WO2025169047A1

WO2025169047A1 - Improved production of angptl3 mimetics

Info

Publication number: WO2025169047A1
Application number: PCT/IB2025/051119
Authority: WO
Inventors: Mathias AEHLE; David Auslaender; Katrin HARDANK; Nicolas LEBESGUE; Petr Obrdlik; Dominik Georg SAUER; Joël Aloïs René TAPPAREL; Nina WAGNER
Original assignee: Novartis AG
Current assignee: Novartis AG
Priority date: 2024-02-05
Filing date: 2025-02-03
Publication date: 2025-08-14
Anticipated expiration: 2026-08-05

Abstract

The present invention pertains to the use of gene editing and miRNA technologies for improving recombinant production of ANGPTL3 mimetics in CHO cells. The gene expression modifications are used for knock-out/knock-down of the endogenous protein CCL2 of the CHO cells which is difficult to separate from ANGPTL3 mimetics during purification.

Description

IMPROVED PRODUCTION OF ANGPTL3 MIMETICS

FIELD OF THE INVENTION

The present invention pertains to the field of recombinant protein production. The present invention provides methods for producing ANGPTL3 mimetics wherein the level of a host cell protein is reduced which interferes with purification of ANGPTL3 mimetics. The production of the interfering host cell protein is reduced using artificial miRNAs targeting the host cell protein and gene knock out technologies.

BACKGROUND OF THE INVENTION

The generation of recombinant cell lines for production of secreted proteins requires the transfection of a DNA vector into host cells and uses selection markers to enrich stable transfectants. The secreted proteins may affect cell parameters, such as growth, viability and/or productivity, which often requires cell line engineering methods to achieve high- expressing stable cell lines.

Similarly, the quality of the secreted recombinant protein can be affected by intrinsic cell- derived factors. Often, an endogenously expressed protein, such as a cell surface receptor, an enzyme or a protease (host cell proteins, HOP), can be identified as the root cause of the undesired effect impacting the quality of the recombinant protein (see, e.g., WO 2014/097113 A2). In addition, host cell proteins are well known process-related impurities of biologies. When present in drug products, these impurities can cause adverse effects due to increased drug immunogenicity, inflammation and other activities associated with the specific residual host cell protein function (Vanderlaan et al., 2018, Biotechnology Progress 34, 828-837). The composition and abundance of HCPs present in various steps of manufacturing processes and in the final drug substance depend on many factors. These factors can be quite difficult to predict a priori and are often learned only by testing during process development.

Respective undesired effects include enzymatic cleavage of the polypeptide chain of the recombinant protein, such as digestion of the entire protein or amino acid clipping, i.e. removal of one or several amino acids from the N or C terminal end of the protein of interest. Other effects are unwanted post-translational modifications - or removal of desired modifications. Furthermore, endogenous gene products of the host cell may specifically interact with the protein of interest. Such an interaction may recruit the protein of interest from the supernatant of the cell culture, render it difficult to remove the endogenous protein during purification, or even lead to activation of signaling pathways in the host cells which result in reduced growth, viability and/or productivity of the cells. In other cases, the endogenous gene product might simply have chemical and physical properties which are highly similar to the protein of interest which makes it hard to develop a purification process which efficiently removes the host cell protein without diminishing the yield of the protein of interest. This is especially relevant for therapeutic proteins where a high purity and low residual levels of host cell proteins in the final product are a prerequisite for obtaining and maintaining marketing authorization. Thus, endogenous gene products of the host cell may significantly interfere with the recombinant production of a protein of interest. This is particularly relevant for production of therapeutic proteins used to treat osteoarthritis, because CCL2 is believed to negatively impact the pharmaceutical effect of a given drug by promoting inflammations in osteoarthritis (OA) patients. Raghu et al. have reported that the CCL2/ CCR2 signaling axis preferentially mediates monocyte trafficking and promotes inflammation and tissue damage in OA (Ann Rheum Dis. 2017 May; 76(5): 914-922). The findings of Ishihara et al., suggest that CCL2- CCR2 signaling locally in the joint contributes to knee hyperalgesia in experimental OA, and it is in part mediated through direct stimulation of CCR2 expressed by intra-articular sensory afferents (Ishihara et al. Arthritis Research & Therapy (2021) 23:103). That CCL2 (also called monocyte chemoattractant protein-1 (MCP-1) is a chemotactic factor of monocytes that plays an important role in the initiation of inflammation in osteoarthritis rat models was identified by Na et al (Na et al. Journal of Translational Medicine (2022) 20:428).

In view of the above, there is a need in the art to provide strategies to reduce unwanted influence of host cell proteins on the recombinant production of proteins of interest, particularly for recombinant therapeutic proteins used to treat OA.

SUMMARY OF THE INVENTION

The present inventors aimed at providing a purification method for human ANGPTL3 mimetics produced in CHO cells, especially soluble variants of ANGPTL3 mimetics, particularly c- terminal fragments of ANGPTL3 that have been mutated to reduced protein clipping become protease-resistant (as disclosed in the PCT patent publication WO2014138687). During development of the purification method, CCL2 was identified as a host cell protein which has physicochemical properties highly similar to those of the ANGPTL3 mimetics and therefore, is not significantly removed during the purification steps. The present inventors therefore established a production cell line for the ANGPTL3 mimetics wherein the CCL2 level is reduced by use of biotechnological techniques like miRNA against this host cell protein or gene knock out technologies. Thereby, purity of production batched of ANGPTL3 mimetics could be improved significantly, allowing the production of compositions comprising therapeutic proteins having a reduced risk of triggering undesired biological effects of CCL2 in e.g., the treatment of human patients suffering from OA.

In view of this, in a first aspect, the present invention is directed to a method of producing ANGPTL3 mimetics comprising the steps of (a) providing CHO cells capable of producing ANGPTL3 mimetics;

(b) cultivating the CHO cells in a cell culture under conditions which allow for the production of ANGPTL3 mimetics;

(c) obtaining ANGPTL3 mimetics from the cell culture; and

(d) optionally processing ANGPTL3 mimetics; wherein the CHO cells are engineered to reduce the production of CCL2 in the CHO cell.

In a second aspect, the present invention provides a CHO cell which is capable of producing ANGPTL3 mimetics and which is engineered to reduce the production of CCL2 in the CHO cell.

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

In a third aspect, the present invention provides a method of improving production of ANGPTL3 mimetics in a CHO cell, comprising the steps of

(a-i) providing a CHO cell capable of producing ANGPTL3 mimetics;

(a-ii) engineering the CHO cell so as to reduce the production of CCL2 in the CHO cell.

In certain embodiments, step (a-ii) of engineering the CHO cell to reduce the production of CCL2 includes introducing a vector nucleic acid according to the fourth aspect into the CHO cell.

In a fourth aspect, the present invention provides a method for producing a CHO cell which is capable of expressing ANGPTL3 mimetics, comprising introducing a vector nucleic acid comprising an expression cassette for expression of a miRNA in a CHO cell, comprising a template sequence for a pri-miRNA, wherein the pri-miRNA is suitable to be processed in a CHO cell to form a miRNA targeting CCL2 into a CHO cell. The vector nucleic acid may additionally comprise a coding sequence encoding ANGPTL3 mimetics, or another nucleic acid comprising a coding sequence encoding ANGPTL3 mimetics is present in or introduced into the CHO cell.

In a fifth aspect, the present invention provides a composition comprising ANGPTL3 mimetics and CCL2, wherein the composition is obtained by production using CHO cells according to the second aspect, and wherein (i) the amount of CCL2 in the composition is at least 10-times lower compared to the same composition obtained by production using CHO cells which were not engineered to reduce the production of CCL2 in the CHO cells; and/or

(ii) the composition comprises CCL2 in a concentration of 100 ppm or lower, preferably 25 ppm or lower, more preferably 10 ppm or lower.

In a sixth aspect, the present invention provides a CHO cell which is engineered to (i) express and produce an ANGPTL3 mimetic and (ii) to reduce the production of CCL2 in the CHO cell. In certain embodiments, a CHO cell of the sixth’s aspect produces an ANGPTL3 mimetic comprising a protein sequence according to any of the SEQ IDs No. 29-50 and a miRNA targeting CCL2. In another embodiment, a CHO cell of the sixth’s aspect produces an ANGPTL3 mimetic comprising a protein sequence according to any of SEQ IDs No. 29, 31 , 34, 36, 39, 41 , 43, 45, 47 and 50 and a miRNA targeting CCL2. In a different embodiment, a CHO cell of the sixth’s aspect produces the ANGPTL3 mimetic protein comprising the amino acid sequence of SEQ ID NO: 43 and a miRNA targeting CCL2. In an additional embodiment, a CHO cell of the sixth’s aspect produces the ANGPTL3 mimetic protein consisting of the amino acid sequence of SEQ ID NO: 43 and a miRNA targeting CCL2. In particular, the CHO cell of the sixth’s aspect is engineered by introduction of a vector nucleic acid comprising an expression cassette for expression of a miRNA in a CHO cell, comprising a template sequence for a pri-miRNA, wherein the pri-miRNA is suitable to be processed in a CHO cell to form a miRNA targeting CCL2.

In a seventh aspect the present invention provides a CHO cell in which (i) the CCL2 gene has been knock-out using gene-editing technologies and which is engineered to (ii) express and produce an ANGPTL3 mimetic. Gene-editing technologies represent alternative methods of the vectorized RNAi approach using artificial intronic miRNAs. A gene knockout on the DNA level which encodes the CCL2 gene leads to a complete functional deactivation due to loss of essential gene information. Cas-CLOVER™ gRNA pairs for targeting the CCL2 gene as outlined in example 4. In certain embodiments, a CHO cell of the seventh’s aspect produces an ANGPTL3 mimetic comprising a protein sequence according to any of the SEQ ID NOs: 29-50. In another embodiment, a CHO cell of the seventh’s aspect produces an ANGPTL3 mimetic comprising a protein sequence according to any of SEQ ID NOs: 29, 31 , 34, 36, 39, 41 , 43, 45, 47 and 50. In a different embodiment, a CHO cell of the seventh’s aspect produces the ANGPTL3 mimetic protein comprising the amino acid sequence of SEQ ID NO:43. In an additional embodiment, a CHO cell of the seventh's aspect produces the ANGPTL3 mimetic protein consisting of the amino acid sequence of SEQ ID NO: 43. In particular, the CHO cell of the seventh’s aspect is engineered by using the Cas-CLOVER™ technology as described in example 4.

In an eight’s aspect the present invention provides the composition according to the previous aspects, e.g., the fifth’s aspect, for use in the treatment of arthritis or cartilage damage in a human subject, wherein the composition is administered intra-articularly. In one embodiment the composition for use according to the eight’s aspect relates to a composition comprising a dose of about 20-40 mg of an ANGPTL3 mimetic comprising a protein sequence according to any of the SEQ IDs No. 29-50. In another embodiment the composition for use according to the eight’s aspect relates to a composition comprising a dose of about 20-40 mg of an ANGPTL3 mimetic comprising a protein sequence according to SEQ ID NO: 43. In an additional embodiment of the eight’s aspect the composition comprises a dose of about 20-40 mg of an ANGPTL3 mimetic consisting of a protein sequence according to SEQ ID NO: 43.

Other objects, features, advantages and aspects of the present invention will become apparent to those skilled in the art from the following description and appended claims. It should be understood, however, that the following description, appended claims, and specific examples, which indicate preferred embodiments of the application, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following.

DEFINITIONS

As used herein, the following expressions are generally intended to preferably have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise.

The expression "comprise", as used herein, besides its literal meaning also includes and specifically refers to the expressions "consist essentially of" and "consist of'. Thus, the expression "comprise" refers to embodiments wherein the subject-matter which "comprises" specifically listed elements does not comprise further elements as well as embodiments wherein the subject-matter which "comprises" specifically listed elements may and/or indeed does encompass further elements. Likewise, the expression "have" is to be understood as the expression "comprise", also including and specifically referring to the expressions "consist essentially of" and "consist of'. The term "consist essentially of", where possible, in particular refers to embodiments wherein the subject-matter comprises 20% or less, in particular 15% or less, 10% or less or especially 5% or less further elements in addition to the specifically listed elements of which the subject-matter consists essentially of.

The term "nucleic acid" includes single-stranded and double-stranded nucleic acids and ribonucleic acids as well as deoxyribonucleic acids. It may comprise naturally occurring as well as synthetic nucleotides and can be naturally or synthetically modified, for example by methylation, 5'- and/or 3'-capping. In specific embodiments, a nucleic acid refers to a doublestranded deoxyribonucleic acid. The term "expression cassette" in particular refers to a nucleic acid construct which is capable of enabling and regulating the expression of a coding nucleic acid sequence and/or template nucleic acid sequence introduced therein. An expression cassette may comprise promoters, ribosome binding sites, enhancers and other control elements which regulate transcription of a gene or translation of an mRNA. The exact structure of an expression cassette may vary as a function of the species or cell type, but generally comprises 5'-untranscribed and 5'- and 3'- untranslated sequences which are involved in initiation of transcription and translation, respectively, such as TATA box, capping sequence, CAAT sequence, and the like. More specifically, 5'-untranscribed expression control sequences comprise a promoter region which includes a promoter sequence for transcriptional control of the operatively connected nucleic acid. Expression cassettes may also comprise enhancer sequences or upstream activator sequences. Some expression cassettes are only used for transcription of a template nucleic acid sequence into an RNA product such as a pri-miRNA. Such expression cassettes do not necessarily comprise regulatory elements for translation.

