternative embodiments in which the vector nucleic acid does not comprise a coding sequence encoding a polypeptide of interest, the expression cassette according to the fourth aspect of the invention comprises a coding sequence encoding a selective marker.

[0151] In certain embodiments, the vector nucleic acid comprises a coding sequence encoding a polypeptide of interest. The coding sequence encoding the polypeptide of interest may be present in an expression cassette according to the fourth aspect of the present invention, or may be present in another expression cassette. In a specific embodiment, the vector nucleic acid comprises at least two expression cassettes, a first expression cassette for expressing a polypeptide of interest and a second expression cassette for expressing a selective marker, wherein the first or second expression cassette is an expression cassette according to the fourth aspect of the present invention. In an alternative embodiment, the vector nucleic acid comprises at least three expression cassettes, a first expression cassette is an expression cassette according to the fourth aspect of the present invention, a second expression cassette for expressing a polypeptide of interest, and a third expression cassette for expressing a selective marker. In particular, the polypeptide of interest may be CD39 or a variant thereof, in particular CD39 or a variant thereof as described herein.

[0152] 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. These expression cassettes may each contain template sequences for the same or different pri-miRNAs, in particular for different pri-miRNAs. In particular, the miRNAs produced by the pri-miRNAs may all target CCL2, but bind to different parts of the mRNA or pre-mRNA of CCL2.

[0153] 5. Methods for Producing CHO Cells

[0154] In a sixth aspect, the present invention provides a method for producing a CHO cell capable of expressing CD39 or a variant thereof, the method comprising introducing the vector nucleic acid according to the fifth aspect into a CHO cell. The vector nucleic acid may additionally 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 a CHO cell or introduced into a CHO cell.

[0155] Therefore, in certain embodiments, the method for producing CHO cells includes the step of 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 its variant in an expression cassette expressing a miRNA targeting CCL2 or in another expression cassette. In alternative embodiments, the method for producing CHO cells includes the step of introducing the vector nucleic acid according to the fifth aspect into CHO cells, wherein the vector nucleic acid does not comprise a coding sequence of CD39 or its variant, and introducing another vector nucleic acid suitable for recombinant expression of CD39 or its variant into the CHO cells, wherein different vector nucleic acids can be introduced into the CHO cells simultaneously or continuously in any order. In even further embodiments, the method for producing CHO cells includes the steps of: (a) providing a CHO cell capable of expressing CD39 or its variant, and (b) introducing the vector nucleic acid according to the fifth aspect into CHO cells. In these embodiments, the vector nucleic acid according to the fifth aspect preferably does not comprise a coding sequence of CD39 or its variant.

[0156] In particular, the CD39 or a variant thereof is CD39 or a variant thereof as described herein.

[0157] The carrier nucleic acid is artificially introduced into the CHO cell. In particular, the carrier nucleic acid is introduced by transfection. In this respect, the transfection can be transient or stable, especially using stable transfection. Therefore, in certain embodiments, the CHO cell produced comprises an expression cassette according to the fifth aspect described in its genome that is stably integrated.

[0158] In a further aspect, the present invention provides the use of the expression cassette according to the fourth aspect, the vector nucleic acid according to the fifth aspect, or the CHO cell according to the sixth aspect for producing CD39 or a variant thereof. The features, embodiments and examples of the methods for producing CD39 or a variant thereof described herein also apply to this use.

[0159] The present invention further provides the use of the expression cassette according to the fourth aspect or the vector nucleic acid according to the fifth aspect for improving the production of CD39 or its variants by CHO cells, comprising introducing the expression cassette or vector nucleic acid into CHO cells capable of producing CD39 or its variants. In certain embodiments, the vector nucleic acid introduced into the CHO cells does not contain the coding sequence of CD39 or its variants. In particular, improving the production of CD39 or its variants includes increasing the purity of CD39 or its variants. The features, embodiments and examples of the methods for improving the production of CD39 or its variants in CHO cells described herein are also applicable to this use.

[0160] 6. Compositions comprising CD39 or variants thereof

[0161] In a seventh aspect, the present invention provides a composition comprising CD39 or a variant thereof and CCL2, wherein the composition is obtained by producing using the CHO cells according to the second aspect, and wherein:

[0162] (i) the amount of CCL2 in the composition is at least 10-fold lower than the same composition obtained by production using CHO cells that have not been engineered to reduce production of CCL2 in CHO cells; and / or

[0163] (ii) The composition comprises CCL2 at a concentration of 100 ppm or less, preferably 25 ppm or less, more preferably 10 ppm or less.

[0164] In certain embodiments, according to the first aspect of the present invention, the composition is obtained by step (c1) of the method of producing CD39 or its variants. In these embodiments, in particular, the composition can be a cell culture supernatant or a cell-free bulk harvest of a cell culture. In particular, the composition according to these embodiments can contain CCL2 at a concentration of 100 ppm or less, preferably 25 ppm or less, more preferably 10 ppm or less.

[0165] In other embodiments, according to the first aspect of the present invention, the composition is obtained by step (c4) of the method for producing CD39 or its variants. In these embodiments, in particular, the composition can be a purified composition substantially free of proteins other than CD39 or its variants. A composition substantially free of proteins other than CD39 or its variants, in particular, relative to the total amount of protein in the composition, the composition comprises 5% or less, preferably 2% or less, more preferably 1% or less of proteins other than CD39 or its variants. In particular, the composition according to these embodiments may include a concentration of 25ppm or less, preferably 10ppm or less, more preferably 5ppm or less of CCL2.

[0166] In further embodiments, according to the first aspect of the present invention, the composition is obtained by step (d) of the method of producing CD39 or its variants. In these embodiments, in particular, the composition may be a pharmaceutical product composition suitable for use in treating patients (especially human patients). In particular, the composition according to these embodiments may contain CCL2 at a concentration of 25ppm or less, preferably 10ppm or less, more preferably 5ppm or less.

[0167] 7. Specific Embodiments

[0168] In the following, specific embodiments of the invention are described. These embodiments can be combined with other embodiments, features and examples described herein.

[0169] Example 1. A method for producing CD39 or a variant thereof, the method comprising the following steps:

[0170] (a) providing a CHO cell capable of producing CD39 or a variant thereof;

[0171] (b) cultivating the CHO cells in cell culture under conditions that allow production of CD39 or a variant thereof;

[0172] (c) obtaining CD39 or a variant thereof from the cell culture; and

[0173] (d) optionally processing CD39 or a variant thereof;

[0174] The CHO cells were engineered to reduce the production of CCL2 in the CHO cells.

[0175] Embodiment 2. The method according to embodiment 1, wherein the production of CCL2 in the CHO cells is reduced by knocking down or knocking out CCL2 expression or degrading CCL2 protein.

[0176] Embodiment 3. The method according to embodiment 1 or 2, wherein the CHO cells are engineered to produce miRNA targeting endogenous CCL2 of the CHO cells.

[0177] 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.

