Methods for determining the biological activity of ANGPTL polypeptides

Abstract

Provided herein are methods for assaying the chondrogenic inducing activity of therapeutic agents by measuring the expression and / or secretion levels of chondrogenic biomarkers.

Description

[Technical field]

[0001] The present disclosure relates to methods for determining the biological activity of compounds capable of inducing chondrogenesis. [Background technology]

[0002] Cartilage is formed through a process called chondrogenesis. The formation of cartilage in vivo first involves the differentiation of mesenchymal stem cells into chondrocytes, which then secrete molecules, such as collagen and proteoglycans, that form the extracellular matrix that comprises cartilage. Cartilage can be damaged like any other tissue; however, unlike other tissues, cartilage has a very limited ability to repair itself. As a result, damaged cartilage will gradually deteriorate over many years. Indeed, a hallmark of osteoarthritis (OA), the most common degenerative joint disease affecting over 300 million people worldwide, is the progressive destruction of articular cartilage. The progression of OA is mediated by both enzymatic degradation of the cartilage matrix and by impaired cartilage matrix formation. Since the only treatment options to date are pain management and / or joint replacement, considerable research efforts have been directed toward improving the self-repair of cartilage by developing therapeutics that can induce chondrogenesis, for example, by developing therapeutics that stimulate the differentiation of endogenous mesenchymal stem cells into chondrocytes, thereby resulting in the regeneration of articular cartilage. However, identifying therapeutics that induce chondrogenesis can be difficult and time-consuming. There is therefore a need to develop methods that can rapidly and accurately determine the chondrogenic inducing activity of such therapeutic agents. Summary of the Invention

[0003] The present disclosure provides a method for determining the chondrogenic induction activity of an ANGPTL polypeptide. In some embodiments, the method comprises exposing a cell culture to an ANGPTL polypeptide, measuring the expression and / or secretion level of a chondrogenic biomarker, and comparing the expression and / or secretion level with a cell culture that is not exposed to the ANGPTL polypeptide. In some embodiments, the expression and / or secretion level of the biomarker is increased compared to a cell culture that is not exposed to the ANGPTL polypeptide. In other embodiments, the expression and / or secretion level of the biomarker is decreased compared to a cell culture that is not exposed to the ANGPTL polypeptide.

[0004] In some embodiments, the ANGPTL polypeptide is ANGPTL2, ANTPTL3, ANGPTL4, or derivatives thereof. In a preferred embodiment, the ANGPTL polypeptide is SEQ ID NO:2. In one embodiment, the cell culture used is comprised of chondrocytes. In a particular embodiment, the chondrocytes are immortalized chondrocytes. In another embodiment, the chondrocytes are C-28 / 12 cells. In another embodiment, the cell culture is comprised of mesenchymal stem cells. In a preferred embodiment, the mesenchymal stem cells are human.

[0005] In one embodiment, the expression and / or secretion level of the chondrogenic biomarker is measured by a method capable of quantifying the expression of the chondrogenic biomarker. In one embodiment, the method for quantifying the expression of the biomarker is an immunosorbent assay. In a particular embodiment, the immunosorbent assay is an ELISA or a Western blot. In another embodiment, the chondrogenic biomarker is Annexin A6, CD44, CD151, ITM2A, FAM20B, FoxC1, FoxC2, SOX5, SOX6, SOX9, ACAN, Cathepsin B, CHAD, CHADL, Chondroadherin, Collagen II, Collagen IV, Collagen IX, CRTAC1, DSPG3, Decorin, IBSP / Sialoprotein II, Matrilin-1, Matrilin-3, Matrilin-4, MIA, Otraplin / OTOR, URB, DKK1, FBN2, LEP, ALPL, CORIN, CLEC3b, or COMP. In a preferred embodiment, the chondrogenic biomarker is DKK1.

[0006] In another embodiment, the method for determining the activity of ANGPTL polypeptide to induce chondrogenesis is provided herein.In this embodiment, the method comprises adding an amount of ANGPTL polypeptide to cell culture and measuring the amount of secretion of DKK1, wherein the amount of secretion of DKK1 after exposure to the polypeptide is increased compared to the amount of secretion of DKK1 of cell culture that does not add the polypeptide.In some embodiments, the ANGPTL polypeptide is ANGPTL2, ANGPTL3, ANGPTL4, or their derivatives.In a preferred embodiment, the ANGPTL polypeptide is SEQ ID NO:2.