A template nucleic acid is understood according to this application as a DNA which is transcribed into a functional RNA product or a precursor thereof, especially a pri-miRNA. A functional RNA product in particular has a biological activity, alone or in combination with other RNA products and/or proteins, such as the activity of a miRNA (in combination with the proteins of the RNA-induced silencing complex (RISC)) to interfere with expression of a target gene.

A target amino acid sequence is "derived" from or "corresponds" to a reference amino acid sequence if the target amino acid sequence shares an identity over its entire length with the reference amino acid sequence of at least 75%, more preferably at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98% or at least 99%. In particular embodiments, a target amino acid sequence which is "derived" from or "corresponds" to a reference amino acid sequence is 100% identical over its entire length with the reference amino acid sequence. Similarly, a target nucleotide sequence is "derived" from or "corresponds" to a reference nucleotide sequence if the target nucleotide sequence shares an identity over its entire length with the reference nucleotide sequence of at least 75%, more preferably at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98% or at least 99%. In particular embodiments, a target nucleotide sequence which is "derived" from or "corresponds" to a reference nucleotide sequence is 100% identical over its entire length with the reference nucleotide sequence. An "identity" of an amino acid sequence or nucleotide sequence is preferably determined according to the invention over the entire length of the reference sequence.

As used herein, a "microRNA", abbreviated "miRNA", is a single-stranded non-coding RNA molecule which plays a role in RNA silencing and post-transcriptional regulation of gene expression. miRNAs generally consist of 19 to 24 nucleotides, in particular about 22 nucleotides, especially 22 nucleotides. miRNA molecules are capable of silencing mRNAs comprising a complementary nucleotide sequence. Silencing of the target mRNA may occur by cleavage of the mRNA, destabilization of the mRNA or blockage of translation of the mRNA. Silencing of a target mRNA results in reduced or abolished production of the protein encoded by the target mRNA. It is generally understood in the art that the miRNA associates with dicer and argonaute proteins, forming an RNA-induced silencing complex (RISC) which binds to the target mRNA. miRNAs are produced by transcription of a template DNA sequence into a miRNA precursor (pri-miRNA). The pri-miRNA contains a hairpin stem-loop structure with a double-stranded stem connected to a loop on one side and flanked by single-stranded 5' and 3' extensions on the other side. The double-stranded stem contains the guide strand, which forms the miRNA once processed, and the passenger strand which is essentially complementary to the guide strand. Especially, the passenger strand and the guide strand are complementary to each other except for the nucleotide pair at the end of the hairpin stem-loop structure, i.e. the nucleotide pair of passenger and guide strand which is furthest from the loop structure. Guide strand and passenger strand generally each have a length of about 19 to 24 nucleotides, especially of 22 nucleotides. The remaining parts of the pri-miRNA are referred to herein as miRNA scaffold. From 5' to 3', the pri-miRNA thus comprises (i) the 5' miRNA scaffold stem, consisting of the 5' single-stranded extension and the 5' part of the stem structure up to the passenger strand; (ii) the passenger strand; (iii) the miRNA scaffold loop; (iv) the guide strand; and (v) the 3' miRNA scaffold stem, consisting of the 3' part of the stem structure following the guide strand and the 3' single-stranded extension. Positions of the passenger strand and the guide strand may also be switched.

The pri-miRNA is processed by cleaving off the 5' and 3' miRNA scaffold stems, resulting in a hairpin structure termed pre-miRNA. From 5' to 3', the pre-miRNA consists of the passenger strand, the miRNA scaffold loop, and the guide strand; wherein the positions of the passenger strand and the guide strand may also be switched. Then the loop structure is cleaved off and the resulting RNA duplex is separated into the 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 generally does not have any function.

The cells referred to herein in particular are host cells. According to the invention, the term "host cell" relates to any cell which can be transformed or transfected with an exogenous nucleic acid. Particular preference is given to mammalian cells such as cells from humans, mice, hamsters, pigs, goats, or primates. The cells may be derived from a multiplicity of tissue types and comprise primary cells and cell lines. A nucleic acid may be present in the host cell in the form of a single copy or of two or more copies and, in one embodiment, is expressed in the host cell. A host cell in particular refers to a cell present in cell culture, especially a cell not present in a living multicellular organism. The term "ANGPTL3 mimetics" as used herein refers to ANGPTL3 polypeptides comprising sequence modifications compared to the ANGPTL3 polypeptide sequence of SEQ ID NO:55.

A “ANGPTL3 polypeptide” refers to a naturally occurring ANGPTL3 protein or a fragment or variant thereof. For the purposes of the present disclosure, the numbering of an amino acid is typically determined with reference to the full-length wildtype human ANGPTL3 polypeptide sequence (SEQ ID NO:55). Thus, in embodiments in which a polypeptide of the invention contains only a C-terminal portion of full-length ANGPTL3, but not the N-terminal portion, although the peptide is less than 460 amino acids in length, the numbering of the positions is based on SEQ ID NO:55. For example, reference to position 423 of an ANGPTL3 polypeptide of the invention refers to position 423 of SEQ ID NO:55, even though the ANGPTL3 polypeptide of the invention itself may only be 200 amino acids in length. In determining an amino acid in a sequence of interest that “corresponds to” a position in a reference sequence, such as SEQ ID NO:55, this is performed by optimally aligning the sequences, e.g., using the default CLUSTAL alignment parameters or default BLAST 2 alignment parameters and comparing the sequences. For example, position 423 in a sequence of interest that is “determined with reference to SEQ ID NO:55”, or an amino acid that “corresponds to” position 423 of SEQ ID NO:55, means the amino acid that aligns with position 423 of SEQ ID NO:55 when the sequence of interest is optimally aligned with SEQ ID NO:55.

The term "CCL2" as used herein refers to the Chinese hamster protein CCL2 (C-C motif chemokine receptor ligand 2), also known as monocyte chemoattractant protein 1 (MCP1). CCL2 in particular has the amino acid sequence of SEQ ID NO: 24. Amounts and concentrations of CCL2 in a composition as referred to herein are in particular determined by ELISA (enzyme-linked immune adsorbent assay) or LC-MS (liquid chromatography-mass spectrometry, especially by LC-MS. Respective measurements are preferably performed as described in the example sections.

The term "pharmaceutical composition" or "pharmaceutical formulation" particularly refers to a composition suitable for administering to a human or animal, i.e., a composition containing components which are pharmaceutically acceptable. Preferably, a pharmaceutical composition comprises an active compound or a salt or prodrug thereof together with a carrier, diluent or pharmaceutical excipient such as buffer, preservative and tonicity modifier.

The numbers given herein may in certain embodiments be understood as approximate numbers. In particular, the numbers preferably may 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 specific embodiments, the numbers given herein are not approximate numbers and may only vary within the inaccuracy of the technical measurement.

Numeric ranges described herein are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects or embodiments of this invention which can be read by reference to the specification as a whole. According to one embodiment, subject-matter described herein as comprising certain steps in the case of methods or as comprising certain ingredients in the case of compositions refers to subjectmatter consisting of the respective steps or ingredients. It is preferred to select and combine preferred aspects and embodiments described herein and the specific subject-matter arising from a respective combination of preferred embodiments also belongs to the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the finding that the endogenous CHO protein CCL2 causes major problems during purification of recombinantly produced therapeutic ANGPTL3 mimetics. Typically, residual host proteins are removed from the drug by adapting or by optimizing downstream purification process, e.g. by introducing novel or optimized affinity purification steps. As observed during the purification process, the host cell protein CCL2 is not separated from the ANGPTL3 mimetics by the established chromatography steps, especially by anion exchange chromatography or hydrophobic interaction chromatography. It is assumed that this lack of separation is caused by highly similar physicochemical properties of CCL2 and the ANGPTL3 mimetics, in particular by a similar charge distribution.

The present inventors solved this problem by knocking down and knocking out CCL2 in the CHO host cells using the gene editing technology Cas-CLOVER™ (but other technologies like homologous recombination, site-specific nucleases, CRISPR/Cas or TALENs could be used to achieve the same result) and biotechnological technologies like an miRNA approach. Such approaches have the advantage that the presence of the problematic protein can be avoided from the very beginning of the production process and do not require further timely and potentially costly adaptions of downstream process. Transfecting the CHO host cell with a vector producing e.g., a miRNA targeting the CCL2 mRNA as well as knocking down the CCL2 gene showed a strong efficacy and succeeded in significantly reducing the CCL2 level in the cell culture supernatant. Thereby, in the final composition of the purified ANGPTL3 mimetics the CCL2 was lowered to an acceptable residual amount. Furthermore, the method of producing ANGPTL3 mimetics as described herein, allow to produce a pure, and safe ANGPTL3 mimetics, suitable for use as a drug.

1. Production methods for ANGPTL3 mimetics

In a first aspect, the present invention provides a method of producing an ANGPTL3 mimetic, comprising the steps of

(a) providing CHO cells capable of producing the ANGPTL3 mimetic; (b) cultivating the CHO cells in a cell culture under conditions which allow for the production of the ANGPTL3 mimetic;

(c) obtaining the ANGPTL3 mimetic from the cell culture; and

(d) optionally further processing the ANGPTL3 mimetic; wherein the CHO cells are engineered to have a reduced production of CCL2 in the CHO cell.

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

In specific embodiments, the CHO cells are engineered to have a reduced production/expression of CCL2 in the CHO cell. The CHO cells may be engineered in any suitable way to reduce the production/expression of CCL2. Reducing the production of CCL2 in particular refers to reducing the expression of CCL2. In general, cell line engineering, especially genetic engineering, is used. In certain embodiments, production of CCL2 in the CHO cells is reduced by reducing CCL2 expression by knockdown of CCL2 gene expression products or knockout of the CCL2 gene as disclosed in the example section.

Knockdown of CCL2 expression can be done by reducing the amount of functional mRNA encoding CCL2 in the cell and/or reducing the rate of translation of the mRNA encoding CCL2 into CCL2 protein. In specific embodiments, the CHO cells are engineered to produce a miRNA targeting the endogenous CCL2 of the CHO cells as disclosed in the example section.

Knockout of CCL2 expression may be performed by deleting one or both alleles of the gene encoding CCL2 in the genome of the CHO cell or mutating one or both of these alleles so that they cannot be transcribed into functional mRNA. Knockout of CCL2 expression may be done using, for example, homologous recombination, site-specific nucleases, CRISPR/Cas, TALENs or Cas-CLOVER™. A specific example using the Cas-CLOVER™ technology is provided in the example section.

Reducing of the production of CCL2 in particular means that after engineering of the CHO cell, the amount of CCL2 in the cell is 50% or less of the amount of CCL2 in the cell prior to engineering. In certain embodiments, the amount of CCL2 in the cell is 25% or less, especially 10% or less or 5% or less of the amount of CCL2 in the cell prior to engineering.

The host cell may be any cell suitable for producing ANGPTL3 mimetics, especially human ANGPTL3 mimetics, and endogenously expressing CCL2. Especially, the host cell is a mammalian cell. It may be selected from, but not limited to, the group consisting of cells derived from mice, such as COP, L, C127, Sp2/0, NSO, NS1 , At20 and NIH3T3; rats, such as PC12, PC12h, GH3, MtT, YB2/0 and Y0; hamsters, such as BHK, CHO and DHFR gene defective 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 specific embodiments, the host cell is a Chinese hamster ovary (CHO) cell. The host cell may be suitable for suspension cultures and/or adherent cultures, and in particular can be used in suspension cultures. The features, embodiments and examples of the method of producing ANGPTL3 mimetics according to the first aspect of the invention also likewise apply to the method according to this further aspect of the invention.

1.1 The miRNA targeting CCL2

In certain embodiments, the CHO cells are engineered to produce a miRNA targeting the endogenous CCL2 of the CHO cells. It is understood that the miRNA targeting CCL2 is at least partially complementary to and is capable of binding to and initiating silencing of the mRNA or pre-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 part of the 5' UTR, to a part of the coding region, to a part of an intron, and/or to a part of the 3' UTR of the CCL2 mRNA. In particular, the miRNA targeting CCL2 is complementary to a part of the 3'UTR of the CCL2 mRNA. Exemplary miRNAs targeting CCL2 have a nucleotide sequence selected from the group consisting of SEQ ID NO: 26.

In specific embodiments, the CHO cells comprise an expression cassette for production of the 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 to be processed in the CHO cell to form the miRNA targeting CCL2. The expression cassette enables transcription of the template sequence into the pri-RNA. the template sequence may be present anywhere within the transcribed region of the expression cassette.

In certain embodiments, the template sequence is present within an intronic sequence. Upon transcription, the intronic sequence is excised from the transcribed RNA, thereby forming the pri-miRNA.

Since the expression cassette comprises the template sequence for the pri-miRNA within an intronic sequence, it may contain further sequences for expression of other products, such as coding sequences for the production of a polypeptide of interest, coding sequences for the production of selectable marker, and template sequence for other RNA products, especially other pri-miRNAs. Alternatively, the expression cassette may be used exclusively for production of the miRNA targeting CCL2.