[0178] Embodiment 5. The method according to embodiment 3 or 4, wherein the CHO cells comprise an expression cassette for producing the miRNA targeting CCL2.

[0179] Embodiment 6. The method according to embodiment 5, wherein the expression cassette comprises an intron sequence, the intron sequence comprises a template sequence of a pri-miRNA, wherein the pri-miRNA is suitable for processing in the CHO cell to form the miRNA targeting CCL2.

[0180] Embodiment 7. The method according to embodiment 6, wherein the pri-miRNA comprises from 5' to 3':

[0181] (i) a 5' miRNA scaffold stem, the 5' miRNA scaffold stem optionally comprising a nucleotide sequence of SEQ ID NO: 1-4,

[0182] (ii) a passenger strand having a nucleotide sequence complementary to the sequence of the miRNA, optionally comprising one or two mismatches,

[0183] (iii) a miRNA scaffold loop, the miRNA scaffold loop optionally comprising the nucleotide sequence of SEQ ID NO: 8,

[0184] (iv) a guide strand having the nucleotide sequence of the miRNA, and (v) a 3' miRNA scaffold stem, the 3' miRNA scaffold stem optionally comprising the nucleotide sequence of SEQ ID NOs: 11-14;

[0185] The positions of the passenger strand and the guide strand can be switched, and wherein 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.

[0186] 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-miRNAs, wherein these pri-miRNAs are suitable for processing in the CHO cell to form miRNAs targeting CCL2, wherein the miRNAs processed from these pri-miRNAs bind to different parts of the RNA, especially mRNA or pre-mRNA, of CCL2.

[0187] Embodiment 9. The method according to embodiment 8, wherein the different miRNAs targeting CCL2 have a nucleotide sequence selected from the group consisting of SEQ ID NOs: 26 and 28.

[0188] Example 10. A method for producing CD39 or a variant thereof, the method comprising the following steps:

[0189] (a) providing a CHO cell capable of producing CD39 or a variant thereof;

[0190] (b) cultivating the CHO cells in cell culture under conditions that allow production of CD39 or a variant thereof;

[0191] (c) obtaining CD39 or a variant thereof from the cell culture; and

[0192] (d) optionally processing CD39 or a variant thereof.

[0193] 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.

[0194] Embodiment 12. The method according to any one of embodiments 1 to 11, wherein step (c) comprises:

[0195] (c1) isolating the CHO cells from the cell culture supernatant containing CD39 or its variants;

[0196] (c2) isolating CD39 or its variant from the cell culture supernatant using anion exchange chromatography;

[0197] (c3) further purifying CD39 or its variant using hydrophobic interaction chromatography; and

[0198] (c4) further purifying CD39 or its variant using anion exchange chromatography.

[0199] 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.

[0200] Embodiment 14. The method according to any one of embodiments 1 to 13, wherein the method is used to generate a variant of CD39 having one or more of the following characteristics:

[0201] (i) is a soluble variant lacking the membrane anchor of human CD39;

[0202] (ii) compared with human CD39, it has an N-terminal deletion, a C-terminal deletion, and a central deletion;

[0203] (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;

[0204] (iv) contain up to 5 point mutations compared to human CD39;

[0205] (v) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-49.

[0206] Embodiment 15. The method according to any one of embodiments 1 to 14, wherein the method is used to produce a variant of CD39 comprising the amino acid sequence of SEQ ID NO: 49.

[0207] Embodiment 16. The method according to any one of embodiments 1 to 15, wherein the method is used to produce a variant of CD39 consisting of the amino acid sequence of SEQ ID NO: 49.

[0208] Embodiment 17. The method according to any one of embodiments 1 to 16, wherein the CD39 or variant thereof obtained after step (c) is present in a composition comprising CCL2 at a concentration of 100 ppm or less, preferably 25 ppm or less, more preferably 10 ppm or less.

[0209] Embodiment 18. The method according to any one of Embodiment 12, wherein the CD39 or variant thereof obtained after step (c1) is present in a composition comprising CCL2 at a concentration of 100 ppm or less, preferably 25 ppm or less, more preferably 10 ppm or less.

[0210] Embodiment 19. The method according to any one of Embodiment 12, wherein the CD39 or its variant obtained after step (c4) is present in a composition comprising CCL2 at a concentration of 25 ppm or less, preferably 10 ppm or less, more preferably 5 ppm or less.

[0211] Embodiment 20. The method according to any one of embodiments 1 to 19, wherein the CD39 or variant thereof obtained after step (d) is present in a composition comprising CCL2 at a concentration of 25 ppm or less, preferably 10 ppm or less, more preferably 5 ppm or less.

[0212] Embodiment 21. A method according to any one of embodiments 1 to 20, wherein the CD39 or variant thereof obtained after step (c) is present in a composition comprising a concentration of CCL2 that is at least 10 times lower, preferably 25 times lower, more preferably 100 times lower, compared to a composition obtained by the same method after step (c) using CHO cells that have not been engineered to reduce CCL2 production in CHO cells.

[0213] Embodiment 22. A method according to any one of embodiments 1 to 20, wherein the CD39 or variant thereof obtained after step (c1) is present in a composition comprising a concentration of CCL2 at least 10 times lower, preferably 25 times lower, more preferably 100 times lower, compared to a composition obtained by the same method after step (c1) using CHO cells that have not been engineered to reduce CCL2 production in CHO cells.

[0214] Embodiment 23. A method according to any one of embodiments 1 to 20, wherein the CD39 or variant thereof obtained after step (c4) is present in a composition comprising a concentration of CCL2 at least 10 times lower, preferably 25 times lower, more preferably 100 times lower, compared to a composition obtained by the same method after step (c4) using CHO cells that have not been engineered to reduce CCL2 production in CHO cells.

[0215] Embodiment 24. A method according to any one of embodiments 1 to 20, wherein the CD39 or variant thereof obtained after step (d) is present in a composition comprising a concentration of CCL2 at least 10 times lower, preferably 25 times lower, more preferably 100 times lower, compared to a composition obtained by the same method after step (d) using CHO cells that have not been engineered to reduce CCL2 production in CHO cells.

[0216] Embodiment 25. A CHO cell capable of producing CD39 or a variant thereof, and the CHO cell is engineered to reduce the production of CCL2 in the CHO cell.

[0217] Embodiment 26. The cell according to embodiment 25, wherein the production of CCL2 is reduced by knocking down or knocking out CCL2 expression or degrading CCL2 protein.

[0218] Embodiment 27. The cell of embodiment 25 or 26, which is engineered to produce a miRNA targeting endogenous CCL2 of the CHO cells.

[0219] 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 NO: 26 and 28.

[0220] Embodiment 29. The cell according to embodiment 27 or 28, comprising an expression cassette for producing the miRNA targeting CCL2.

[0221] Embodiment 30. The cell according to embodiment 29, wherein the expression cassette comprises an intron sequence comprising a template sequence for a pri-miRNA, wherein the pri-miRNA is suitable for processing in the CHO cell to form a miRNA targeting CCL2.