[0007] In another embodiment, a method for determining the activity of ANGPTL polypeptides inducing chondrogenesis is provided herein.In this embodiment, the method comprises adding an amount of ANGPTL polypeptide to a cell culture and measuring the amount of expression and / or secretion of SOX9, ACAN, COMP, LEP, ALPL, CLEC3b, CORIN or FBN2, wherein (i) the amount of expression / secretion of SOX9, ACAN and COMP after exposure to the polypeptide is increased compared to the amount of expression / secretion of SOX9, ACAN and COMP of a cell culture to which the polypeptide is not added, and (ii) the amount of expression / secretion of LEP, ALPL, CLEC3b, CORIN and FBN2 after exposure to the polypeptide is decreased compared to the amount of expression / secretion of LEP, ALPL, CLEC3b, CORIN and FBN2 of a cell culture to which the polypeptide is not added.

[0008] In some embodiments, when measuring expression and / or secretion of SOX9, ACAN, COMP, LEP, ALPL, CLEC3b, CORIN or FBN2 in the assays disclosed herein, the ANGPTL polypeptide is ANGPTL2, ANGPTL3, ANGPTL4, or a derivative thereof. In a preferred embodiment, the ANGPTL polypeptide is SEQ ID NO:2.

[0009] In some embodiments, provided herein is a method for identifying a substance having a chondrogenic inductive effect, comprising the steps of culturing cells capable of expressing a chondrogenic biomarker, adding the substance to the cell culture, and measuring secretion of the chondrogenic biomarker following addition of the substance. In one embodiment, a substance has a chondrogenic inductive effect if the level of the chondrogenic biomarker following addition of the substance is altered compared to the level of the biomarker in a cell culture not exposed to the substance. In certain embodiments, the level of the chondrogenic biomarker is increased. In other embodiments, the level of the chondrogenic biomarker is decreased. In certain embodiments, the chondrogenesis biomarker is annexin A6, CD44, CD151, ITM2A, FAM20B, FoxC1, FoxC2, SOX5, SOX6, SOX9, ACAN, cathepsin B, CHAD, CHADL, chondroadherin, collagen II, collagen IV, collagen IX, CRTAC1, DSPG3, decorin, IBSP / sialoprotein II, matrilin-1, matrilin-3, matrilin-4, MIA, otraprin / OTOR, URB, DKK1, FBN2, LEP, ALPL, CORIN, CLEC3b, or COMP. [Brief description of the drawings]