The expression cassette may comprise the template sequence for the pri-miRNA within an intronic sequence. Upon expression, a pre-mRNA is formed which contains the intronic sequence. The intronic sequence is then spliced out of the pre-mRNA, thereby forming the pri- miRNA which thereafter is further processed to ultimately provide the miRNA. The formed pre- mRNA does not have to comprise any sequences coding for a polypeptide. In certain embodiments, the expression cassette further comprises a polymerase II promoter. This promoter is functionally linked to the template sequence for the pri-miRNA and controls expression of the pri-miRNA. The promoter may be any RNA polymerase II promoter suitable for expression of a gene in a host cell, especially a CHO cell. For example, the promoter may be selected from the group consisting of cytomegalovirus (CMV) promoter, simian virus 40 (SV40) promoter, ubiquitin C (UBC) promoter, elongation factor 1 alpha (EF1A) promoter, phosphoglycerate kinase (PGK) promoter, Rous sarcoma virus (RSV) promoter, BROAD3 promoter, murine rosa 26 promoter, pCEFL promoter, chicken p-actin promoter (CBA), p-actin promoter coupled with CMV early enhancer (CAGG), a-1-antitrypsin promoter, and inducible promoters such as tetracycline-inducible promoters (e.g. pTRE), and vanillic acid inducible promoters. In specific embodiments, the promoter is a CMV promoter or a SV40 promoter, especially a CMV promoter.

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

The template sequence for the pri-miRNA is in particular located between the promoter and the terminator of the expression cassette.

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

The intronic sequence comprising the template sequence for the pri-miRNA in particular comprises a splice donor site upstream of the pri-miRNA and a corresponding splice acceptor site downstream of the pri-miRNA. With these splice donor and acceptor sites, the pri-miRNA is spliced out of the pre-m RNA after transcription.

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

In certain embodiments, the expression cassette comprises only one intronic sequence with one or more template sequences for a pri-miRNA. In alternative embodiments, the expression cassette comprises two or more intronic sequence with one or more template sequences for a pri-miRNA.

The pri-miRNAs of the two or more template sequences present within the same or different intronic sequences in particular are different from each other. In specific embodiments, the miRNAs produced from the pri-miRNAs all target CCL2, but bind to different parts of the mRNA or pre-mRNA of CCL2.

The expression cassette may comprise a coding sequence which encodes a polypeptide. The coding sequence may in particular code for a selectable marker. The coding sequence preferably is functionally linked to the polymerase II promoter and the terminator of the expression cassette. In embodiments wherein the expression cassette comprises the template sequence for the pri-miRNA and the coding sequence for the selectable marker, the expression of the miRNA is linked to the selectable marker expression. Thus, by increasing the selection pressure during clone selection, also the miRNA level is increased.

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

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

In certain embodiments, the pri-miRNA comprises a passenger strand and a guide strand. The guide strand in particular comprises or consists of the miRNA formed after processing of the pri-miRNA by the host cell. The pri-miRNA furthermore, 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 specific embodiments, the pri-miRNA comprises, from 5' to 3', a 5' miRNA scaffold stem, a passenger strand, a miRNA scaffold loop, a guide strand, and a 3' miRNA scaffold stem. In alternative embodiments, the pri-miRNA comprises, from 5' to 3', a 5' miRNA scaffold stem, a guide strand, a miRNA scaffold loop, a passenger strand, and a 3' miRNA scaffold stem. Embodiments wherein the passenger strand is positioned upstream of the guide strand are preferred. The passenger strand and the guide strand of the pri-miRNA in particular have artificial sequences. An artificial sequence in this respect refers to a sequence which is not present as passenger or guide strand in naturally occurring miRNAs. In particular, the sequences of the passenger strand and the guide strand are not found in naturally occurring miRNAs.

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.

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

In specific embodiments, all of the scaffold sequences of the pri-miRNA share a nucleotide sequence identity with the corresponding scaffold sequences of a naturally occurring pri- miRNA of at least 80%, especially at least 90%, in particular at least 95% over their entire length. In certain embodiments, the 5' miRNA scaffold stem of the pri-miRNA shares a nucleotide sequence identity with the corresponding scaffold sequence of a naturally occurring pri-miRNA of at least 80%, especially at least 85%, in particular at least 90% over its entire length. In certain embodiments, the 3' miRNA scaffold stem of the pri-miRNA shares a nucleotide sequence identity with the corresponding scaffold sequence of a naturally occurring pri-miRNA of at least 80%, especially at least 90%, in particular at least 95% over its entire length. In certain embodiments, the miRNA scaffold loop of the pri-miRNA shares a nucleotide sequence identity with the corresponding scaffold sequence of a naturally occurring pri-miRNA of at least 60%, especially at least 70%, in particular at least 75% over its entire length. In these embodiments, the naturally occurring pri-miRNA may in particular be miR-30A. In certain embodiments, the 5' miRNA scaffold stem of the pri-miRNA comprises the nucleotide sequence of any one of SEQ ID NOs: 1-7 or a sequence derived therefrom. In particular, the 5' miRNA scaffold stem of the pri-miRNA comprises the nucleotide sequence of any one of SEQ ID NOs: 1-7 or a sequence sharing a nucleotide sequence identity therewith of at least 90%, preferably at least 95%, more preferably at least 98%, and most preferably 100%. Especially, the 5' miRNA scaffold stem of the pri-miRNA consists of the nucleotide sequence of any one of SEQ I D NOs: 1 -4, in particular SEQ I D NO: 1.

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

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

In certain embodiments, the template sequence for the pri-miRNA comprises at least one recognition site, especially two recognition sites, for a DNA restriction enzyme. In particular, the two recognition sites are for different DNA restriction enzymes and generate different overhangs after cleavage. The two recognition sites preferably flank the pre-miRNA part of the pri-miRNA - which comprises the guide strand, the passenger strand and the miRNA scaffold loop - on both sides. In particular, one of the recognition sites is located within the sequence which is transcribed into the 5' miRNA scaffold stem and the other recognition site is located within the sequence which is transcribed into the 3' miRNA scaffold stem. In specific embodiments, the recognition sites are located within the sequences which are transcribed into the single-stranded parts of the 5' and 3' miRNA scaffold stems. The recognition sites in particular are unique recognition sites within the expression cassette, especially within the entire vector harboring the expression cassette.

1.2 Knockout of the CCL2 gene

In certain embodiments, the CHO cells are engineered by knockout of the CCL2 gene. Knockout of the CCL2 gene refers to deletion or inactivation of the CCL2 gene. In specific embodiments, both alleles of the CCL2 gene in the CHO cells are knocked out. The CCL2 gene may be completely removed, or its transcription may be impaired or the translation of a complete CCL2 protein may be inhibited. In particular, a mutation may be introduced into the gene which results in a frameshift of the coding sequence of the CCL2 mRNA.

Knockout of CCL2 expression may be done using, for example, homologous recombination, site-specific nucleases, CRISPR/Cas, TALENs or Cas-CLOVER™. The Cas-CLOVER™ technology is a highly specific gene editing technology with low/no detectable off-target events (Li et al (2018) Cas-CLOVER™: A High-Fidelity Genome Editing System for Safe and Efficient Modification of Cells for Immunotherapy. Precision CRISPR congress poster; Chi X, Zheng Q, Jiang R, Chen-Tsai RY, Kong L-J (2019) A system for site-specific integration of transgenes in mammalian cells. PLoS ONE 14(7): e0219842). In particular, production of CCL2 knockout cells using Cas-CLOVER™ may be performed using the gRNA pair with a gRNA comprising the nucleotide sequence of SEQ ID NO: 53 and another gRNA comprising the nucleotide sequence of SEQ ID NO: 54 as disclosed in example 4.

1.3 The production and purification steps

In certain embodiments, the method further comprises between steps (a) and (b) the steps of

(a1) inoculating a cell culture medium with the CHO cells to provide a cell culture, and

(a2) cultivating the CHO cells in the cell culture under conditions which allow for increasing the number of CHO cells in the cell culture.

Suitable conditions for cultivating the host cells, increasing their cell number and expressing ANGPTL3 mimetics can be readily determined by the skilled person and are also already known in the art. In certain embodiments, a vector nucleic acid encoding ANGPTL3 mimetics in the CHO cells comprises one or more selectable marker genes. In these embodiments, the culturing conditions in step (a2) and/or (b) may include the presence of corresponding selection agent(s) in the cell culture medium.

Obtaining ANGPTL3 mimetics from the cell culture in step (c) in particular includes isolating ANGPTL3 mimetics from the cell culture. Isolation of ANGPTL3 mimetics in particular refers to the separation of ANGPTL3 mimetics from the remaining components of the cell culture. The term "cell culture" as used herein in particular includes the cell culture medium and the CHO cells. In certain embodiments, ANGPTL3 mimetics is secreted by the CHO cells. In these embodiments, ANGPTL3 mimetics is isolated from the cell culture medium. Separation of ANGPTL3 mimetics from the cell culture medium may be performed, for example, by chromatographic methods and/or filtration methods.

Suitable methods and means for isolating ANGPTL3 mimetics are known in the art and can be readily applied by the skilled person. Exemplary isolation methods for example include filtration steps such as tangential flow filtration, alternating flow filtration, depth filtration, ultrafiltration and diafiltration, and/or chromatography steps such as affinity chromatography, anion- and/or cation exchange chromatography, hydrophilic interaction chromatography, size exclusion chromatography and reverse phase chromatography. The step of obtaining ANGPTL3 mimetics from the cell culture may in particular include a filtration step, a bind/elute chromatography step and one or more polishing chromatography steps. In addition, step (c) may further include one or more virus inactivation steps.

In specific embodiments, step (c) comprises performing cation exchange chromatography and/or hydrophobic interaction chromatography. In particular, step (c) comprises:

(c1) separating the CHO cells from cell culture supernatant containing ANGPTL3 mimetics;

(c2) separating ANGPTL3 mimetics from the cell culture supernatant using cation exchange chromatography;

(c3) further purifying ANGPTL3 mimetics using anion exchange chromatography; and

(c4) further purifying ANGPTL3 mimetics using hydrophobic interaction chromatography.

Step (c1) may in particular be performed using a filtration method such as tangential flow filtration or alternating flow filtration, wherein the CHO cells are held back in the retentate and the cell culture supernatant containing ANGPTL3 mimetics pass through the filter to the permeate. Furthermore, step (c1) may also be performed using a centrifugation method. In addition to steps (c1) to (c4), step (c) may also further comprise one or more ultrafiltration and/or diafiltration steps for buffer exchange between or after the chromatography steps as well as virus inactivation and/or virus retention steps. Virus inactivation may in particular be achieved using detergent for a time sufficient to inactivate substantially all viruses, and virus retention may be achieved using sterile filtration.

The obtained ANGPTL3 mimetics may optionally be subject to further processing steps (d) such as e.g. modification and/or formulation steps in order to produce ANGPTL3 mimetics in the desired quality and composition. Such further processing steps and methods are generally known in the art. Formulation steps may include buffer exchange, addition of formulation components, pH adjustment, and concentration adjustment. Any combination of these and further steps may be used.

In certain embodiments, the method for producing ANGPTL3 mimetics further comprises as step (d) or part of step (d) the step of providing a pharmaceutical formulation comprising ANGPTL3 mimetics. Providing a pharmaceutical formulation comprising ANGPTL3 mimetics or formulating ANGPTL3 mimetics as a pharmaceutical composition in particular comprises exchanging the buffer solution or buffer solution components of the composition comprising ANGPTL3 mimetics. Furthermore, this step may include lyophilization of ANGPTL3 mimetics. In particular, ANGPTL3 mimetics is transferred into a composition only comprising pharmaceutically acceptable ingredients.

The method of producing ANGPTL3 mimetics in particular provides the ANGPTL3 mimetics with a higher purity compared to the same method using CHO cells which are not engineered to reduce the production of CCL2 in the CHO cells. In certain embodiments, ANGPTL3 mimetics obtained after step (c) is in a composition which comprises CCL2 in a concentration which is at least 10-times lower, preferably 25-times lower, more preferably 100-times lower, compared to a composition obtained with the same method after step (c) using CHO cells which are not engineered to reduce the production of CCL2 in the CHO cells. In particular, ANGPTL3 mimetics obtained after step (c) is in a composition which comprises CCL2 in a concentration of 100 ppm or lower, preferably 25 ppm or lower, more preferably 10 ppm or lower.

In certain embodiments, ANGPTL3 mimetics obtained after step (c1) is in a composition which comprises CCL2 in a concentration which is at least 10-times lower, preferably 25-times lower, more preferably 100-times lower, compared to a composition obtained with the same method after step (c1) using CHO cells which are not engineered to reduce the production of CCL2 in the CHO cells. In particular, ANGPTL3 mimetics obtained after step (c1) is in a composition which comprises CCL2 in a concentration of 100 ppm or lower, preferably 25 ppm or lower, more preferably 10 ppm or lower.

In certain embodiments, ANGPTL3 mimetics obtained after step (c4) is in a composition which comprises CCL2 in a concentration which is at least 10-times lower, preferably 25-times lower, more preferably 100-times lower, compared to a composition obtained with the same method after step (c4) using CHO cells which are not engineered to reduce the production of CCL2 in the CHO cells. In particular, ANGPTL3 mimetics obtained after step (c4) is in a composition which comprises CCL2 in a concentration of 25 ppm or lower, preferably 10 ppm or lower, more preferably 5 ppm or lower.

In certain embodiments, ANGPTL3 mimetics obtained after step (d) is in a composition which comprises CCL2 in a concentration which is at least 10-times lower, preferably 25-times lower, more preferably 100-times lower, compared to a composition obtained with the same method after step (d) using CHO cells which are not engineered to reduce the production of CCL2 in the CHO cells. In particular, ANGPTL3 mimetics obtained after step (d) is in a composition which comprises CCL2 in a concentration of 25 ppm or lower, preferably 10 ppm or lower, more preferably 5 ppm or lower.