[0222] Embodiment 31. The cell according to embodiment 30, wherein the pri-miRNA comprises from 5' to 3':

[0223] (i) a 5' miRNA scaffold stem, the 5' miRNA scaffold stem optionally comprising a nucleotide sequence of SEQ ID NO: 1-4,

[0224] (ii) a passenger strand having a nucleotide sequence complementary to the sequence of the miRNA, optionally comprising one or two mismatches,

[0225] (iii) a miRNA scaffold loop, the miRNA scaffold loop optionally comprising the nucleotide sequence of SEQ ID NO: 8,

[0226] (iv) a guide strand having the nucleotide sequence of the miRNA, and (v) a 3' miRNA scaffold stem, the 3' miRNA scaffold stem optionally comprising the nucleotide sequence of SEQ ID NOs: 11-14;

[0227] The positions of the passenger strand and the guide strand can be switched, and wherein 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.

[0228] Embodiment 32. A cell according to any one of embodiments 29 to 31, wherein the expression cassette comprises two or more template sequences for pri-miRNAs, wherein these pri-miRNAs are suitable for processing in the CHO cell to form miRNAs targeting CCL2, wherein the miRNAs processed from these pri-miRNAs bind to different parts of the RNA, particularly mRNA or pre-mRNA, of CCL2.

[0229] Embodiment 33. The cell according to embodiment 32, wherein the different miRNAs targeting CCL2 have a nucleotide sequence selected from the group consisting of SEQ ID NOs: 26 and 28.

[0230] Embodiment 34. A cell according to any one of embodiments 25 to 33, which cell is capable of producing a variant of CD39 having one or more of the following characteristics:

[0231] (i) is a soluble variant lacking the membrane anchor of human CD39;

[0232] (ii) compared with human CD39, it has an N-terminal deletion, a C-terminal deletion, and a central deletion;

[0233] (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;

[0234] (iv) contain up to 5 point mutations compared to human CD39;

[0235] (v) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-49.

[0236] Embodiment 35. A cell according to any one of embodiments 25 to 34, which is capable of producing a variant of CD39 comprising the amino acid sequence of SEQ ID NO: 49.

[0237] Embodiment 36. A 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.

[0238] Example 37. A method for improving the production of CD39 or a variant thereof in CHO cells, the method comprising the following steps:

[0239] (ai) providing CHO cells capable of producing CD39 or a variant thereof;

[0240] (a-ii) engineering the CHO cell to reduce the production of CCL2 in the CHO cell.

[0241] Embodiment 38. The method according to embodiment 37, wherein in step (a-ii) the CHO cell is engineered by knocking down or knocking out CCL2 expression or inducing degradation of CCL2 protein in the CHO cell.

[0242] Embodiment 39. The method according to embodiment 37 or 38, wherein the CHO cell is engineered in step (a-ii) to produce a miRNA targeting endogenous CCL2 of the CHO cell.

[0243] 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 NO: 26 and 28.

[0244] Embodiment 41. The method according to embodiment 39 or 40, wherein in step (a-ii) an expression cassette for producing the miRNA targeting CCL2 is introduced into the CHO cell.

[0245] Embodiment 42. The method according to embodiment 41, wherein the expression cassette comprises an intron sequence, the intron sequence comprises a template sequence of a pri-miRNA, wherein the pri-miRNA is suitable for processing in the CHO cell to form the miRNA targeting CCL2.

[0246] Embodiment 43. The method according to embodiment 42, wherein the pri-miRNA comprises from 5' to 3':

[0247] (i) a 5' miRNA scaffold stem, the 5' miRNA scaffold stem optionally comprising a nucleotide sequence of SEQ ID NO: 1-4,

[0248] (ii) a passenger strand having a nucleotide sequence complementary to the sequence of the miRNA, optionally comprising one or two mismatches,

[0249] (iii) a miRNA scaffold loop, the miRNA scaffold loop optionally comprising a nucleotide sequence of SEQ ID NO: 8,

[0250] (iv) a guide strand having the nucleotide sequence of the miRNA, and (v) a 3' miRNA scaffold stem, the 3' miRNA scaffold stem optionally comprising the nucleotide sequence of SEQ ID NOs: 11-14;

[0251] The positions of the passenger strand and the guide strand can be switched, and wherein 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.

[0252] Embodiment 44. A method according to any one of embodiments 41 to 43, wherein the expression cassette comprises two or more template sequences for pri-miRNAs, wherein these pri-miRNAs are suitable for processing in the CHO cell to form miRNAs targeting CCL2, wherein the miRNAs processed from these pri-miRNAs bind to different parts of the RNA, especially mRNA or pre-mRNA, of CCL2.

[0253] Embodiment 45. The method according to embodiment 44, wherein the different miRNAs targeting CCL2 have a nucleotide sequence selected from the group consisting of SEQ ID NOs: 26 and 28.

[0254] Embodiment 46. The method according to any one of embodiments 41 to 45, wherein step (a-ii) comprises introducing a vector nucleic acid into the CHO cell, wherein the vector nucleic acid comprises an expression cassette for expressing the miRNA targeting CCL2.

[0255] Embodiment 47. The method according to any one of embodiments 37 to 46, wherein engineering the CHO cell reduces the level of CCL2 mRNA in the CHO cell by at least 5-fold, preferably at least 10-fold, more preferably at least 25-fold compared to the same CHO cell before step (a-ii).

[0256] Embodiment 48. The method according to any one of embodiments 37 to 47, wherein engineering the CHO cell reduces the level of CCL2 protein in the CHO cell by at least 5-fold, preferably at least 10-fold, more preferably at least 25-fold compared to the same CHO cell before step (a-ii).

[0257] Embodiment 49. The method according to any one of embodiments 37 to 48, wherein the method further comprises the following steps:

[0258] (b) culturing the CHO cells obtained in step (a-ii) in a cell culture under conditions that allow the CHO cells to proliferate and simultaneously and / or subsequently produce CD39 or a variant thereof;

[0259] (c) obtaining the CD39 or its variant from the cell culture; and

[0260] (d) optionally processing CD39 or a variant thereof.

[0261] Embodiment 50. The method according to embodiment 49, wherein step (c) comprises performing anion exchange chromatography and / or hydrophobic interaction chromatography.

[0262] Embodiment 51. The method according to embodiment 49 or 50, wherein step (c) comprises

[0263] (c1) isolating the CHO cells from a cell culture supernatant containing CD39 or its variants;

[0264] (c2) isolating CD39 or its variant from the cell culture supernatant using anion exchange chromatography;

[0265] (c3) further purifying CD39 or its variant using hydrophobic interaction chromatography; and

[0266] (c4) further purifying CD39 or its variant using anion exchange chromatography.

[0267] Embodiment 52. The method of any one of embodiments 49 to 51, wherein step (d) comprises providing a pharmaceutical formulation comprising CD39 or a variant thereof.