[0010] [Figure 1] Compound 1 induces secretion of DKK1 (Dickkopf-related protein 1) by chondrocytes. ELISA of DKK1 in supernatants from C-28 / I2 cells cultured for 24 hours in C-28 / I2 test medium supplemented with compound 1. Levels are expressed in pg / ml. Values ​​are the mean + / - SD of measurements from triplicate wells. ****One-way ANOVA with p<0.0001. [Diagram 2]Secretion of DKK1 is stimulated by compound 1, ANGPTL3, ANGPTL2 and ANGPTL4 treatment in chondrocytes. ELISA of DKK1 in supernatants from C-28 / I2 cells cultured for 24 hours in C-28 / I2 test medium supplemented with compound 1 or ANGPTL. Toxicity scale based on cell morphology assessment in bright field microscopy: + first slight signs of toxicity; ++: only a few intact cells remaining; +++: many particles / cell debris present. Levels are expressed in pg / ml. Values ​​are the mean + / - SD of measurements from triplicate wells. One-way ANOVA with **p<0.05, ***p<0.001 and ****p<0.0001. [Diagram 3] Secretion of DKK1 is stimulated by compound 1 and the truncated form (C-terminal part) of ANGPTL2 in chondrocytes. Secretion of DKK1 by C-28 / I2 cells upon treatment with compound 1 and C-terminal ANGPTL2 (260-493). ELISA of DKK1 in supernatants from C-28 / I2 cells cultured for 24 hours in C-28 / I2 test medium supplemented with compound 1 or C-terminal ANGPTL2. Toxicity scale based on cell morphology assessment in bright field microscopy: + first slight signs of toxicity; ++: only a few intact cells remaining; +++: many particles / cell debris present. Levels are expressed in pg / ml. Values ​​are the mean + / - SD of measurements obtained from triplicate wells. One-way ANOVA with **p<0.05, ***p<0.001 and ****p<0.0001. [Figure 4] Compound 1 induces secretion of DKK1 by human MSCs. Accumulated DKK1 secretion between days 8-11, 15-18, and 22-25 of 3D hMSC pellet culture was quantified by ELISA. Comparable effects of Compound 1 were detected in hMSCs from two different donors (out of four donors tested). Levels are expressed in pg / ml. Values ​​are the mean + / - SD of measurements from triplicate wells. One-way ANOVA with ***p<0.001 and ****p<0.0001. [Diagram 5]Compound 1 induces the production of lubricin protein by human MSCs. Lubricin immunohistochemical staining of human MSC pellets treated for 28 days with increasing concentrations of Compound 1. Isotype control shows staining specificity; bar=100 mm. Quantification of immunostaining is expressed as sum of lubricin intensity. Values ​​are the mean + / - SD of duplicate measurements obtained with cells isolated from one donor. [Figure 6-1] Figure 6: Compound 1 reduces alkaline phosphatase (ALPL) and leptin (LEP) transcripts and leptin protein expression in human MSCs. Dose-response effect of compound 1 on relative expression (2-ΔCt) of alkaline phosphatase (ALPL) and leptin (LEP) in 3D pellets of hMSCs after 28 days of culture and on secreted leptin (pg / ml) in the supernatant (ELISA). Values ​​are the mean + / - SD of measurements from triplicate wells. **p<0.01, and ****p<0.0001 by one-way ANOVA. [Figure 6-2] (As stated above.) [Figure 7] Compound 1 increases the expression of COMP (cartilage oligomeric matrix protein) in UE7T-13 cells. Dose-response effect of compound 1 on relative expression (2-ΔCt) of COMP transcripts and secreted COMP protein (ng / ml, ELISA) in UE7T-13 cells cultured in chondrogenic medium under inflammatory conditions after 3, 7, 11 and 14 days of culture. Values ​​are the mean + / - SD of measurements from triplicate wells. One-way ANOVA with *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. [Figure 8]Compound 1 increases the expression of the transcription factor SOX9 in UE7T-13. Dose-response effect of compound 1 on the relative expression (2-ΔCt) of SOX9 transcripts in UE7T-13 cells cultured in chondrogenic medium under inflammatory conditions after 3, 7, 11 and 14 days of culture. Values ​​are the mean + / - SD of measurements obtained from triplicate wells. One-way ANOVA with **p<0.01, ***p<0.001 and ****p<0.0001. [Figure 9] Compound 1 increases expression of Acan (the gene encoding aggrecan, also known as cartilage-specific proteoglycan core protein or chondroitin sulfate proteoglycan 1) in UE7T-13. Dose-response effect of compound 1 on the relative expression (2-ΔCt) of Acan transcripts in UE7T-13 cells cultured in chondrogenic medium under inflammatory conditions after 3, 7, 11 and 14 days of culture. Values ​​are the mean + / - SD of measurements from triplicate wells. One-way ANOVA with ***p<0.001 and ****p<0.0001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Modified human ANGPTL3 polypeptides have previously been shown to exhibit chondrogenic and chondroprotective effects. Examples of such modified human ANGPTL3 polypeptides have already been described in WO 2014 / 138687, the contents of which are fully incorporated by reference. However, there is still a need for improved methods of determining the biological activity of such polypeptides to ensure that they remain effective and / or retain potency after manufacturing, batch storage, stability testing, or any other instance where it is necessary to evaluate the biological activity of such polypeptides. The inventors have discovered an effective method to meet this need by developing an assay to measure the expression or secretion of biomarkers associated with chondrogenesis. In particular, the inventors have discovered that DKK1 is an accurate and reliable indicator of the chondrogenic induction activity of a given compound, such as modified ANGPTL3 polypeptides.