1.4 ANGPTL3 mimetics

The ANGPTL3 mimetics produced by the method in particular is a human ANGPTL3 mimetic. In certain embodiments, the ANGPTL3 mimetics variant has an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 31 , 34, 36, 39, 41 , 43, 45, 47 and 50. In particular, the ANGPTL3 mimetic comprises the amino acid sequence of SEQ ID NO: 43, and especially consists of the amino acid sequence of SEQ ID NO: 43.

2. CHO host cells

In a second aspect, the present invention provides a CHO cell which is capable of producing ANGPTL3 mimetic and which is engineered to reduce the production of CCL2 in the CHO cell. The CHO cell in particular may be engineered as described herein above with respect to the method of producing an ANGPTL3 mimetic.

In a further aspect, the present invention provides a host cell which is capable of producing an ANGPTL3 mimetic and which is engineered to reduce the production of CCL2 in the host cell. The host cell in particular is a host cell as described herein above with respect to the method of producing an ANGPTL3 mimetic and may be engineered as described herein above with respect to the method of producing the ANGPTL3 mimetic. The features and embodiments described herein for the CHO cell likewise also apply to the host cell in general.

The host cell may be any cell suitable for producing an ANGPTL3 mimetic, especially human ANGPTL3 mimetics, and endogenously expressing CCL2 when not engineered to reduce the production of CCL2. Especially, the host cell is a mammalian cell. It may be selected from, but not limited to, the group consisting of cells derived from mice, such as COP, L, C127, Sp2/0, NSO, NS1 , At20 and NIH3T3; rats, such as PC12, PC12h, GH3, MtT, YB2/0 and Y0; hamsters, such as BHK, CHO and DHFR gene defective 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 specific embodiments, the host cell is a Chinese hamster ovary (CHO) cell. The host cell may be suitable for suspension cultures and/or adherent cultures, and in particular can be used in suspension cultures.

The CHO cell or host cell may contain further exogenous nucleic acids in addition to the herein disclosed expression cassettes or the herein disclosed vector nucleic acids. In particular, the CHO cell or host cell may contain an expression cassette for expression of an ANGPTL3 mimetic. Said expression cassette for expression of the ANGPTL3 mimetic may be present on a further vector nucleic acid or integrated into the genome of the CHO cell or host cell.

In certain embodiments, the coding sequence of the ANGPTL3 mimetic is present in the CHO cell or host cell:

(i) within the expression cassette which expresses the miRNA targeting CCL2,

(ii) within a further expression cassette on the same vector nucleic acid as the expression cassette which expresses the miRNA targeting CCL2, or (iii) on a further vector nucleic acid or in the genome of the CHO cell or host cell.

The ANGPTL3 mimetics in particular is a ANGPTL3 mimetic as described herein, e.g., comprising or consisting of an amino acid sequence of SEQ ID NO: 29-50.

In a further aspect, the present invention provides a mammalian cell, especially a CHO cell, which is engineered to reduce the production of CCL2 in the mammalian cell. The mammalian cell may be engineered as described herein above with respect to the method of producing ANGPTL3 mimetics.

3. Methods for improving production of ANGPTL3 mimetics

In one aspect, the present invention provides a method of improving production of ANGPTL3 mimetics in a CHO cell, comprising the steps of

(a-i) providing a CHO cell capable of producing ANGPTL3 mimetics;

Engineering of the CHO cell may in particular be performed as described herein above or in the examples with respect to the method of producing ANGPTL3 mimetics. In certain embodiments, the CHO cell is engineered in step (a-ii) by knockdown or knockout of CCL2 expression or inducing degradation of CCL2 protein in the CHO cell. Especially, the CHO cell is engineered in step (a-ii) to produce a miRNA targeting the endogenous CCL2 of the CHO cells. In specific embodiments, step (a-ii) of engineering the CHO cell to reduce the production of CCL2 includes introducing a vector nucleic acid encoding a miRNA targeting CCL2 into the CHO cell, especially a vector nucleic acid which comprises an expression cassette for expression of the miRNA targeting CCL2. In particular, a vector nucleic acid as described herein is used.

The embodiments, features and examples of the method of producing ANGPTL3 mimetics as described herein also likewise apply to the method of improving production of ANGPTL3 mimetics in a CHO cell. In particular, the CHO cell, the ANGPTL3 mimetics, the miRNA targeting CCL2, the gene editing constructs and/or the expression cassette may be as defined herein above with respect to the method of producing ANGPTL3 mimetics.

In certain embodiments, step (a-ii) of engineering the CHO cell results in an engineered CHO cell wherein 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 prior to step (a-ii). In specific embodiments, step (a-ii) of engineering the CHO cell results in an engineered CHO cell wherein 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 prior to step (a-ii). The method of improving production of ANGPTL3 mimetics in a CHO cell may further comprise the steps of:

(b) cultivating the CHO cell obtained in step (a-ii) in a cell culture under conditions which allow for proliferation of the CHO cell and simultaneous and/or subsequent production of ANGPTL3 mimetics;

(c) obtaining said ANGPTL3 mimetics from the cell culture; and

(d) optionally processing ANGPTL3 mimetics.

The embodiments, features and examples of steps (a), (b), (c) and (d) of the method of producing ANGPTL3 mimetics as described herein also likewise apply to steps (a-i), (b), (c) and (d), respectively, of the method of improving production of ANGPTL3 mimetics in a CHO cell.

4. Expression cassettes and vector nucleic acids

In one aspect, the present invention provides an expression cassette for expression of a miRNA in a CHO cell, comprising a template sequence for a pri-miRNA, wherein the pri-miRNA is suitable to be processed in a CHO cell to form a miRNA targeting CCL2.

The features, embodiments and examples of the expression cassette described herein above with respect to the method of producing ANGPTL3 mimetics also likewise apply to the expression cassette according to additional aspect of the invention.

In one aspect, the present invention provides a vector nucleic acid for transfection of a CHO cell, comprising the expression cassettes described herein. The vector nucleic acid may be any vector nucleic acid suitable for transfection of a CHO cell. In certain embodiments, the vector nucleic acid is a plasmid. In other embodiments, the vector nucleic acid is a viral vector.

The vector nucleic acid may comprise further elements in addition to the expression cassette. For example, the vector nucleic acid may comprise an origin of replication (ORI), a coding sequence encoding a polypeptide of interest, a selectable marker gene, and/or an antibiotics resistance gene. The polypeptide of interest may in particular be ANGPTL3 mimetics, especially ANGPTL3 mimetics as described herein.

In certain embodiments, the vector nucleic acid further comprises a coding sequence encoding ANGPTL3 mimetics, especially ANGPTL3 mimetics as described herein. The coding sequence encoding ANGPTL3 mimetics may be present within the same expression cassette as the template sequence for the pri-miRNA, or may be present within a further expression cassette present within the vector nucleic acid. In certain embodiments, the vector nucleic acid does not comprise a coding sequence encoding a polypeptide of interest. In specific embodiments where the vector nucleic acid does not comprise a coding sequence encoding the polypeptide of interest, the expression cassette according to the fourth aspect of the invention does not comprise a coding sequence for a polypeptide. In these embodiments, the vector nucleic acid in particular comprises a further expression cassette comprising a selectable marker gene. In alternative embodiments where 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 which encodes a selectable marker.

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 within the expressing cassette according to the fourth aspect of the invention or may be present in a further expression cassette. In specific embodiments, the vector nucleic acid comprises at least two expression cassettes, a first expression cassette for expression of the polypeptide of interest and a second expression cassette for the expression of a selectable marker, with either the first or the second expression cassette being an expressing cassette according to the fourth aspect of the invention. In alternative embodiments, the vector nucleic acid comprises at least three expression cassettes, a first expression cassette being an expressing cassette according to the fourth aspect of the invention, a second expression cassette for expression of the polypeptide of interest, and a third expression cassette for the expression of a selectable marker. The polypeptide of interest may in particular be an ANGPTL3 mimetic, especially an ANGPTL3 mimetic as described herein.

In certain embodiments, two or more of the expression cassettes of the vector nucleic acid are expression cassettes according to the fourth aspect of the invention. These expression cassettes may each comprise template sequences for the same or different pri-miRNAs, in particular for different pri-miRNAs. The miRNAs produced from the pri-miRNAs may in particular all target CCL2, but bind to different parts of the mRNA or pre-mRNA of CCL2.

5. Methods for producing a CHO cell

In another aspect, the present invention provides a method for producing a CHO cell which is capable of expressing ANGPTL3 mimetics, comprising introducing a vector nucleic acid as disclosed herein into a CHO cell. The vector nucleic acid may additionally comprise a coding sequence encoding ANGPTL3 mimetics, or another nucleic acid comprising a coding sequence encoding ANGPTL3 mimetics is present in or introduced into the CHO cell.

Thus, in certain embodiments the method for producing a CHO cell comprises the step of introducing a vector nucleic acid as disclosed herein into a CHO cell, wherein the vector nucleic acid comprises a coding sequence for ANGPTL3 mimetics, either within the expression cassette which expresses the miRNA targeting CCL2, or within a further expression cassette. In alternative embodiments, the method for producing a CHO cell comprises the step of introducing a vector nucleic acid as disclosed herein into a CHO cell, wherein the vector nucleic acid does not comprise a coding sequence for ANGPTL3 mimetics, and introducing a further vector nucleic acid suitable for recombinant expression of ANGPTL3 mimetics into the CHO cell, wherein the different vector nucleic acids may be introduced into the CHO cell simultaneously or consecutively, in any order. In even further embodiments, the method for producing a CHO cell comprises the steps of (a) providing a CHO cell which is capable of expressing ANGPTL3 mimetics, and (b) introducing a vector nucleic acid according to the fifth aspect into the CHO cell. In these embodiments, the vector nucleic acid as disclosed herein preferably does not comprise a coding sequence for ANGPTL3 mimetics.

The ANGPTL3 mimetic in particular is a ANGPTL3 mimetic as described herein, e.g., comprising or consisting of an amino acid sequence of SEQ ID NO: 29-50.

The vector nucleic acid is artificially introduced into the CHO cell. In particular, the vector nucleic acid is introduced by transfection. Transfection in this respect may be transient or stable, and especially stable transfection is used. Hence, in certain embodiments the produced CHO cell comprises the expression cassette according to the fifth aspect stably integrated into its genome.

In a further aspect, the present invention provides the use of the expression cassette as disclosed herein or a vector nucleic acid as disclosed herein or the CHO cell as disclosed herein for the production of ANGPTL3 mimetics. The features, embodiments and examples of the method for producing ANGPTL3 mimetics described herein likewise apply to this use.

The present invention further provides the use of the expression cassette according to the fourth aspect or the vector nucleic acid as disclosed herein for improving production of ANGPTL3 mimetics by a CHO cell, including introducing the expression cassette or vector nucleic acid into a CHO cell capable of producing ANGPTL3 mimetics. In certain embodiments, the vector nucleic acid introduced into the CHO cell does not comprise a coding sequence for ANGPTL3 mimetics. Improving production of an ANGPTL3 mimetic in particular includes increasing the purity of the ANGPTL3 mimetic. The features, embodiments and examples of the method of improving production of an ANGPTL3 mimetic in a CHO cell described herein likewise apply to this use.

6. Compositions comprising ANGPTL3 mimetics

In one aspect, the present invention provides a composition comprising an ANGPTL3 mimetic and CCL2, wherein the composition is obtained by production using CHO cells according to the second aspect, and wherein: (i) the amount of CCL2 in the composition is at least 10-times lower compared to the same composition obtained by production using CHO cells which were not engineered to reduce the production of CCL2 in the CHO cells; and/or

In certain embodiments, the composition is obtained by step (c1) of the method of producing an ANGPTL3 mimetics according to the first aspect of the invention. In these embodiments, the composition in particular may be a cell culture supernatant or a cell-free bulk harvest of a cell culture. The composition according to these embodiments in particular may comprise CCL2 in a concentration of 100 ppm or lower, preferably 25 ppm or lower, more preferably 10 ppm or lower.

In further embodiments, the composition is obtained by step (c4) of the method of producing an ANGPTL3 mimetic according to the first aspect of the invention. In these embodiments, the composition in particular may be a purified composition essentially free of proteins other than the ANGPTL3 mimetic. A composition essentially free of proteins other than the ANGPTL3 mimetic in particular comprises 5% or less, preferably 2% or less, more preferably 1% or less of proteins other than a ANGPTL3 mimetic relative to the entire amount of protein in the composition. The composition according to these embodiments in particular may comprise CCL2 in a concentration of 25 ppm or lower, preferably 10 ppm or lower, more preferably 5 ppm or lower.

In further embodiments, the composition is obtained by step (d) of the method of producing an ANGPTL3 mimetic according to the first aspect of the invention. In these embodiments, the composition in particular may be a drug product composition suitable for use in the treatment of patients, especially human patients. The composition according to these embodiments in particular may comprise CCL2 in a concentration of 25 ppm or lower, preferably 10 ppm or lower, more preferably 5 ppm or lower.