[0268] Embodiment 53. A method according to any one of embodiments 49 to 52, wherein the CD39 or variant thereof obtained after step (c) is present in a composition comprising CCL2 at a concentration of 100 ppm or less, preferably 25 ppm or less, more preferably 10 ppm or less.

[0269] Embodiment 54. A method according to any one of embodiments 51 to 53, wherein the CD39 or variant thereof obtained after step (c1) is present in a composition comprising CCL2 at a concentration of 100 ppm or less, preferably 25 ppm or less, more preferably 10 ppm or less.

[0270] Embodiment 55. A method according to any one of embodiments 51 to 54, wherein the CD39 or variant thereof obtained after step (c4) is present in a composition comprising CCL2 at a concentration of 25 ppm or less, preferably 10 ppm or less, more preferably 5 ppm or less.

[0271] Embodiment 56. A method according to any one of embodiments 49 to 55, wherein the CD39 or variant thereof obtained after step (d) is present in a composition comprising CCL2 at a concentration of 25 ppm or less, preferably 10 ppm or less, more preferably 5 ppm or less.

[0272] Embodiment 57. A method according to any one of embodiments 49 to 56, wherein the CD39 or variant thereof obtained after step (c) is present in a composition comprising a concentration of CCL2 that is at least 10 times lower, preferably 25 times lower, more preferably 100 times lower, compared to a composition obtained by the same method after step (c) using CHO cells that have not been engineered to reduce CCL2 production in CHO cells.

[0273] Embodiment 58. A method according to any one of embodiments 51 to 57, wherein the CD39 or variant thereof obtained after step (c1) is present in a composition comprising a concentration of CCL2 that is at least 10 times lower, preferably 25 times lower, more preferably 100 times lower, compared to a composition obtained by the same method after step (c1) using CHO cells that have not been engineered to reduce CCL2 production in CHO cells.

[0274] Embodiment 59. A method according to any one of embodiments 51 to 58, wherein the CD39 or variant thereof obtained after step (c4) is present in a composition comprising a concentration of CCL2 that is at least 10 times lower, preferably 25 times lower, more preferably 100 times lower, compared to a composition obtained by the same method after step (c4) using CHO cells that have not been engineered to reduce CCL2 production in CHO cells.

[0275] Embodiment 60. A method according to any one of embodiments 49 to 59, wherein the CD39 or variant thereof obtained after step (d) is present in a composition comprising a concentration of CCL2 at least 10 times lower, preferably 25 times lower, more preferably 100 times lower, compared to a composition obtained by the same method after step (d) using CHO cells that have not been engineered to reduce CCL2 production in CHO cells.

[0276] Embodiment 61. The method according to any one of embodiments 37 to 60, wherein the method is used to improve the production of a variant of CD39 having one or more of the following characteristics:

[0277] (i) is a soluble variant lacking the membrane anchor of human CD39;

[0278] (ii) compared with human CD39, it has an N-terminal deletion, a C-terminal deletion, and a central deletion;

[0279] (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;

[0280] (iv) contain up to 5 point mutations compared to human CD39;

[0281] (v) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-49.

[0282] Embodiment 62. A method according to any one of embodiments 37 to 61, wherein the method is used to improve the production of a variant of CD39 comprising the amino acid sequence of SEQ ID NO: 49.

[0283] Embodiment 63. A method according to any one of embodiments 37 to 62, wherein the method is used to improve the production of a variant of CD39 consisting of the amino acid sequence of SEQ ID NO: 49.

[0284] Embodiment 64. An expression cassette for expressing a miRNA in a CHO cell, the expression cassette comprising a template sequence of a pri-miRNA, wherein the pri-miRNA is suitable for processing in a CHO cell to form a miRNA targeting CCL2.

[0285] Embodiment 65. The expression cassette of embodiment 64, wherein the miRNA targeting CCL2 has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 26 and 28.

[0286] Embodiment 66. An expression cassette according to embodiment 64 or 65, wherein the template sequence of the pri-miRNA is present within an intron sequence.

[0287] Embodiment 67. The expression cassette of any one of embodiments 64 to 66, wherein the pri-miRNA comprises from 5' to 3':

[0288] (i) a 5' miRNA scaffold stem, the 5' miRNA scaffold stem optionally comprising a nucleotide sequence of SEQ ID NO: 1-4,

[0289] (ii) a passenger strand having a nucleotide sequence complementary to the sequence of the miRNA, optionally comprising one or two mismatches,

[0290] (iii) a miRNA scaffold loop, the miRNA scaffold loop optionally comprising the nucleotide sequence of SEQ ID NO: 8,

[0291] (iv) a guide strand having the nucleotide sequence of the miRNA, and (v) a 3' miRNA scaffold stem, the 3' miRNA scaffold stem optionally comprising the nucleotide sequence of SEQ ID NOs: 11-14;

[0292] The positions of the passenger strand and the guide strand can be switched, and wherein 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.

[0293] Embodiment 68. An expression cassette according to any one of embodiments 64 to 67, comprising two or more template sequences for pri-miRNAs, wherein these pri-miRNAs are suitable for processing in CHO cells to form miRNAs targeting CCL2, wherein the miRNAs processed from these pri-miRNAs bind to different parts of the RNA of CCL2, especially mRNA or pre-mRNA.

[0294] Embodiment 69. The expression cassette of embodiment 68, wherein the different miRNAs targeting CCL2 have a nucleotide sequence selected from the group consisting of SEQ ID NOs: 26 and 28.

[0295] Embodiment 70. A vector nucleic acid for transfecting CHO cells, the vector nucleic acid comprising the expression cassette according to any one of embodiments 64 to 69.

[0296] Embodiment 71. According to embodiment 70, the vector nucleic acid further comprises a coding sequence encoding CD39 or a variant thereof.

[0297] Embodiment 72. The vector nucleic acid according to embodiment 71, wherein the coding sequence encoding CD39 or its variant is present in the same expression cassette as the template sequence of pri-miRNA.

[0298] Embodiment 73. 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, and the additional expression cassette is present in the vector nucleic acid.

[0299] Embodiment 74. The vector nucleic acid of any one of embodiments 71 to 73, wherein the coding sequence encodes a variant of CD39 having one or more of the following characteristics:

[0300] (i) is a soluble variant lacking the membrane anchor of human CD39;

[0301] (ii) compared with human CD39, it has an N-terminal deletion, a C-terminal deletion, and a central deletion;

[0302] (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;

[0303] (iv) contain up to 5 point mutations compared to human CD39;

[0304] (v) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-49.

[0305] Embodiment 75. The vector nucleic acid of any one of embodiments 71 to 74, wherein the coding sequence encodes a variant of CD39 comprising the amino acid sequence of SEQ ID NO:49.

[0306] Embodiment 76. The vector nucleic acid of 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.

[0307] Embodiment 77. A method for producing CHO cells capable of expressing CD39 or a variant thereof, the method comprising introducing the vector nucleic acid according to any one of embodiments 70 to 76 into CHO cells.