[0012] As used herein, the terms "a" and "an" and "the" and similar references in the context of describing the invention should be construed to encompass both the singular and the plural, unless otherwise stated herein or the context clearly contradicts. When the plural is used for compounds, salts, and the like, this is understood to mean a single compound, salt, or the like.

[0013] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless context clearly dictates otherwise.

[0014] "About" and "approximately" are generally intended to mean an acceptable degree of error of the measured quantity given the nature or precision of the measurement. Exemplary degrees of error are within 20 percent (%), typically within 10%, and more typically within 5% of a given value or range of values.

[0015] "ANGPTL2" refers to a member of the angiopoietin protein family. The amino acid sequence of ANGPTL2 (GenBank Accession No. NP_036230.1) is set forth in SEQ ID NO: 3. "ANGPTL2 polypeptide" refers to a naturally occurring expressed polypeptide. For purposes of this disclosure, amino acid numbering is generally determined with reference to the full-length wild-type human ANGPTL2 polypeptide sequence (SEQ ID NO: 3). Thus, in embodiments where a polypeptide contains only the C-terminal portion of full-length ANGPTL2, and not the N-terminal portion, the peptide is less than 493 amino acids long, but the position numbering is based on SEQ ID NO: 3. For example, a reference to position 350 of an ANGPTL2 polypeptide refers to position 350 of SEQ ID NO: 3, even though the ANGPTL2 polypeptide itself may only be 200 amino acids long. When determining the amino acid "corresponding" to a position in a reference sequence, such as SEQ ID NO: 3, in a sequence of interest, this is performed by optimally aligning the sequences and comparing the sequences, for example, using default CLUSTAL alignment parameters or default BLAST2 alignment parameters. For example, if a subject sequence is optimally aligned with SEQ ID NO:3, then position 350 in the subject sequence "determined with reference to SEQ ID NO:3", or an amino acid "corresponding to" position 350 of SEQ ID NO:3, means the amino acid that is aligned with position 350 of SEQ ID NO:3.

[0016] "ANGPTL3" refers to a member of the angiopoietin protein family. The amino acid sequence of ANGPTL3 (GenBank Accession No. NP_055310.1) is set forth in SEQ ID NO:1. "ANGPTL3 polypeptide" refers to a naturally occurring expressed polypeptide. For purposes of this disclosure, amino acid numbering is generally determined with reference to the full-length wild-type human ANGPTL3 polypeptide sequence (SEQ ID NO:1). Thus, in embodiments where a polypeptide contains only the C-terminal portion of full-length ANGPTL3, but not the N-terminal portion, the peptide is less than 460 amino acids long, but the position numbering is based on SEQ ID NO:1. For example, a reference to position 423 of an ANGPTL3 polypeptide refers to position 423 of SEQ ID NO:1, even though the ANGPTL3 polypeptide itself may only be 200 amino acids long. When determining the amino acid "corresponding" to a position in a reference sequence, such as SEQ ID NO:1, in a sequence of interest, this is performed by optimally aligning the sequences and comparing the sequences, for example, using default CLUSTAL alignment parameters or default BLAST2 alignment parameters. For example, if a subject sequence is optimally aligned with SEQ ID NO:1, then position 423 in the subject sequence "determined with reference to SEQ ID NO:1", or an amino acid "corresponding to" position 423 of SEQ ID NO:1, means the amino acid that is aligned with position 423 of SEQ ID NO:1.