In some embodiments, provided herein are compositions comprising an ANGPTL3 mimetic, e.g., an ANGPTL3 mimetic of SEQ ID NO: 43, obtained according to the teaching of the invention disclosed herein comprising about 20-60mg, about 10mg, about 11 mg, about 12mg, about 13mg, about 14mg, about 15mg, about 16mg, about 17mg, about 18mg, about 19mg, about 20mg, about 21 mg, about 22mg, about 23mg, about 24mg, about 25mg, about 26mg, about 27mg, about 28mg, about 29mg, about 30mg, about 31 mg, about 32mg, about 33mg, about 34mg, about 35mg, about 36mg, about 37mg, about 38mg, about 39mg, about 40mg, about 41 mg, about 42mg, about 43mg, about 44mg, about 45mg, about 46mg, about 47mg, about 48mg, about 49mg, about 50mg, about 51 mg, about 52mg, about 53mg, about 54mg, about 55mg, about 56mg, about 57mg, about 58mg, about 59mg, about 60mg of an ANGPTL3 mimetic for use in the treatment of arthritis or cartilage damage in a human subject. In some embodiments the herein provided composition comprising an ANGPTL3 mimetic, e.g., an ANGPTL3 mimetic of SEQ ID NO: 43, obtained according to the teaching of the invention disclosed herein, can be used in the treatment of arthritis or cartilage damage in a human subject, wherein said composition is administered intra-articularly and comprises a dose of about 20-40 mg of said ANGPTL3 mimetic.

In another embodiment the disclosed composition can be used in the treatment of arthritis or cartilage damage in a human subject, wherein said composition is administered intra-articularly and comprises a dose of about 20 mg of the ANGPTL3 mimetic consisting of a protein sequence of SEQ ID NO: 43.

In an additional embodiment the disclosed composition can be used in the treatment of arthritis or cartilage damage in a human subject, wherein said composition is administered intraarticularly and comprises a dose of about 40 mg of the ANGPTL3 mimetic consisting of a protein sequence of SEQ ID NO: 43.

7. Specific embodiments

In the following, specific embodiments of the present invention are described. These embodiments can be combined with the further embodiments, features and examples described herein.

Embodiment 1. A method of producing an ANGPTL3 mimetics, comprising the steps of:

(a) providing CHO cells capable of producing anANGPTL3 mimetic;

(b) cultivating the CHO cells in a cell culture under conditions which allow for the production of theANGPTL3 mimetic;

(c) obtaining the ANGPTL3 mimetic from the cell culture; and

(d) optionally further processing the ANGPTL3 mimetic; wherein the CHO cells are engineered to reduce the production of CCL2 in the CHO cells.

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

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

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

Embodiment 6. The method according to embodiment 5, wherein the expression cassette comprises an intronic sequence comprising a template sequence for a pri-miRNA, wherein the pri-miRNA is suitable to be processed in the CHO cell to form the miRNA targeting CCL2.

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

(i) a 5' miRNA scaffold stem, optionally comprising the 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, optionally comprising the nucleotide sequence of SEQ ID NO: 8,

(iv) a guide strand, having the nucleotide sequence of the miRNA, and

(v) a 3' miRNA scaffold stem, optionally comprising the nucleotide sequence of SEQ ID NO: 11-14; wherein the positions of the passenger strand and guide strand may 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.

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 the pri-miRNAs are suitable to be processed in the CHO cell to form miRNAs targeting CCL2, wherein the miRNAs processed from the pri-miRNAs bind to different parts of the RNA, especially the mRNA or pre-mRNA, of CCL2.

Embodiment 9. The method according to embodiment 8, wherein the different miRNAs targeting CCL2 have nucleotide sequences selected from the group consisting of SEQ ID NO: 26.

Embodiment 10. A method of producing an ANGPTL3 mimetic, comprising the steps of: (a) providing CHO cells capable of producing an ANGPTL3 mimetic;(b) cultivating the CHO cells of step (a) in a cell culture under conditions which allow for the production of the ANGPTL3 mimetic;

(c) obtaining the ANGPTL3 mimetic from the cell culture of step (b); and

(d) optionally processing the ANGPTL3 mimetic obtained in step (c).

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

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

(c1) separating the CHO cells from cell culture supernatant containing the ANGPTL3 mimetic;

(c2) separating the ANGPTL3 mimetic from the cell culture supernatant using cation exchange chromatography;

(c3) further purifying ANGPTL3 mimetics or the variant thereof using anion exchange chromatography; and

(c4) further purifying the ANGPTL3 mimetic using hydrophobic interaction chromatography.

Embodiment 13. The method according to any one of embodiments 1 to 12, wherein step (d) comprises providing a pharmaceutical formulation comprising the produced ANGPTL3 mimetic.

Embodiment 14. The method according to any one of embodiments 1 to 13, wherein the method is for producing a variant of ANGPTL3 mimetic which has one or more of the following characteristics:

(i) it has an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-50.

Embodiment 15. The method according to any one of embodiments 1 to 14, wherein the method is for producing a ANGPTL3 mimetic comprising the amino acid sequence of SEQ ID NO:43.

Embodiment 16. The method according to any one of embodiments 1 to 15, wherein the method is for producing a variant of ANGPTL3 mimetics consisting of the amino acid sequence of SEQ ID NO: 43. Embodiment 17. The method according to any one of embodiments 1 to 16, wherein the ANGPTL3 mimetic obtained after step (c) is in a composition which comprises CCL2 in a concentration of 100 ppm or lower, preferably 25 ppm or lower, more preferably 10 ppm or lower.

Embodiment 18. The method according to any one of embodiment 12, wherein the ANGPTL3 mimetic obtained after step (c1) is in a composition which comprises CCL2 in a concentration of 100 ppm or lower, preferably 25 ppm or lower, more preferably 10 ppm or lower.

Embodiment 19. The method according to any one of embodiment 12, wherein the ANGPTL3 mimetic obtained after step (c4) is in a composition which comprises CCL2 in a concentration of 25 ppm or lower, preferably 10 ppm or lower, more preferably 5 ppm or lower.

Embodiment 20. The method according to any one of embodiments 1 to 19, wherein the ANGPTL3 mimetic obtained after step (d) is in a composition which comprises CCL2 in a concentration of 25 ppm or lower, preferably 10 ppm or lower, more preferably 5 ppm or lower.

Embodiment 21. The method according to any one of embodiments 1 to 20, wherein the ANGPTL3 mimetics obtained after step (c) is in a composition which comprises CCL2 in a concentration which is at least 10-times lower, preferably 25-times lower, more preferably 100- times lower, compared to a composition obtained with the same method after step (c) using CHO cells which are not engineered to reduce the production of CCL2 in the CHO cells.

Embodiment 22. The method according to any one of embodiments 1 to 20, wherein the ANGPTL3 mimetic obtained after step (c1) is in a composition which comprises CCL2 in a concentration which is at least 10-times lower, preferably 25-times lower, more preferably 100- times lower, compared to a composition obtained with the same method after step (c1) using CHO cells which are not engineered to reduce the production of CCL2 in the CHO cells.

Embodiment 23. The method according to any one of embodiments 1 to 20, wherein the ANGPTL3 mimetic obtained after step (c4) is in a composition which comprises CCL2 in a concentration which is at least 10-times lower, preferably 25-times lower, more preferably 100- times lower, compared to a composition obtained with the same method after step (c4) using CHO cells which are not engineered to reduce the production of CCL2 in the CHO cells.

Embodiment 24. The method according to any one of embodiments 1 to 20, wherein the ANGPTL3 mimetic obtained after step (d) is in a composition which comprises CCL2 in a concentration which is at least 10-times lower, preferably 25-times lower, more preferably 100- times lower, compared to a composition obtained with the same method after step (d) using CHO cells which are not engineered to reduce the production of CCL2 in the CHO cells.

Embodiment 25. A CHO cell which is capable of producing an ANGPTL3 mimetic and which is engineered to reduce the production of CCL2 in the CHO cell. Embodiment 26. The cell according to embodiment 25, wherein production of CCL2 is reduced by knockdown or knockout of CCL2 expression or degradation of CCL2 protein.

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

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.

Embodiment 29. The cell according to embodiment 27 or 28, comprising an expression cassette for production of the miRNA targeting CCL2.

Embodiment 30. The cell according to embodiment 29, wherein the expression cassette comprises an intronic sequence comprising a template sequence for a pri-miRNA, wherein the pri-miRNA is suitable to be processed in the CHO cell to form the miRNA targeting CCL2.

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

(iv) a guide strand, having the nucleotide sequence of the miRNA, and

Embodiment 32. The cell according to any one of embodiments 29 to 31 , wherein the expression cassette comprises two or more template sequences for pri-miRNAs, wherein the pri-miRNAs are suitable to be processed in the CHO cell to form miRNAs targeting CCL2, wherein the miRNAs processed from the pri-miRNAs bind to different parts of the RNA, especially the mRNA or pre-mRNA, of CCL2. Embodiment 33. The cell according to embodiment 32, wherein the different miRNAs targeting CCL2 have nucleotide sequences selected from the group consisting of SEQ ID NO: 26.

Embodiment 34. The cell according to any one of embodiments 25 to 33, capable of producing an ANGPTL3 mimetic which has one or more of the following characteristics:

(i) it has an amino acid sequence selected from the group consisting of SEQ ID NOs: 29- 50.

Embodiment 35. The cell according to any one of embodiments 25 to 34, capable of producing the ANGPTL3 mimetic comprising the amino acid sequence of SEQ ID NO: 43

Embodiment 36. The cell according to any one of embodiments 25 to 35, capable of producing the ANGPTL3 mimetic consisting of the amino acid sequence of SEQ ID NO: 43

Embodiment 37. A method of improving production of an ANGPTL3 mimetic in a CHO cell, comprising the steps of

(a-i) providing a CHO cell capable of producing an ANGPTL3 mimetic;

Embodiment 38. The method according to embodiment 37, wherein the CHO cell is engineered in step (a-ii) by knockdown or knockout of CCL2 expression or inducing degradation of CCL2 protein in the CHO cell.

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 the endogenous CCL2 of the CHO cells.

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.

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

Embodiment 42. The method according to embodiment 41 , wherein the expression cassette comprises an intronic sequence comprising a template sequence for a pri-miRNA, wherein the pri-miRNA is suitable to be processed in the CHO cell to form the miRNA targeting CCL2.

Embodiment 43. The method according to embodiment 42, wherein the pri-miRNA comprises, from 5' to 3', (i) a 5' miRNA scaffold stem, optionally comprising the nucleotide sequence of SEQ ID NO: 1-4,

(iv) a guide strand, having the nucleotide sequence of the miRNA, and

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

Embodiment 45. The method according to embodiment 44, wherein the different miRNAs targeting CCL2 have nucleotide sequences selected from the group consisting of SEQ ID NO: 26.

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

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 prior to step (a-ii).

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 prior to step (a-ii). Embodiment 49. The method according to any one of embodiments 37 to 48, wherein the method further comprises the steps of:

(b) cultivating the CHO cell obtained in step (a-ii) in a cell culture under conditions which allow for proliferation of the CHO cell and simultaneous and/or subsequent production of an ANGPTL3 mimetic;

(c) obtaining said ANGPTL3 mimetic from the cell culture; and

(d) optionally further processing the ANGPTL3 mimetic.

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

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

(c1) separating the CHO cells from cell culture supernatant containing an ANGPTL3 mimetic;

(c3) further purifying the ANGPTL3 mimetic using anion exchange chromatography; and

Embodiment 52. The method according to any one of embodiments 49 to 51 , wherein step (d) comprises providing a pharmaceutical formulation comprising an ANGPTL3 mimetic.

Embodiment 53. The method according to any one of embodiments 49 to 52, wherein the ANGPTL3 mimetic obtained after step (c) is in a composition which comprises CCL2 in a concentration of 100 ppm or lower, preferably 25 ppm or lower, more preferably 10 ppm or lower.

Embodiment 54. The method according to any one of embodiments 51 to 53, wherein the ANGPTL3 mimetic obtained after step (c1) is in a composition which comprises CCL2 in a concentration of 100 ppm or lower, preferably 25 ppm or lower, more preferably 10 ppm or lower.

Embodiment 55. The method according to any one of embodiments 51 to 54, wherein the ANGPTL3 mimetic obtained after step (c4) is in a composition which comprises CCL2 in a concentration of 25 ppm or lower, preferably 10 ppm or lower, more preferably 5 ppm or lower. Embodiment 56. The method according to any one of embodiments 49 to 55, wherein the ANGPTL3 mimetic obtained after step (d) is in a composition which comprises CCL2 in a concentration of 25 ppm or lower, preferably 10 ppm or lower, more preferably 5 ppm or lower.

Embodiment 57. The method according to any one of embodiments 49 to 56, wherein the ANGPTL3 mimetic obtained after step (c) is in a composition which comprises CCL2 in a concentration which is at least 10-times lower, preferably 25-times lower, more preferably 100- times lower, compared to a composition obtained with the same method after step (c) using a CHO cell which is not engineered to reduce the production of CCL2 in the CHO cell.

Embodiment 58. The method according to any one of embodiments 51 to 57, wherein the ANGPTL3 mimetic obtained after step (c1) is in a composition which comprises CCL2 in a concentration which is at least 10-times lower, preferably 25-times lower, more preferably 100- times lower, compared to a composition obtained with the same method after step (c1) using CHO cells which are not engineered to reduce the production of CCL2 in the CHO cells.

Embodiment 59. The method according to any one of embodiments 51 to 58, wherein the ANGPTL3 mimetic obtained after step (c4) is in a composition which comprises CCL2 in a concentration which is at least 10-times lower, preferably 25-times lower, more preferably 100- times lower, compared to a composition obtained with the same method after step (c4) using CHO cells which are not engineered to reduce the production of CCL2 in the CHO cells.