[0308] Embodiment 78. A method according to embodiment 77; wherein the CHO cell does not contain a coding sequence encoding CD39 or a variant thereof prior to introduction of the vector nucleic acid; and wherein the vector nucleic acid according to any one of embodiments 71 to 76 is introduced into the CHO cell.

[0309] Embodiment 79. A method according to embodiment 77; wherein the vector nucleic acid according to embodiment 70 is introduced into the CHO cells, the vector nucleic acid does not contain a coding sequence encoding CD39 or a variant thereof; and wherein the CHO cells contain a coding sequence encoding CD39 or a variant thereof before the introduction of the vector nucleic acid.

[0310] Embodiment 80. A method according to embodiment 77; wherein the vector nucleic acid according to embodiment 70 is introduced into the CHO cells, and the vector nucleic acid does not contain a coding sequence encoding CD39 or a variant thereof; and wherein an additional vector nucleic acid is introduced into the CHO cells, and the additional vector nucleic acid contains a coding sequence encoding CD39 or a variant thereof; wherein the two different vector nucleic acids are introduced simultaneously or subsequently in any order.

[0311] Embodiment 81. The method according to any one of embodiments 77 to 80, wherein the CHO cell is capable of expressing a variant of CD39 having one or more of the following characteristics:

[0312] (i) is a soluble variant lacking the membrane anchor of human CD39;

[0313] (ii) compared with human CD39, it has an N-terminal deletion, a C-terminal deletion, and a central deletion;

[0314] (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;

[0315] (iv) contain up to 5 point mutations compared to human CD39;

[0316] (v) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-49.

[0317] Embodiment 82. A method according to any one of embodiments 77 to 81, wherein the CHO cell is capable of expressing a variant of CD39 comprising the amino acid sequence of SEQ ID NO:49.

[0318] Embodiment 83. A method according to any one of embodiments 77 to 82, wherein the CHO cell is capable of expressing a variant of CD39 consisting of the amino acid sequence of SEQ ID NO:49.

[0319] Embodiment 84. A composition comprising CD39 or a variant thereof and CCL2, wherein the composition is obtained by production using the CHO cells according to any one of embodiments 25 to 36, and wherein the amount of CCL2 in the composition is at least 10 times lower, preferably 25 times lower, more preferably 100 times lower, compared to the same composition obtained by production using CHO cells that have not been engineered to reduce CCL2 production in CHO cells.

[0320] Embodiment 85. A composition comprising CD39 or a variant thereof and CCL2, wherein the composition is obtained by production using CHO cells, and wherein the composition comprises CCL2 at a concentration of 100 ppm or less, preferably 25 ppm or less, more preferably 10 ppm or less.

[0321] Embodiment 86. A composition according to embodiment 84, which is a composition according to embodiment 85.

[0322] Embodiment 87. The composition according to any one of embodiments 84 to 86, obtained by steps (a), (b) and (c1) of the method described in embodiment 12.

[0323] Embodiment 88. The composition of embodiment 87, which is a cell culture supernatant or a cell-free bulk harvest of a cell culture.

[0324] Embodiment 89. The composition of embodiment 87 or 88, wherein the composition comprises CCL2 at a concentration of 100 ppm or less, preferably 25 ppm or less, more preferably 10 ppm or less.

[0325] Embodiment 90. The 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.

[0326] 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 described in embodiment 12.

[0327] Embodiment 92. The composition of embodiment 90 or 91, which is a purified composition substantially free of proteins other than CD39 or a variant thereof.

[0328] Embodiment 93. The composition of any one of embodiments 84 to 86, obtained by steps (a), (b), (c) and (d) of the method of any one of embodiments 1 to 24.

[0329] Embodiment 94. A composition according to any one of embodiments 90 to 93, which is a purified composition substantially free of proteins other than CD39 or a variant thereof.

[0330] Embodiment 95. A composition according to any one of Embodiments 90 to 94, wherein the composition comprises 5% or less, preferably 2% or less, more preferably 1% or less of proteins other than CD39 or its variants relative to the total amount of protein in the composition.

[0331] Embodiment 96. The composition of any one of embodiments 90 to 95, wherein the composition comprises CCL2 at a concentration of 25 ppm or less, preferably 10 ppm or less, more preferably 5 ppm or less.

[0332] Embodiment 97. A CHO cell engineered to reduce the production of CCL2 in the CHO cell.

[0333] Embodiment 98. The cell according to embodiment 97, wherein the production of CCL2 is reduced by knocking down or knocking out CCL2 expression or degrading CCL2 protein.

[0334] Embodiment 99. The cell of embodiment 97 or 98, which is engineered to produce a miRNA targeting endogenous CCL2 of the CHO cells.

[0335] Embodiment 100. The cell according to embodiment 99, wherein the miRNA targeting CCL2 has a nucleotide sequence selected from the group consisting of SEQ ID NO: 26 and 28.

[0336] Embodiment 101. The cell according to embodiment 99 or 100, comprising an expression cassette for producing the miRNA targeting CCL2.

[0337] Embodiment 102. The cell according to embodiment 101, wherein the expression cassette comprises an intron sequence comprising a template sequence for a pri-miRNA, wherein the pri-miRNA is suitable for processing in the CHO cell to form the miRNA targeting CCL2.

[0338] Embodiment 103. The cell according to embodiment 102, wherein the pri-miRNA comprises from 5' to 3':

[0339] (i) a 5' miRNA scaffold stem, the 5' miRNA scaffold stem optionally comprising a nucleotide sequence of SEQ ID NO: 1-4,

[0340] (ii) a passenger strand having a nucleotide sequence complementary to the sequence of the miRNA, optionally comprising one or two mismatches,

[0341] (iii) a miRNA scaffold loop, the miRNA scaffold loop optionally comprising a nucleotide sequence of SEQ ID NO: 8,

[0342] (iv) a guide strand having the nucleotide sequence of the miRNA, and (v) a 3' miRNA scaffold stem, the 3' miRNA scaffold stem optionally comprising the nucleotide sequence of SEQ ID NOs: 11-14;

[0343] The positions of the passenger strand and the guide strand can be switched, and wherein 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.

[0344] Embodiment 104. A cell according to any one of embodiments 101 to 103, wherein the expression cassette comprises two or more template sequences for pri-miRNAs, wherein these pri-miRNAs are suitable for processing in the CHO cell to form miRNAs targeting CCL2, wherein the miRNAs processed from these pri-miRNAs bind to different parts of the RNA, particularly mRNA or pre-mRNA, of CCL2.

[0345] Embodiment 105. The cell of Embodiment 104, wherein the different miRNAs targeting CCL2 have a nucleotide sequence selected from the group consisting of SEQ ID NOs: 26 and 28. BRIEF DESCRIPTION OF THE DRAWINGS

[0346] Figure 1 The production and purification process of CD39* is shown.

[0347] Figure 2 The titers of CD39* and CCL2 in the cell culture supernatant during the culture of host cells and the production of CD39* are shown. CCL2 levels were detected by CCL2 ELISA.