[0017] "ANGPTL4" refers to a member of the angiopoietin protein family. The amino acid sequence of ANGPTL4 (GenBank Accession No. NP_647475.1) is set forth in SEQ ID NO:5. "ANGPTL4 polypeptide" refers to a naturally occurring expressed polypeptide. For purposes of this disclosure, amino acid numbering is generally determined with reference to the full-length wild-type human ANGPTL4 polypeptide sequence (SEQ ID NO:5). Thus, in embodiments where a polypeptide contains only the C-terminal portion of full-length ANGPTL4, but not the N-terminal portion, the peptide is less than 406 amino acids long, but the position numbering is based on SEQ ID NO:5. For example, a reference to position 400 of an ANGPTL4 polypeptide refers to position 400 of SEQ ID NO:5, even though the ANGPTL4 polypeptide itself may only be 200 amino acids long. When determining the amino acid in a sequence of interest that "corresponds" to a position in a reference sequence, such as SEQ ID NO:5, this is performed by optimally aligning the sequences and comparing the sequences, for example, using default CLUSTAL alignment parameters or default BLAST2 alignment parameters. For example, if a subject sequence is optimally aligned with SEQ ID NO:5, then position 400 in the subject sequence "determined with reference to SEQ ID NO:5", or an amino acid "corresponding to" position 400 of SEQ ID NO:5, means the amino acid that is aligned with position 400 of SEQ ID NO:5.

[0018] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to naturally occurring and non-naturally occurring amino acid polymers, as well as to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid.

[0019] The term "biological activity" or "biologically active" refers to the ability of a compound to alter the physical, chemical, or biochemical properties of a biological system, pathway, tissue, cell, or molecule. Such alteration can be, for example, an increase, decrease, maintenance, or modulation of the concentration or amount of a protein, polypeptide, peptide, DNA, RNA, saccharide, sugar, metabolite, precursor, cofactor, or other biological molecule, whether in vivo, in vitro, or ex vivo. Such alteration can be determined qualitatively or quantitatively.

[0020] The terms "assay" or "assaying" are used to refer to the act of identifying, screening, probing, or determining, which may be performed by any conventional means. For example, by using ELISA assays, Western blots, Northern blots, Southern blots, mass spectrometry, reverse transcription quantitative polymerase chain reaction (RT-QPCR), imaging, etc., a sample can be assayed for the presence of a particular biomarker to detect whether the biomarker is present in the sample. Furthermore, as used herein, the terms "assaying" and "determining" are used to mean to test and / or measure.

[0021] Previous studies with modified ANGPTL3 polypeptides, such as those described in WO 2014 / 138687, the contents of which are fully incorporated by reference, have shown that such polypeptides have chondrogenic and chondroprotective effects. Compound 1 (SEQ ID NO: 2) is an example of such a modified ANGPTL3 polypeptide. The chondrogenic induction effect of Compound 1 has been repeatedly established through in vitro studies, in vivo animal studies, and clinical trials. Despite the body of evidence establishing the chondrogenic effect of Compound 1, conventional assays to confirm such biological activity can be time-consuming and difficult to evaluate. There was a need for an improved method to determine the chondrogenic activity of such polypeptides. The inventors hypothesized that the relative expression levels of various biomarkers known to be involved in chondrogenesis could be utilized to develop the required method.

[0022] Chondrogenesis is a complex and dynamic cellular process involving the up- and down-regulation of several different genes, along with increased or decreased expression and / or secretion of various proteins. These changes in expression and / or secretion levels can be detected using a variety of standard techniques, and therefore these genes and / or proteins with changes in expression and / or secretion levels resulting from chondrogenesis have the potential to be used as biomarkers to detect the onset of chondrogenesis. The inventors have determined that biomarkers that play an important role in chondrogenesis, such as biomarkers that induce chondrogenesis, inhibit cartilage anabolic inhibitors, or inhibit cartilage catabolic activity, have the potential to be reliable biomarkers for assaying chondrogenic activity. For example, inhibition of WNT signaling has been reported to have chondrogenic and anti-hypertrophic effects, thus promoting the regeneration of hyaline cartilage, so the WNT signaling pathway is believed to have an important role in the progression of OA. DKK1 is a known inhibitor of WNT signaling. DKK1 has also been shown to be negatively correlated with the severity of OA, for example, overexpression of DKK1 in chondrocytes inhibits experimental OA cartilage destruction in mice. DKK-1 also inhibits hypertrophic differentiation and IL1β-induced MMP expression in articular chondrocytes, which may contribute to the development of OA. Therefore, we hypothesized that the secretion level of DKK1 can serve as a surrogate for assaying the chondrogenic activity of compounds, such as compound 1. As shown in Figure 1, the secretion of DKK1 increased in a dose-dependent manner after exposure to compound 1. This indicates that DKK1 can be used as a surrogate marker for determining chondrogenic activity.