Embodiment 60. The method according to any one of embodiments 49 to 59, wherein the ANGPTL3 mimetic obtained after step (d) is in a composition which comprises CCL2 in a concentration which is at least 10-times lower, preferably 25-times lower, more preferably 100- times lower, compared to a composition obtained with the same method after step (d) using CHO cells which are not engineered to reduce the production of CCL2 in the CHO cells.

Embodiment 61. The method according to any one of embodiments 37 to 60, wherein the method is for improving production of an ANGPTL3 mimetic which has one or more of the following characteristics:

Embodiment 62. The method according to any one of embodiments 37 to 61 , wherein the method is for improving production of an ANGPTL3 mimetic comprising the amino acid sequence of SEQ ID NO: 43.

Embodiment 63. The method according to any one of embodiments 37 to 62, wherein the method is for improving production of an ANGPTL3 mimetic consisting of the amino acid sequence of SEQ ID NO: 43. Embodiment 64. An expression cassette for expression of a miRNA in a CHO cell, comprising a template sequence for a pri-miRNA, wherein the pri-miRNA is suitable to be processed in a CHO cell to form a miRNA targeting CCL2, wherein the miRNA targeting CCL2 has a nucleotide sequence selected from the group consisting of SEQ ID NO: 26 and, wherein the template sequence for the pri-miRNA is present within an intronic sequence and, wherein the pri-miRNA comprises, from 5' to 3',

(iv) a guide strand, having the nucleotide sequence of the miRNA, and

(v) a 3' miRNA scaffold stem, optionally comprising the nucleotide sequence of SEQ ID NO: 11-14; wherein the positions of the passenger strand and guide strand may 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 and wherein the vector nucleic acid according to embodiment 70, further comprising a coding sequence encoding an ANGPTL3 mimetic.

Embodiment 65. The vector nucleic acid according to embodiment 64, wherein the coding sequence encoding the ANGPTL3 mimetic is present within the same expression cassette as the template sequence for the pri-miRNA.

Embodiment 66. The vector nucleic acid according to embodiment 65, wherein the coding sequence encoding the ANGPTL3 mimetic is present within a further expression cassette present within the vector nucleic acid.

Embodiment 67. The vector nucleic acid according to any one of embodiments 64 to 66, wherein the coding sequence encodes a variant of ANGPTL3 mimetics which has one or more of the following characteristics:

(i) it has an amino acid sequence selected from the group consisting of SEQ ID NOs: 29- 50. Embodiment 68. The vector nucleic acid according to any one of embodiments 64 to 67, wherein the coding sequence encodes an ANGPTL3 mimetic comprising the amino acid sequence of SEQ ID NO: 43.

Embodiment 69. The vector nucleic acid according to any one of embodiments 64 to 67, wherein the coding sequence encodes an ANGPTL3 mimetic consisting of the amino acid sequence of SEQ ID NO: 43.

Embodiment 70. A method for producing a CHO cell which is capable of expressing an ANGPTL3 mimetic, comprising introducing a vector nucleic acid according to any one of embodiments 64 to 69 into a CHO cell.

Embodiment 71. The method according to embodiment 70; wherein the CHO cell does not comprise a coding sequence encoding an ANGPTL3 mimetic prior to introduction of the vector nucleic acid; and wherein a vector nucleic acid according to any one of embodiments 65 to 69 is introduced into the CHO cell.

Embodiment 73. The method according to embodiment 70; wherein a vector nucleic acid according to embodiment 64 which does not comprise a coding sequence encoding an ANGPTL3 mimetic is introduced into the CHO cell; and wherein the CHO cell comprises a coding sequence encoding an ANGPTL3 mimetic prior to introduction of the vector nucleic acid.

Embodiment 74. The method according to embodiment 70; wherein a vector nucleic acid according to embodiment 64 which does not comprise a coding sequence encoding an ANGPTL3 mimetic is introduced into the CHO cell; and wherein a further vector nucleic acid comprising a coding sequence encoding an ANGPTL3 mimetic is introduced into the CHO cell; wherein introduction of said two different vector nucleic acids is performed either concomitantly or subsequently, in any order.

Embodiment 75. The method according to any one of embodiments 70 to 74, wherein the CHO cell is capable of expressing an ANGPTL3 mimetic which has one or more of the following characteristics:

(i) it has an amino acid sequence selected from the group consisting of SEQ ID

NOs: 29-50.

Embodiment 76. The method according to any one of embodiments 70 to 75, wherein the CHO cell is capable of expressing an ANGPTL3 mimetic comprising the amino acid sequence of SEQ ID NO: 43. Embodiment 77. The method according to any one of embodiments 70 to 76, wherein the CHO cell is capable of expressing an ANGPTL3 mimetic consisting of the amino acid sequence of SEQ ID NO: 43.

Embodiment 78. A composition comprising an ANGPTL3 mimetic and CCL2, wherein the composition is obtained by production using 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 which were not engineered to reduce the production of CCL2 in the CHO cells.

Embodiment 79. A composition comprising an ANGPTL3 mimetic and CCL2, wherein the composition is obtained by production using CHO cells, and wherein the composition comprises CCL2 in a concentration of 100 ppm or lower, preferably 25 ppm or lower, more preferably 10 ppm or lower.

Embodiment 80. The composition according to embodiment 78, being a composition according to embodiment 79.

Embodiment 81. The composition according to any one of embodiments 78 to 80, being obtained by steps (a), (b) and (c1) of the method according to embodiment 12.

Embodiment 82. The composition according to embodiment 81 , being a cell culture supernatant or a cell-free bulk harvest of a cell culture.

Embodiment 83. The composition according to embodiment 81 or 82, wherein the composition comprises CCL2 in a concentration of 100 ppm or lower, preferably 25 ppm or lower, more preferably 10 ppm or lower.

Embodiment 84. The composition according to any one of embodiments 78 to 80, being obtained by steps (a), (b) and (c) of the method according to any one of embodiments 1 to 24.

Embodiment 85. The composition according to any one of embodiments 78 to 80, being obtained by steps (a), (b), (c1), (c2), (c3) and (c4) of the method according to embodiment 12.

Embodiment 86. The composition according to embodiment 84 or 85, being a purified composition essentially free of proteins other than an ANGPTL3 mimetic.

Embodiment 87. The composition according to any one of embodiments 78 to 80, being obtained by steps (a), (b), (c) and (d) of the method according to any one of embodiments 1 to 24. Embodiment 88. The composition according to any one of embodiments 84 to 87, being a purified composition essentially free of proteins other than an ANGPTL3 mimetic.

Embodiment 89. The composition according to any one of embodiments 84 to 88, wherein the composition comprises 5% or less, preferably 2% or less, more preferably 1% or less of proteins other than an ANGPTL3 mimetic relative to the entire amount of protein in the composition.

Embodiment 90. The composition according to any one of embodiments 84 to 89, wherein the composition comprises CCL2 in a concentration of 25 ppm or lower, preferably 10 ppm or lower, more preferably 5 ppm or lower.

Embodiment 91 . A CHO cell which is engineered to reduce the production of CCL2 in the CHO cell.

Embodiment 92. The cell according to embodiment 91 , wherein production of CCL2 is reduced by knockdown or knockout of CCL2 expression or degradation of CCL2 protein.

Embodiment 93. The cell according to embodiment 91 or 92, which is engineered to produce a miRNA targeting the endogenous CCL2 of the CHO cells.

Embodiment 94. The cell according to embodiment 93, wherein the miRNA targeting CCL2 has a nucleotide sequence selected from the group consisting of SEQ ID NO: 26.

Embodiment 95. The cell according to embodiment 93 or 94, comprising an expression cassette for production of the miRNA targeting CCL2.

Embodiment 96. The cell according to embodiment 95, wherein the expression cassette comprises an intronic sequence comprising a template sequence for a pri-miRNA, wherein the pri-miRNA is suitable to be processed in the CHO cell to form the miRNA targeting CCL2.

Embodiment 97. The cell according to embodiment 96, wherein the pri-miRNA comprises, from 5' to 3',

(iv) a guide strand, having the nucleotide sequence of the miRNA, and (v) a 3' miRNA scaffold stem, optionally comprising the nucleotide sequence of SEQ ID NO: 11-14; wherein the positions of the passenger strand and guide strand may 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.

Embodiment 98. The cell according to any one of embodiments 95 to 97, wherein the expression cassette comprises two or more template sequences for pri-miRNAs, wherein the pri-miRNAs are suitable to be processed in the CHO cell to form miRNAs targeting CCL2, wherein the miRNAs processed from the pri-miRNAs bind to different parts of the RNA, especially the mRNA or pre-mRNA, of CCL2.

Embodiment 99. The cell according to embodiment 98, wherein the different miRNAs targeting CCL2 have nucleotide sequences selected from the group consisting of SEQ ID NO: 26.

Embodiment 100. The composition according to any one of embodiments 84 to 90, wherein the composition is for use in the treatment of arthritis or cartilage damage in a human subject, wherein the composition is administered intra-articularly.

Embodiment 101 : The composition for use in the treatment of arthritis or cartilage damage according to embodiment 100, wherein the subject has arthritis, e.g., osteoarthritis, trauma arthritis or autoimmune arthritis.

Embodiment 102: A method of treating arthritis or cartilage damage in a subject (e.g., a human subject) comprising administering to a joint of the subject an intra-articular dose of a composition according to any one of embodiments 84 to 90.

Embodiment 103: The method according to embodiment 102, wherein the subject has arthritis, e.g., osteoarthritis, trauma arthritis or autoimmune arthritis.

Embodiment 104: A method of improving production of an ANGPTL3 mimetic in a CHO cell, comprising the steps of

(a-i) providing a CHO cell capable of producing an ANGPTL3 mimetic;

(a-ii) engineering the CHO cell so as to reduce the production of CCL2 in the CHO cell, wherein the CHO cell is engineered in step (a-ii) by knockout of CCL2 expression. Embodiment 105: A method of improving production of an ANGPTL3 mimetic in a CHO cell according to embodiment 104, comprising the use of single gRNA pairs for the targeting of a dCas9 protein fused to the nuclease Clo51 (Cas-CLOVER™) to the CCL2 gene on exons 1 and 2.

Embodiment 106: A method according to embodiments 104 and 105, wherein gRNAs comprising the nucleic acid sequences of SEQ ID NO: 51-54 (gRNA CCL2-01 , -02, -03, -04) respectively, are used.

Embodiment 107: A method according to embodiments 104 and 105, wherein the gRNAs gSA099 (SEQ ID NO: 61), gSA100 (SEQ ID NO: 62), gSA101 (SEQ ID NO: 63), gSA102 (SEQ ID NO: 64), gSA103 (SEQ ID NO: 65), gSA104 (SEQ ID NO: 66), gSA105 (SEQ ID NO: 67), gSA106 (SEQ ID NO: 68) targeting dCas9 protein fused to the nuclease Clo51 (Cas- CLOVER™) to the 5' or 3' flanking regions or to the 5'IITR or 3'IITR of the CCL2 gene.

FIGURES

Figure 1 shows the production and purification process of ANGPTL3 mimetic consisting of protein sequence of SEQ ID NO: 43.

Figure 2 shows the elution profile of the ANGPTL3 mimetic consisting of protein sequence of SEQ ID NO: 43 and CCL2 from the cation exchange capture chromatography step. CCL2 levels were detected by CCL2 ELISA. Triangles are used to describe the ANGPTL3 mimetic titer. Squares are used to describe the CCL2 concentrations.

Figure 3 shows cell line engineering and development strategy for the CCL2 knockdown approach.

Figure 4 shows cell viabilities (dotted lines) and viable cell densities of the different host cells during an a fed-batch cultivation in 7L bioreactors. The CCL2 knockdown pool (CCL2_A) shows similar viable cell densities as compared to control (ANGPTL3 mimetic consisting of protein sequence of SEQ ID NO: 43 cell line).

Figure 5 shows ANGPTL3 mimetic consisting of protein sequence of SEQ ID NO: 43 titers in the different host cells. Harvest titer of CCL2 KD (knock-down) pool is comparable to control cell line.

Figure 6 shows relative mRNA expression of ANGPTL3 mimetic consisting of protein sequence of SEQ ID NO: 43, CCL2 and control gene. At day 10 of the 24 deep-well plate fed- batch, samples were taken and RNA was purified. ANGPTL3 mimetic consisting of protein sequence of SEQ ID NO: 43, CCL2 and control gene transcripts were quantified using qPCR. CCL2 KD pools show significantly lower CCL2 transcript levels as compared to controls.

Figure 7A shows CCL2 protein levels (concentration) quantified using an CCL2 ELISA. CCL2 KD pools reveal lower CCL2 protein levels in non-purified harvest at day 10 of a 24 deep-well plate fed-batch run.

Figure 7B shows CCL2 protein levels (ppm) quantified using an CCL2 ELISA. CCL2 KD pools reveal lower CCL2 protein levels in non-purified harvest at day 14 of 7L bioreactor fed-batch run.

Figure 8 shows ANGPTL3 mimetic consisting of protein sequence of SEQ ID NO: 43 titers of CCL2 KD single cell clones. CCL2_A-encoding pools were used for single cell cloning. 120 clones were inoculated into a 24dwp fed-batch process and ANGPTL3 mimetic consisting of protein sequence of SEQ ID NO: 43 harvest titers were assessed at day 14. As expected, some clones show higher and others lower titers as compared to originating pools and the ANGPTL3 mimetic consisting of protein sequence of SEQ ID NO: 43 cell line controls.