[0348] Figure 3 Elution profiles of CD39* and CCL2 during the anion exchange capture chromatography step are shown. CCL2 levels were detected by CCL2 ELISA.

[0349] Figure 4 Cell line engineering and development strategies for the CCL2 knockdown approach are shown.

[0350] Figure 5 Cell viability (dashed line) and viable cell density of different host cells during optimized fed-batch are shown. CCL2 knockdown pools (CCL2_A and CCL2_B) showed similar viable cell density compared to controls (CD39* cell line, control miRNA and empty CHO cell line).

[0351] Figure 6 CD39* titers in different host cells are shown. Titers of the CCL2 KD pool were comparable to controls (optimized fed-batch).

[0352] Figure 7 Relative mRNA expression of CD39*, CCL2 and control proteins is shown. RNA was sampled and purified on day 10 of the optimized fed-batch. CD39*, CCL2 and control gene transcripts were quantified using qPCR. Both CCL2 KD pools showed significantly lower CCL2 transcript levels compared to the control.

[0353] Figure 8 CCL2 protein levels quantified using CCL2 ELISA are shown. The CCL2 KD pool shows low CCL2 protein levels in the non-purified harvest at day 10 of the optimized fed-batch run.

[0354] Fig. 9CD39* titers of CCL2 KD pools and single cell clones are shown. The CCL2_A encoding pool was used for single cell cloning. 96 clones were inoculated into a 24dwp standard fed batch and CD39* titers were assessed. As expected, some clones showed higher titers compared to the original pool and CD39* cell line control, while others had lower titers.

[0355] Fig.10 Shown are CCL2 mRNA levels of CCL2 KD single cell clones. The CCL2_A coding pool was used for single cell cloning. 96 clones were inoculated into 24dwp standard feed batches and qPCR was used to quantify CCL2 mRNA levels. Most clones showed significantly reduced CCL2 transcript levels compared to CD39* cell line controls (normalized to 1).

[0356] Fig.11 Shown are the characteristics of the top 3 CCL2 KD clones compared to the CD39* cell line. A: CD39* titers derived from the 7L bioreactor were significantly higher in the CCL2 KD clones. B: CCL2 mRNA levels were greatly reduced on day 10 of the standard fed batch run in the 7L bioreactor compared to the CD39* cell line (normalized to 1). Clone B showed a more than 100-fold reduction in IGP transcripts. C: CCL2 protein levels in non-purified harvests from the 7L bioreactor run (measured by CCL2 ELISA) showed that the CCL2 levels of the top 3 clones were below LOQ (<2ppm) compared to the CD39* cell line. 2ppm corresponds to 2ng CCL2 / mg CD39.

[0357] Examples

[0358] Example 1: Identification of CCL2 as a key impurity in a human CD39 variant produced recombinantly in CHO cells.

[0359] A human CD39 variant having the amino acid sequence of SEQ ID NO: 49 (hereinafter referred to as "CD39*") was produced by a CHO production cell line ("CD39* cell line") and isolated from the cell culture using a standard purification process. The purification process for the CD39* drug product follows a 3-chromatographic step process including anion exchange, hydrophobic interaction and multimodal anion exchange chromatography, low pH viral inactivation, virus removal filtration, and two ultrafiltration / diafiltration steps ( Figure 1 ).

[0360] Application of a purification process targeting CD39* demonstrated depletion of host cell proteins (HCPs) using ELISA assays specific for multiple HCPs. Analysis of individual HCPs using an orthogonal LC-MS approach revealed one prominent protein: CCL2 (CC-chemokine ligand 2; also known as MCP-1 (monocyte chemoattractant protein 1)), which was not detected by ELISA.

[0361] Table 1: Amount of CCL2 in the purified product

[0362] CCL2[ppm] Batch 1 366.9 Batch 2 368.5 Batch 3 410.8

[0363] It was also observed that CCL2 was not removed during the purification process, as the concentration of CCL2 in the product remained almost constant after each purification step.

[0364] Further analysis of the upstream processes (USP) and downstream processes (DSP) of CD39* production showed that CCL2 formation in cell culture increased with increasing culture time ( Figure 2 ), and the elution curve of CCL2 significantly overlapped with that of CD39* in the AEC capture step ( Figure 3 ).

[0365] Example 2: miRNA vector design.

[0366] The intron-miRNA encoding vectors (pCMV04_A and pCMV04_B) are based on a standard vector (pCMV). The vectors were modified by inserting the intron miRNA sequence into the cloning site of the expression cassette driven by the CMV promoter. 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 be optimally processed for the resulting miRNA sequence. However, the sequence was further modified: i) the EcoRI restriction site was replaced by a BglII restriction site, ii) the sequence downstream of the XhoI restriction site was replaced by a CHO-derived sequence, and iii) additional miR-30A scaffolds were added upstream and downstream of the published sequence. Two different miRNA sequences targeting endogenous CCL2 of CHO cells were tested: CCL2_A AIM (artificial intron miRNA), which comprises a guide strand having a nucleotide sequence of SEQ ID NO: 26 and a passenger strand having a nucleotide sequence of SEQ ID NO: 25, and CCL2_B AIM, which comprises a guide strand having a nucleotide sequence of SEQ ID NO: 28 and a passenger strand having a nucleotide sequence of SEQ ID NO: 27. In addition, a vector with AIM (targeting an unrelated endogenous protein of CHO cells) was used as a control.

[0367] Example 3: Generation of CCL2 knockdown cell lines.

[0368] The strategy for generating CCL2 KD cell lines is as follows Figure 4 As shown. The CHO parental cell line was transfected with the vector pCMV03 encoding a human CD39 variant (hereinafter referred to as "CD39*") and pooled using MTX in a low folic acid medium. The pool was single-cell cloned and a monoclonal cell line expressing CD39* was selected, referred to as the CD39* cell line. Subsequently, the primary seed batch (PSL) of the CD39* cell line was transfected with a vector encoding an artificial intronic miRNA targeting endogenous CCL2, and a pool was generated using puromycin.

[0369] Two different miRNAs targeting CHO CCL2 mRNA were generated, called CCL2_A and CCL2_B, both targeting the 3'UTR of the transcript. For pool generation, miRNAs targeting different genes (control AIM), parental CD39* cell line as well as empty parental cell line (CHO) were included as controls. All samples (triplicate pool generation of knockdown approach) were inoculated into optimized fed-batch runs and cell growth, gene expression and CD39* titer were assessed on different days ( Figures 5 to 7In addition, CCL2 protein levels at harvest level were assessed using CCL2 ELISA ( Figure 8 ):

[0370] After pool generation and confirmation of effective CCL2 knockdown at the mRNA level, the CCL2_A pool was selected for single-cell cloning and 96 expanded clones were inoculated into a 24 dwp standard fed-batch to assess CCL2 knockdown efficiency and CD39* productivity ( Fig. 9 and Fig.10 ).