[0023] Other members of the ANGPTL protein family, namely ANGPTL2 and ANGPTL4, which have been reported to be involved in chondrogenesis and cartilage remodeling, were also evaluated. When compound 1 was compared with ANGPTL3, ANGPTL2 and ANGPTL4 in the assay, it was observed that all of these molecules had the ability to induce the secretion of DKK1 into the supernatant (Figure 2). However, unlike compound 1, ANGPTL2 and ANGPTL4 proteins also induced cytotoxicity at low concentrations. The results of this experiment validate the robustness of the assay described herein and demonstrate that it can be used to rapidly screen ANGPTL polypeptides for potential chondrogenic activity.

[0024] The assay described herein was also used to screen for truncated forms of ANGPTL2 (FIG. 3). The results of this experiment demonstrate that C-terminal ANGPTL2 (260-493; SEQ ID NO: 4) is capable of inducing secretion levels of DKK1 comparable to compound 1. This suggests that truncated ANGPTL2 retains chondrogenic potential and could potentially be developed as a therapeutic agent to induce cartilage repair.

[0025] [Table 1]

[0026] The above experiments demonstrate that DKK1 can be used as an alternative biomarker to evaluate the biological activity of ANGPTL polypeptides, such as compound 1. Other biomarkers involved in chondrogenesis are also suitable for use in the disclosed methods. Non-limiting examples of such biomarkers include Annexin A6, CD44, CD151, ITM2A, FAM20B, FoxC1, FoxC2, SOX5, SOX6, SOX9, ACAN, cathepsin B, CHAD, CHADL, chondroadherin, collagen II, collagen IV, collagen IX, CRTAC1, DSPG3, decorin, IBSP / sialoprotein II, matrilin-1, matrilin-3, matrilin-4, MIA, otraprin / OTOR, URB, DKK1, FBN2, LEP, ALPL, CORIN, CLEC3b, or COMP. Those skilled in the art will further recognize that other biomarkers that have been shown to correlate with chondrogenic activity are also suitable for use in the disclosed assays and methods.

[0027] The assays and methods provided herein can also be used to screen and / or identify potential therapeutics with chondrogenic induction activity. Those skilled in the art will recognize that different compounds may induce chondrogenesis through different pathways or mechanisms. Thus, in some embodiments, multiple biomarkers can be used to screen potential therapeutics for chondrogenic induction activity. It will be recognized that the biomarkers used to screen one potential therapeutic can be the same or different from the biomarkers used to screen another potential therapeutic.

[0028] Conventional methods for detecting and quantifying selected biomarkers, such as DKK1, can be used with the methods of the present disclosure. Examples of such conventional methods include immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) or Western blot analysis, and mass spectrometry. However, those skilled in the art will recognize that any method capable of detecting a specific protein in a cell culture is acceptable for use with the methods described. In some embodiments, the detection method is ELISA, and in other embodiments, the method is sandwich ELISA.

[0029] Methods for detecting gene expression are also suitable for use in the disclosed assays and methods.Non-limiting examples of methods for detecting gene expression include PCR, quantitative PCR, RNA-seq analysis, or microarray.Those skilled in the art will also understand that other methods for detecting nucleic acid levels are acceptable.

[0030] Chondrocytes are suitable for use with the assays and methods disclosed herein because they are already further differentiated into cells that express typical chondrogenic markers and can secrete extracellular matrix proteins, including cartilage. Because chondrocytes are further differentiated, the biological activity of the assayed chondrogenic-inducing or hypertrophy-inhibiting compounds can be determined more quickly compared to non-differentiated cells. In addition, chondrocyte cell lines can be propagated many times, making it possible to work with the same batch of cells. Examples of chondrocyte cell lines that can be used with the present method include, but are not limited to, C-28 / I2 and T / C28a2.