Figure 9 shows CCL2 mRNA levels of CCL2 KD single cell clones. CCL2_A-encoding pools were used for single cell cloning. 120 clones were inoculated into a 24dwp fed-batch process and at day 10 of the fed-batch, CCL2 mRNA levels were quantified using qPCR. The majority of clones show significantly reduced levels of CCL2 transcripts as compared to the ANGPTL3 mimetic consisting of protein sequence of SEQ ID NO: 43 cell line control (normalized to 1).

Figure 10 shows a schematic description of the wildtype and expected knockout CCL2 genotypes annotated with used gRNA pairs (gSA101/102 and gSA103/SA104) binding the 5'UTR and 3' flanking region of the CCL2 gene. A) The wildtype CCL2 gene consists of three exons encoding the CCL2 gene and resulting in functional CCL2 gene expression. B) The expected CCL2 knockout genotype consists only a small fragment of the 5'UTR eliminating the expression of functional CCL2 gene.

Figure 11 shows CCL2 concentration in supernatant of knockout clones and control clone (non-engineered ANGPTL3 Master Cell Bank (MCB) during fed-batch production process at day 7. Asterisks mark clones with CCL2 concentrations below the limit of quantification of < 5ng/mL.

Figure 12: ANGPTL3 mimetic (protein sequence of SEQ ID NO: 43) titers were assessed at day 10 during fed-batch production process in supernatant of knockout clones and control clone (non-engineered ANGPTL3 MCB).

Figure 13: Chromatogram of the elution phase of the pseudo-affinity resin (Cellufine DexS- HbP) and CCL2 content pattern in fractions. EXAMPLES

Example 1 : Identification of CCL2 as critical impurity of a human ANGPTL3 mimetics variant recombinantly produced in CHO cells.

A variant of human ANGPTL3 mimetic having the amino acid sequence of SEQ ID NO: 43 in the following "ANGPTL3 mimetic of SEQ ID NO: 43") was produced by a CHO production cell line (“ANGPTL3 mimetic of SEQ ID NO: 43 cell line”) and isolated from the cell culture using a standard purification process. The ANGPTL3 mimetic of SEQ ID NO: 43 drug product purification process followed a 3-chromatography step process, which includes cation, anion exchange chromatography and hydrophobic interaction chromatography, virus inactivation by detergent, virus removal filtration and two ultrafiltration/diafiltration steps (Figure 1).

Applying the purification process for ANGPTL3 mimetic of SEQ ID NO: 43 showed depletion of host cell proteins (HCPs) using an ELISA assay specific for multiple HCPs. Analysis of the individual HCPs using an orthogonal LC-MS method revealed one prominent protein: CCL2 (CC-chemokine ligand 2; also known as MCP-1 (monocyte chemoattractant protein 1)), which was not detected by ELISA.

Table 1 : Amount of CCL2 in the purified product

A further analysis of the downstream process (DSP) of ANGPTL3 mimetics of SEQ ID NO: 43 production showed that the elution profile of CCL2 in the CEC capture step overlapped significantly with the elution profile of ANGPTL3 mimetics of SEQ ID NO: 43 (Figure 2).

Example 2: miRNA vector design.

The intronic-miRNA encoding vectors (pCMV04_A and pCMV04_B) were based on a standard vector (pCMV). The vector was modified by insertion of the intronic miRNA sequences into the cloning site of the CMV promoter-driven expression cassette. The sequence environment selected for the miRNA scaffold is similar to the human miR-30A and the miR-E molecules (e.g. Fellmann et al., 2011 , Molecular Cell 41 , 733-746) and is expected to lead to optimal processing of resulting miRNA sequences. However, the sequence was further modified: i) the EcoRI restriction site was replaced with Bglll restriction site, ii) the sequence downstream of the Xhol restriction site was replaced with a CHO-derived sequence and iii) additional miR- 30A scaffold was added up- and downstream of the published sequence. miRNA sequence targeting endogenous CCL2 of the CHO cells was used: CCL2_A AIM (artificial intronic miRNA) comprising a guide strand having the nucleotide sequence of SEQ ID NO: 26 and a passenger strand having the nucleotide sequence of SEQ ID NO: 25. In addition, a vector with an AIM targeting an unrelated endogenous protein of the CHO cells was used as control.

Example 3: CCL2 knockdown cell line generation.

The strategy of CCL2 KD cell line generation is shown in Figure 3. The CHO parental cell line was transfected with an expression vector encoding a ANGPTL3 mimetics of SEQ ID NO: 43 and selection of pools were performed using G418 and MTX. The pools were going into single cell cloning and a monoclonal cell line expressing ANGPTL3 mimetics of SEQ ID NO: 43 was selected, called ANGPTL3 mimetics of SEQ ID NO: 43 cell line. Subsequently, the Master Cell Bank (MCB) of the ANGPTL3 mimetics of SEQ ID NO: 43 cell line was generated and one vial was used to transfect a vector encoding the artificial intronic miRNA targeting endogenous CCL2 and pools were generated using puromycine. miRNAs were generated targeting CHO CCL2 mRNA called CCL2_A, targeting the 3'IITR of the transcript. For pool generations, a miRNA targeting a different gene (control AIM), the parental ANGPTL3 mimetics of SEQ ID NO: 43 cell line as well as the empty parental cell line (CHO) as controls were included. All samples (triplicate pool generations for the knockdown approaches) were inoculated into an optimized fed-batch run and cell growth, gene expression and ANGPTL3 mimetics of SEQ ID NO: 43 titers were assessed at different days (Figures 4 to 6). Also, the CCL2 protein levels on harvest level were assessed using an CCL2 ELISA (Figure 7):

After pool generations and confirmation of an efficient CCL2 knockdown on mRNA level, the CCL2_A pools were used for single cell cloning and 120 expanded clones were inoculated into a 24dwp standard fed-batch to assess CCL2 knockdown efficiencies and ANGPTL3 mimetics of SEQ ID NO: 43 productivities (Figures 8 and 9).

Example 4: ANGPTL3 mimetic CCL2 knockout cell line generation.

Gene-editing technologies represent alternative methods of the vectorized RNAi approach using artificial intronic miRNAs. A gene knockout on the DNA level which encodes the CCL2 gene leads to a complete functional deactivation due to loss of essential gene information. We generated single gRNA pairs for the targeting of Cas-CLOVER™ to the CCL2 gene on exons 1 and 2 (guide CCL2-01 : SEQ ID NO: 51 ; guide CCL2-02: SEQ ID NO: 52; guide CCL2-03: SEQ ID NO: 53; guide CCL2-04: SEQ ID NO: 54). Additionally, we generated single gRNA pairs for targeting of Cas-CLOVER™ to the 5' or 3' flanking regions or to the 5'UTR or 3'UTR of the CCL2 gene (SEQ IDs gSA099, 100, 101 , 102, 103, 104, 105, 106). Cas-CLOVER™, a dCas9 protein fused to the nuclease Clo51 , can homodimerize at the CCL2 locus leading to insertion and deletion (indel) formation and eventually to frameshift mutations of the CCL2 gene. Complete loss of a CCL2-encoding gene sequence can be achieved by targeting Cas- CLOVER™ to two distant sites either within the CCL2 gene or at adjacent flanking sequences 3'or 5' of the CCL2 gene using two different gRNA pairs.

For the generation of an ANGPTL3 mimetic-producing cell line with a CCL2 gene knockout, cells derived from the ANGPTL3 master cell bank (MCB) were thawed. Cells from ANGPTL3 MCB were seeded into 24-well plates and were cotransfected with mRNA encoding Cas- CLOVER™ and two sgRNA pairs gSA101/gSA102 and gSA103/gSA104 targeting the CCL2 gene in the 5'IITR and in the 3' flanking region, respectively (Figure 10). Three days after transfection, the co-transfection of Cas-CLOVER™ mRNA and gRNA pairs was repeated. 4 days after the second transfection, the cells were transferred to shake flasks and frozen as cryovials. Cells were thawed and passaged and the living cell population was single cell sorted using fluorescently-activated cell sorting (FACS). After a 17 days expansion period in 96-well plates 192 clones were transferred into two 96well plates. 3 days after, cells were diluted and one day later, RNA was isolated for a CCL2-specific RT-qPCR experiment using the primers 3520_CCL2_f (SEQ ID NO: 79), 3521_CCL2_r (SEQ ID NO: 80) and 3522_CCL2_s (SEQ ID NO: 81)) binding the DNA region targeted for deletion between the sgRNA pairs encoding the CCL2 gene. Multiple clones revealed no or only a very weak presence of CCL2 mRNA indicating expected deletion of the CCL2 gene in at least one allele. 24 clones were transferred into 24 deep-well plates and genomic DNA of clonal cells were isolated for a PCR reaction using primers (primer sequences ADD SEQ ID: SA440 (SEQ ID NO: 70) and SA446 ((SEQ ID NO: 76) binding the DNA region targeted for deletion between the sgRNA pairs encoding the CCL2 gene, flanking the target sites of the gRNA pairs. A PCR amplicon with the expected size of the deleted CCL2 genotype (approximately 250bp) was detected in multiple clones, while, in addition, in some of those clones the PCR amplicon size (2385bp) representing the wildtype CCL2 gene was missing. This indicated the homozygous deletion of CCL2 gene sequence because of Cas-CLOVER™ gene editing. To confirm absence of CCL2 protein in supernatants of cells, the 24 clones were inoculated into a 14-days 24 deep-well plate fed- batch production process. Samples were taken at day 7 of the fed-batch and CCL2 protein concentration was measured using ELISA. The CCL2 concentration was below the limit of detection (<5ng/mL) in 12 clones, while the non-engineered ANGPTL3 MCB clone produced high CCL2 levels (>2000ng/mL).

In summary, absence of CCL2 mRNA was confirmed via RT-qPCR, absence of wildtype CCL2 gene sequence and presence of expected genetic deletion was confirmed by the generation of different PCR amplicons on genomic DNA and finally, the absence of CCL2 protein in supernatant of cells during a production process as quantified using ELISA strongly indicates the homozygous deletion of CCL2 gene sequences and therefore a knockout of CCL2 gene in multiple ANGPTL3 CCL2 KO clones. 4.1 Upscaling of production batches of CCL2 knock-out cell lines (Mathias)

From the 12 clones which were previously cultured in 24 deep-well plate fed-batch process and showing CCL2 concentrations below the limit of detection (<5ng/mL), one clone was further processed to generate a CCL2 knock-out Master Cell Bank. One cryovial of this MCB was used to establish a CCL2 knock-out Working Cell Bank (WCB) for upscaled ANGPTL3 mimetic production purposes. The upscaled process consisted of a 1000L bioreactor and corresponding upscaled ANGPTL3 mimetic purification equipment with process steps described in section 1.3 above. For two upscaled batches, CCL2 was determined by ELISA at two specific steps, at completion of step (c1) and at completion of step (d). The CCL2 concentrations for both batches are below limit of quantification at completion of step (c1) (< 0.8 ng/mg) and step (d) (< 0.2 ng/mg). Therefore, the absence of CCL2 is already determined after the completion of the cell culture part (c1) of the upscaled process and confirms the homozygous deletion of CCL2 gene sequences and therefore a knockout of CCL2 gene in the final ANGPTL3 CCL2 KO clone employed under manufacturing conditions.

4.2. Pseudo-affinity resigns - Drug substance purification (Dominik)

As observed during the purification process, the host cell protein CCL2 is not separated from the ANGPTL3 mimetics by the established chromatography steps, especially by anion exchange chromatography or hydrophobic interaction chromatography. It is assumed that this lack of separation is caused by highly similar physicochemical properties of CCL2 and the ANGPTL3 mimetics, in particular by a similar charge distribution. Therefore, as an alternative purification method to anion exchange or hydrophobic interaction chromatography was developed. The novel method is based on non-animal derived pseudo-affinity chromatography (ALC) - Cellufine DexS-HbP. Cellufine MAX DexS is a chromatography resin incorporating Dextran sulfate polymer surface modification. This ligand can be used instead of immobilized Heparin for protein purification. Elution pattern of CCL2 in chromatograms are displayed for Cellufine MAX DexS-HbP - see Figure 13 and CCL2 content of eluate fractions are listed in Table 2. CCL2 showed a stronger binding on Cellufine DexS-HbP than the ANGPTL3 mimetics and was eluting at the descending part contrary to the elution behavior on the cation exchange resin in step (c2) (see Figure 2). As load material the cation exchange chromatography eluate of step (c2) was used (content CCL2 400ng/mg). Cellufine DexS-HbP chromatography resulted in a CCL2 content in the eluate of 37.5ng/mg, i.e. a reduction of 90.6% and a yield of 100%. In the ascending elution fractions (1-3) the CCL2 content was <LOQ. By peak cutting optimization a CCL2 depletion of 95.9% (16.4ng/mg in eluate) and a yield of 94.1% was reached. In Table 3 the chromatographic parameters for pseudo-affinity chromatography (ALC) are listed. The pseudo-affinity method confirms to be an effective alternative purification step for CCL2. Figure 13 shows the elution profile of the ANGPTL3 mimetic consisting of protein sequence of SEQ ID NO: 43 and CCL2 from the pseud-affinity chromatography step (Cellufine DexS- HbP). CCL2 levels were detected by CCL2 ELISA. UV280nm signal indicates the eluting ANGPTL3 mimetic. Dots are used to describe the CCL2 concentrations in the eluate fractions.