[0371] The top 30 clones were further characterized. Based on a number of parameters (USP, DSP, CCL2 data, CD39* protein signature), the top 3 clones were selected and inoculated into a 7L bioreactor. CCL2 expression was significantly reduced in the CCL2 knockdown clones, resulting in increased CD39* titers ( Fig.11 ).

[0372] Table 2: Characteristics of selected CCL2 AIM clones

[0373]

[0374] *Normalized to CD39* cell lines

[0375] After harvesting the culture supernatant containing CD39* protein, the concentration of CCL2 in the cell-free supernatant was measured.

[0376] Table 3: Amount of CCL2 in cell-free supernatant

[0377] CCL2[ppm] Batch 1 4.4 Batch 2 <4 Batch 3 <8

[0378] Subsequent application of downstream processes 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):

[0379] Table 4: Amount of CCL2 in the purified drug substance

[0380] CCL2[ppm] Batch 1 <4 Batch 2 <4 Batch 3 <4

[0381] Example 4: Generation of CCL2 knockout cell lines.

[0382] Gene editing technology represents an alternative to the vectorized RNAi method using artificial intronic miRNA. Gene knockout at the DNA level encoding the CCL2 gene results in complete functional inactivation due to the loss of essential gene information. We generated a single gRNA pair for targeting 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 insertions and deletions (indels) formation, and ultimately leading to frameshift mutations in the CCL2 gene. The mRNA encoding Cas-Clover was transfected into MaKo cells seeded in 24-well plates one day before transfection. After 6h, the sgRNA targeting the CCL2 gene was co-transfected into cells expressing Cas-Clover. One day after transfection, single cell sorting was performed on the living cell population using fluorescence activated cell sorting (FACS). After a 16-day expansion period in a 96-well plate, the genomic DNA of the cloned cells was isolated for NGS genotyping. On the MiSeq of Illumina, the PCR amplicon produced by the primers flanking the desired gRNA editing site was sequenced as the Nextera XT library. Sequencing reads were compared with the CCL2 gene sequence to identify insertions and deletions. Clone 048, which was transfected with sgRNA to guide_SA03 and guide_SA04, revealed two deletions (40bp and 15bp-13bp) without detecting wild-type sequences, which confirmed the knockout of the homozygous CCL2 gene in the CHO MaKo cell line.

[0383] Example 5: Materials and methods.

[0384] The following materials and methods were used in Examples 1 to 4.

[0385] 1. Expression vector construction

[0386] The vector used in the example is composed of the following elements: hCMV promoter / enhancer driving the expression of each gene, polyadenylation signal (polyA), folate receptor, DHFR, puromycin and hygromycin resistance genes as selection markers, E. coli replication origin (CoIE ori) and β-lactamase gene providing ampicillin (amp) resistance to start amplification in bacteria. Different plasmid settings were evaluated and more details are provided in the figure.

[0387] 2. Cell lines, culture, transfection and selection

[0388] CHO cells are suspended in a patented chemically defined culture medium in a 24-well plate or shake flask at 300 rpm (24 dwp) or 150 rpm (shake flask), 10% CO2, 36.5°C non-humidified cabinet shaker. Cell viability and growth rate are monitored by an automated system (ViCell, Beckman Coulter) or using analytical flow cytometry (CytoFlex, Beckman Coulter). Cells are passaged 2-3 times per week to fresh culture medium and maintained in the logarithmic growth phase.

[0389] The linearized expression vector was transfected by electroporation (Amaxa Nucleofection System, Lonza, Germany). The transfection reaction was performed in chemically defined medium according to the manufacturer's instructions. The parental CHO cells used for transfection were in exponential growth phase with a cell viability greater than 95%. 5 x 10 6 The cells were transfected. Immediately after transfection, the cells were transferred to a shake flask containing chemically defined medium. Before starting the selection process, the cell pool was incubated at 36.5°C and 10% CO2 for 48 hours.

[0390] As described above, the selection procedure is performed using the selection markers encoded by each expression vector. Both proteins FoIR and DHFR are involved in the same molecular pathway; FolR transports folic acid and the folic acid analog MTX into cells, and DHFR converts it into an important precursor for purine and methionine synthesis. Combining them as a selection principle, a specific strong selection scheme can be used to enrich recombinant cells expressing two recombinant proteins. Puromycin selection is driven by its inhibition of protein synthesis, and the vector encoding the puromycin resistance marker gene enables cells to survive in the presence of puromycin.

[0391] 48h after transfection and growth under low folic acid conditions, additional selection pressure was applied by adding 10nM MTX to a chemically defined medium. Alternatively, puromycin was used as a selection agent. After 48h of transfection, 0.003mg / mL puromycin was added to a chemically defined medium. After the pool was recovered, the cells were frozen in a medium supplemented with 7.5% DMSO, and cell pellets were prepared.

[0392] 3. Gene Expression Analysis by Quantitative Real-time PCR

[0393] RNA extraction was performed using the Qiagen RNeasy micro 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 premix (Applied Biosystems) and 10x diluted cDNA was analyzed in triplicate using the QuantiFast SYBR Green PCR kit (Qiagen) or the TaqMan Primer / Probe system and TaqMan master mix (Applied Biosystems). GAPDH was amplified as an endogenous control for normalization. The ABI Amplification and analysis were performed using the 7900HT Sequence Detection System. To calculate the relative quantity (RQ) of gene expression for sample comparison, a comparative 2 -ΔΔCt method, and the data were normalized.

[0394] 4. Upstream processing

[0395] After selection, material was produced in shake flask fed batch, 24 deep well plate culture or ambr15 bioreactor. Fed batch culture was inoculated with a cell inoculation density of 4E5 vc / ml (a proprietary feed solution was added from day 3, and the culture temperature was changed to 33°C on day 5). In-process controls were performed during the cultivation to monitor the concentration of CD39*. Cell culture samples for RNA isolation were collected on day 10 of the process. Individual cultures were cultivated for a period of 14 days. At the end of the cultivation process, cells were separated from the culture supernatant by centrifugation and / or deep filtration, followed by sterile filtration and further downstream processing.

[0396] 5. CCL2 Protein Analysis by ELISA

[0397] The amount of Chinese hamster (CHO) host cell protein "CC-chemokine ligand 2" (CCL2) is determined using a sandwich ELISA. The sample is added to a microtiter plate coated with an anti-CCL2 antibody (capture antibody). The bound CCL2 is then quantified by incubation with a biotinylated anti-CCL2 antibody (detection antibody), followed by streptavidin-peroxidase and tetramethylbenzidine (TMB) as substrates, and measuring the absorbance at 450 nm. The level of CCL2 in the sample is calculated based on a rat or CHO CCL2 standard, preferably a CHO CCL2 standard.

[0398] 6. CCL2 Assay by LC-MS

[0399] The amount of Chinese hamster (CHO) host cell protein "CC-chemokine ligand 2" (CCL2) was determined using LC-MS absolute quantification. CCL2 heavy labeled peptides derived from the CCL2 sequence were spiked into a tryptic digest of the drug substance. Endogenous CCL2 protein was then quantified by comparing the LC-MS intensity of the endogenous CCL2 non-labeled peptide with the LC-MS intensity of the spiked CCL2 heavy labeled peptide.

[0400] The method demonstrated linear detection of CCL2 heavy labeled peptides in the range between 33 ng / mg DS and 8100 ng / mg DS (R2>0.99). CCL2 quantification in CD39* was not affected by the concentration of heavy labeled peptides used in the experiment (99% agreement), a conclusion confirmed using alternative data analysis (peak area compared to MS intensity). The method demonstrated an accuracy of 9% CV. In summary, this method is considered suitable for quantification of CCL2 in CD39* drug substances.

[0401] Sequence Listing

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Claims

1. A method for producing CD39 or a variant thereof, the method comprising the following steps: (a) providing a CHO cell capable of producing CD39 or a variant thereof; (b) culturing the CHO cells in cell culture under conditions that allow production of CD39 or a variant thereof; (c) obtaining CD39 or a variant thereof from the cell culture; and (d) optionally processing CD39 or a variant thereof; Wherein the CHO cells are engineered to reduce the production of CCL2 in the CHO cells. 2 . The method of claim 1 , wherein the production of CCL2 in the CHO cells is reduced by knocking down or knocking out CCL2 expression or degrading CCL2 protein.

3. The method according to claim 1 or 2, wherein the CHO cells are engineered to produce a miRNA targeting endogenous CCL2 of the CHO cells, wherein the miRNA targeting CCL2 preferably has 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 cell comprises an expression cassette for producing the miRNA targeting CCL2, wherein the expression cassette comprises an intron sequence, the intron sequence comprises a template sequence for the pri-miRNA, wherein the pri-miRNA is suitable for processing in the CHO cell to form the miRNA targeting CCL2.

5. The method according to claim 4, wherein the pri-miRNA comprises from 5' to 3': (i) a 5' miRNA scaffold stem, the 5' miRNA scaffold stem optionally comprising a nucleotide sequence of SEQ ID NO: 1-4, (ii) a passenger strand having a nucleotide sequence complementary to the sequence of the miRNA, optionally comprising one or two mismatches, (iii) a miRNA scaffold loop, the miRNA scaffold loop optionally comprising the nucleotide sequence of SEQ ID NO: 8, (iv) a guide strand having a nucleotide sequence of the miRNA, and (v) a 3' miRNA scaffold stem, the 3' miRNA scaffold stem optionally comprising a nucleotide sequence of SEQ ID NOs: 11-14; The positions of the passenger strand and the guide strand can be switched, and wherein 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.

6. The method according to any one of claims 1 to 5, wherein the method is used to generate a variant of CD39 having one or more of the following characteristics: (i) is a soluble variant lacking the membrane anchor of human CD39; (ii) compared with human CD39, it has an N-terminal deletion, a C-terminal deletion, and a central deletion; (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) contain up to 5 point mutations compared to human CD39; (v) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-49.

7. The method according to any one of claims 1 to 6, wherein the CD39 or variant thereof obtained after step (c) is present in a composition comprising CCL2 at a concentration of 100 ppm or less, preferably 25 ppm or less, more preferably 10 ppm or less, compared to a composition obtained with the same method after step (c) using CHO cells that have not been engineered to reduce CCL2 production in CHO cells, and / or the composition comprises CCL2 at a concentration at least 10-fold lower, preferably 25-fold lower, more preferably 100-fold lower, and / or wherein the CD39 or variant thereof obtained after step (d) is present in a composition comprising CCL2 at a concentration of 25 ppm or less, preferably 10 ppm or less, more preferably 5 ppm or less, compared to a composition obtained with the same method after step (d) using CHO cells that have not been engineered to reduce CCL2 production in CHO cells, and / or the composition comprises CCL2 at a concentration at least 10-fold lower, preferably 25-fold lower, more preferably 100-fold lower.

8. The method according to any one of claims 1 to 7, wherein step (c) comprises: (c1) separating the CHO cells from the cell culture supernatant containing CD39 or its variants; (c2) isolating CD39 or its variant from the cell culture supernatant using anion exchange chromatography; (c3) further purifying CD39 or its variant using hydrophobic interaction chromatography; and (c4) further purifying CD39 or its variant using anion exchange chromatography.

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

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

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

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

13. A method for improving the production of CD39 or a variant thereof in CHO cells, the method comprising the steps of: (ai) providing CHO cells capable of producing CD39 or a variant thereof; (a-ii) engineering the CHO cells to reduce the production of CCL2 in the CHO cells.

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

15. The method according to claim 13 or 14, wherein step (a-ii) comprises introducing a vector nucleic acid into the CHO cells, wherein the vector nucleic acid comprises an expression cassette for expressing a miRNA targeting CCL2.

16. The method according to any one of claims 13 to 15, wherein engineering the CHO cell reduces the CCL2 mRNA level and / or CCL2 protein level in the CHO cell by at least 5-fold, preferably at least 10-fold, more preferably at least 25-fold, compared to the same CHO cell before step (a-ii).

17. The method according to any one of claims 13 to 16, wherein the method further comprises the following steps: (b) culturing the CHO cells obtained in step (a-ii) in a cell culture under conditions that allow the CHO cells to proliferate and simultaneously and / or subsequently produce CD39 or a variant thereof; (c) obtaining the CD39 or variant thereof from the cell culture; and (d) optionally processing CD39 or a variant thereof.

18. The method of claim 17, wherein step (c) comprises: (c1) separating the CHO cells from the cell culture supernatant containing CD39 or its variants; (c2) isolating CD39 or its variant from the cell culture supernatant using anion exchange chromatography; (c3) further purifying CD39 or its variant using hydrophobic interaction chromatography; and (c4) further purifying CD39 or its variant using anion exchange chromatography; and / or Wherein step (d) comprises providing a pharmaceutical formulation comprising CD39 or a variant thereof.

19. An expression cassette for expressing miRNA in CHO cells, the expression cassette comprising a template sequence of a pri-miRNA, wherein the pri-miRNA is suitable for being processed in CHO cells to form a miRNA targeting CCL2.

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

21. A vector nucleic acid for transfecting CHO cells, the vector nucleic acid comprising the expression cassette according to claim 19 or 20.

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

23. A composition comprising CD39 or a variant thereof and CCL2, wherein the composition is obtained by using the CHO cell according to claim 11 or 12, and wherein (i) the amount of CCL2 in the composition is at least 10-fold lower than the same composition obtained by production using CHO cells that have not been engineered to reduce CCL2 production in CHO cells; and / or (ii) The composition comprises CCL2 at a concentration of 100 ppm or less, preferably 25 ppm or less, more preferably 10 ppm or less.

24. A CHO cell engineered to reduce the production of CCL2 in the CHO cell, in particular as defined in any one of claims 2 to 7.

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