Table 2: Amount of CCL2 in eluate fractions (ALC.E) of the pseudo-affinity chromatography (ALC)

Table 3: Chromatographic parameters for pseudo-affinity chromatography (ALC)

Step in unit Buffer Target Comment operation column volume

(CV)

Equilibration Equilibration buffer: 5

20.0 mM succinic acid, 35.1 mM sodium hydroxide, pH 6.0

Load Cation exchange elute; diluted 1 :4 in n.a. Load ratio: 25 gAPi/Lresin

H₂O

Wash Equilibration buffer: 6

20.0 mM succinic acid, 35.1 mM sodium hydroxide, pH 6.0 Step in unit Buffer Target Comment operation column volume

(CV)

Elution Equilibration buffer: 20.0 mM succinic 15 Gradient from 0% acid, 35.1 mM sodium hydroxide, pH 6.0 elution buffer to 100% elution buffer over 15 .. , ,, CV. Peak cut criterion

Elution buffer: for start collection 0.25

20 mM Na succinate (20.0 mM succinic AU₂₁O _NM/_{CM PEAK CUT} acid, 1 .00 M sodium chloride, 38.1 mM criterion for end sodium hydroxide, pH 6.0 collection 0.25

AU28O nm/C

Post elution 1 M sodium chloride 3 wash

CIP 0.5 M NaOH 3

Neutralizatio Equilibration buffer: 20.0 mM succinic 5 acid, 35.1 mM sodium hydroxide, pH 6.0

Storage 20%Et OH+150mM NaCI n.a. not applicable

Example 5: Materials and methods.

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

1 . Expression vector construction The vectors used in the examples consist of following elements: hCMV promoter/enhancer driving expression of the individual genes, polyadenylation signal (polyA), folic acid receptor, DHFR, puromycin and hygromycin resistance genes as selection markers, E.Coli origin (ColE ori) of replication and the beta-lactamase gene for ampicillin (amp) resistance to enable amplification in bacteria. Different plasmid setups were evaluated and more details are provided within the figures.

2. Cell lines, cultivation, transfection and selection

CHO cell lines were cultivated in 24-deep well plates or shake flasks in a non-humidified shaker cabinet at 300 rpm (24dwp) or 150 rpm (shake flasks), 10% CO2 at 36.5°C in suspension in proprietary, chemically defined culture media. Cell viabilities and growth rates were monitored by means of an automated system (ViCell, Beckman Coulter) or using an analytical flow cytometry (CytoFlex, Beckman Coulter). Cells were passaged 2-3 times per week into fresh medium and were maintained in logarithmic growth phase.

Linearized expression vectors were transfected by electroporation (Amaxa Nucleofection system, Lonza, Germany). The transfection reaction was performed in chemically defined cultivation medium, according to the manufactures instructions. The parental CHO cells used for transfection were in exponential growth phase with cell viabilities higher than 95%. Transfections were performed with 5x 10⁶ cells per transfection. Immediately, after transfection cells were transferred into shake flasks, containing chemically defined cultivation medium. Cell pools were incubated for 48 hours at 36.5°C and 10% CO2 before starting the selection process.

A selection procedure was carried out using the selection markers encoded by the individual expression vectors, as described above. The proteins FoIR and DHFR are participating in the same molecular pathway; the FoIR is transporting folic acid as well as the folate analogue MTX into the cell, the DHFR is converting it into vital precursors for purine and methionine synthesis. Combining them as selective principle, a particular strong selective regime can be taken to enrich for recombinant cells expressing both recombinant protein. Puromycin selection is driven by its inhibition of protein synthesis and vectors encoding the puromycin resistance marker gene enable cells to survive in presence of puroymycin.

48 h after transfection and growth under low folate conditions, additional selective pressure was applied by adding 10 nM MTX to the chemically defined cultivation medium. Alternatively, puromycin was used as selection agent. 48h after transfection 0.003mg/mL puromycin was added to the chemically defined cultivation medium. After pool recovery cells were frozen in culture medium, supplemented with 7.5% DMSO and cell pellets prepared.

3. Gene expression analysis by quantitative real-time PCR

RNA extraction was performed using the Qiagen RNeasy Mini Kit according to the manufactures instructions. For real-time qPCR, cDNA was synthesized from 200 ng/pl diluted RNA using the High Capacity RNA-to-cDNA Master Mix (Applied Biosystems) and 10x diluted cDNAs were analyzed in triplicates using the QuantiFast SYBR Green PCR Kit (Qiagen) or TaqMan Primer/Probe system and TaqMan Mastermix (Applied Biosystems). As endogenous control for normalization GAPDH was amplified. Amplification and analysis was performed using the ABI PRISM® 7900HT Sequence Detection System. For calculation of relative quantities (RQ) of gene expression for sample comparison the comparative 2^-AACt method was used and the data normalized. 4. Upstream processing

Subsequent to selection, material was produced either in shake flask fed batch, 24-deep well plate cultures or ambr15 bioreactors. Fed batch cultures were inoculated with a cell seeding density of 4E5 vc/ml (addition of proprietary feed solutions starting on day 3 and cultivation temperature shift to 33°C on day 5). During the cultivation in-process controls were performed to monitor the concentration of ANGPTL3 mimetics of SEQ ID NO: 43. Cell culture samples for RNA isolation were taken at day 10 of the process. The individual culture was cultivated over a period of 14 days. At the end of the cultivation process cells were separated from the culture supernatant by centrifugation and/or depth filtration followed by sterile filtration before further downstream processing.

5. CCL2 protein analysis by ELISA

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

6. CCL2 determination by Gyroslab device

The amount of Chinese hamster (CHO) host cell protein “CC-chemokine ligand 2” (CCL2) was determined using a Gyrolab microfluidic device implementing a sandwich ELISA format. CCL2 was captured on the microfluidic disc system using a biotinylated anti-CCL2 antibody as primary antibody. Bound CCL2 was quantified applying a secondary, fluorophore labelled anti- CCL2 antibody (detection antibody). The CCL2 levels in samples were calculated based on CHO CCL2 standard.

7. CCL2 determination by LC-MS

The amount of Chinese hamster (CHO) host cell protein “CC-chemokine ligand 2” (CCL2) was determined using LC-MS absolute quantification. CCL2 heavy labelled peptides derived from CCL2 sequence are spiked into tryptic digests of drug substance. Endogenous CCL2 protein is then quantified by comparing LC-MS intensities of endogenous CCL2 non-labelled peptides against LC-MS intensities of spiked CCL2 heavy labelled peptides.

The method demonstrated a linear detection of CCL2 heavy labelled peptides over a range between 33 ng I mg DS and 8100 ng I mg DS (R2>0.99). CCL2 quantification in ANGPTL3 mimetics of SEQ ID NO: 43 was not influenced by the concentration of the heavy labelled peptide used in the experiment (99% agreement), which was confirmed using an alternative data analysis (peak area vs. MS intensities). The method demonstrated a precision of 9%CV. By conclusion, the method is considered suitable for quantifying CCL2 in ANGPTL3 mimetics of SEQ ID NO: 43 drug substance.

SEQUENCE LISTING

Claims

1 . A method of producing an ANGPTL3 mimetic, comprising the steps of

(a) providing CHO cells capable of producing the ANGPTL3 mimetic;

(b) cultivating the CHO cells in a cell culture under conditions which allow for the production of the ANGPTL3 mimetic;

(c) obtaining the ANGPTL3 mimetic from the cell culture; and

(d) optionally processing the ANGPTL3 mimetic; wherein the CHO cells are engineered to reduce the production of CCL2 in the CHO cells.

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

3. The method according to claim 1 or 2, wherein the CHO cells are engineered to produce a miRNA targeting the endogenous CCL2 of the CHO cells, wherein the miRNA targeting CCL2 preferably has a nucleotide sequence selected from the group consisting of SEQ ID NO: 26.

4. The method according to claim 3, wherein the CHO cells comprise an expression cassette for production of the miRNA targeting CCL2, wherein the expression cassette comprises an intronic sequence comprising a template sequence for a pri-miRNA, wherein the pri-miRNA is suitable to be processed 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',

(iv) a guide strand, having the nucleotide sequence of the miRNA, and

6. The method according to any one of claims 1 to 5, wherein the method is for producing an ANGPTL3 mimetic comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-50.

7. The method according to any one of claims 1 to 6, wherein the ANGPTL3 mimetic obtained after step (c) is in a composition which comprises CCL2 in a concentration of 100 ppm or lower, preferably 25 ppm or lower, more preferably 10 ppm or lower, and/or which comprises CCL2 in a concentration which is at least 10-times lower, preferably 25-times lower, more preferably 100-times lower, compared to a composition obtained with the same method after step (c) using CHO cells which are not engineered to reduce the production of CCL2 in the CHO cells and/or wherein the ANGPTL3 mimetic obtained after step (d) is in a composition which comprises CCL2 in a concentration of 25 ppm or lower, preferably 10 ppm or lower, more preferably 5 ppm or lower, and/or which comprises CCL2 in a concentration which is at least 10-times lower, preferably 25-times lower, more preferably 100-times lower, compared to a composition obtained with the same method after step (d) using CHO cells which are not engineered to reduce the production of CCL2 in the CHO cells.

8. The method according to any one of claims 1 to 7, wherein step (c) comprises

9. The method according to claim 8, wherein ANGPTL3 mimetics or the variant thereof obtained after step (c1) is in a composition which comprises CCL2 in a concentration of 100 ppm or lower, preferably 25 ppm or lower, more preferably 10 ppm or lower, and/or which comprises CCL2 in a concentration which is at least 10-times lower, preferably 25-times lower, more preferably 100-times lower, compared to a composition obtained with the same method after step (c1) using CHO cells which are not engineered to reduce the production of CCL2 in the CHO cells; and/or wherein ANGPTL3 mimetics or the variant thereof obtained after step (c4) is in a composition which comprises CCL2 in a concentration of 25 ppm or lower, preferably 10 ppm or lower, more preferably 5 ppm or lower, and/or which comprises CCL2 in a concentration which is at least 10-times lower, preferably 25-times lower, more preferably 100-times lower, compared to a composition obtained with the same method after step (c4) using CHO cells which are not engineered to reduce the production of CCL2 in the CHO cells.

10. The method according to any one of claims 1 to 9, wherein step (d) comprises providing a pharmaceutical formulation comprising the ANGPTL3 mimetic.

11. A CHO cell which is capable of producing an ANGPTL3 mimetic and which is 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 to reduce the production of CCL2 in the CHO cell as defined in any one of claims 2 to 7.

13. A method of improving production of an ANGPTL3 mimetic in a CHO cell, comprising the steps of

(a-i) providing a CHO cell capable of producing the ANGPTL3 mimetic;

14. The method according to claim 13, wherein the CHO cell is 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) includes introducing a vector nucleic acid into the CHO cell, wherein the vector nucleic acid comprises an expression cassette for expression of a miRNA targeting CCL2.

16. The method according to any one of claims 13 to 15, wherein engineering the CHO cell reduces the level of CCL2 mRNA and/or 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 prior to step (a-ii).

17. The method according to any one of claims 13 to 16, wherein the method further comprises the steps of

(c) obtaining said ANGPTL3 mimetic from the cell culture; and

(d) optionally processing the ANGPTL3 mimetic.

18. The method according to claim 17, wherein step (c) comprises (c1) separating the CHO cells from cell culture supernatant containing the ANGPTL3 mimetic;

(c4) further purifying ANGPTL3 mimetics using hydrophobic interaction chromatography, and/or wherein step (d) comprises providing a pharmaceutical formulation comprising the ANGPTL3 mimetic.

19. A method for producing a CHO cell which is capable of expressing an ANGPTL3 mimetic, comprising introducing a vector nucleic acid comprising an expression cassette for expression of a miRNA in the CHO cell, comprising a template sequence for a pri-miRNA, wherein the pri-miRNA is suitable to be processed in the CHO cell to form a miRNA targeting CCL2, wherein the expression cassette has the features as defined in any one of claims 3 to 5 into a CHO cell.

20. A composition comprising an ANGPTL3 mimetic and CCL2, wherein the composition is obtained by production using CHO cells according to claim 11 or 12, and wherein

(i) the amount of CCL2 in the composition is at least 10-times lower compared to the same composition obtained by production using CHO cells which were not engineered to reduce the production of CCL2 in the CHO cells; and/or

21. A method according to claims 1 to 10 and 13-19, wherein the ANGPTL3 mimetic comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 43.

22. A CHO cell which is capable of producing an ANGPTL3 mimetic according to claims 11 to 13, wherein said ANGPTL3 mimetic comprises the amino acid sequence SEQ ID NO: 43.

23. A composition according to claim 20, wherein said ANGPTL3 mimetic comprises the amino acid sequence SEQ ID NO: 43.

24. The composition according to claim 20 or 23 for use in the treatment of arthritis or cartilage damage in a human subject, wherein the composition is administered intra-articularly.

25. The composition for use according to claim 24, wherein the composition comprises a dose of about 20-40 mg of the polypeptide consisting of a protein sequence of SEQ ID NO: 43.

26. The composition for use according to claim 25, wherein the composition comprises a dose of about 20 mg of the polypeptide consisting of a protein sequence of SEQ ID NO: 43.

5 27. The composition for use according to claim 24, wherein the composition comprises a dose of about 40 mg of the polypeptide consisting of a protein sequence of SEQ ID NO: 43.