[0031] Mesenchymal stem cells are also suitable for use with the method. Mesenchymal stem cells are primary cells isolated from bone marrow. In some embodiments, the mesenchymal stem cells are human. These cells are not yet differentiated and must be cultured under chondrogenic conditions for some time to reach a differentiation stage comparable to chondrocytes. During their differentiation process into the chondrogenic lineage, mesenchymal stem cells gradually become chondrocytes that express typical chondrogenic markers and can secrete extracellular matrix proteins found in cartilage. Thus, assays in chondrocytes derived from mesenchymal stem cells take longer to complete compared to assays in chondrocytes. In addition, human mesenchymal stem cells can be expanded for only a few passages before losing the ability to differentiate into different cell lineages. However, the advantage of chondrogenically differentiated mesenchymal stem cells is their closer similarity to primary chondrocytes.

[0032] Although chondrocytes and mesenchymal stem cells are well suited for use in the disclosed assays and methods, one of skill in the art will recognize that any cells capable of expressing chondrogenesis-associated markers, in particular DKK1, may be used.

[0033] The method provides a rapid and efficient method for determining the biological activity of compounds capable of inducing chondrogenesis. In some embodiments, the compound is a potential therapeutic agent. In other embodiments, the compound is an ANGPTL polypeptide or variant. In another embodiment, the compound is Compound 1.

[0034] In one aspect, the method involves measuring expression and / or secretion of a biomarker after exposing a cell culture to a compound having chondrogenic inductive activity. In some embodiments, a baseline expression level of the biomarker is established prior to exposure to the compound. In other embodiments, the expression level of the biomarker is compared to a control not exposed to the compound.

[0035] In another aspect, the method involves determining whether a compound has chondrogenic inductive activity by measuring expression and / or secretion of a biomarker after exposing a cell culture to the compound. In some embodiments, expression and / or secretion of the biomarker is increased. In other embodiments, expression and / or secretion of the biomarker is decreased.

[0036] In another aspect, the biomarker used in the assay is a biomarker associated with chondrogenesis. In another aspect, the biomarker is associated with inhibition of cartilage anabolic activity. In some embodiments, the biomarker is Annexin A6, CD44, CD151, ITM2A, FAM20B, FoxC1, FoxC2, SOX5, SOX6, SOX9, ACAN, Cathepsin B, CHAD, CHADL, Chondroadherin, Collagen II, Collagen IV, Collagen IX, CRTAC1, DSPG3, Decorin, IBSP / Sialoprotein II, Matrilin-1, Matrilin-3, Matrilin-4, MIA, Otraplin / OTOR, URB, DKK1, FBN2, LEP, ALPL, CORIN, CLEC3b, or COMP. In another embodiment, the biomarker is DKK1.

[0037] In another aspect, the cell culture is comprised of chondrocytes. In another embodiment, the cell culture is comprised of mesenchymal stem cells. In another embodiment, the mesenchymal stem cells are human.

[0038] In another aspect, the expression and / or secretion levels of biomarkers in cell cultures are determined by detection methods that can quantitatively or qualitatively measure protein and / or gene expression / secretion. In some embodiments, the detection method is Western blot or mass spectrometry. In another embodiment, the detection method is ELISA, and in yet another embodiment, the detection method is sandwich ELISA.

[0039] In another aspect, the method can be used during the manufacturing process to confirm that a compound has chondrogenic inducing activity. Thus, in one aspect, the present disclosure relates to a method for verifying the activity of a compound having chondrogenic activity before release. In another aspect, the method can be used to determine the chondrogenic activity of a compound after storage. In yet another aspect, the method can be used to determine the chondrogenic inducing activity of a compound before administration to a patient.

[0040] In another aspect, the assays and methods described may be used to screen compounds for potential chondrogenic inductive activity. In one embodiment, a compound with potential chondrogenic inductive activity is exposed to a cell culture and the expression and / or secretion levels of chondrogenic biomarkers are measured. In some embodiments, the biomarker levels are elevated compared to cell cultures not exposed to the compound with potential chondrogenic inductive activity. In another embodiment, the biomarker levels are decreased compared to cell cultures not exposed to the compound with potential chondrogenic inductive activity. EXAMPLES

[0041] Example 1: ELISA assay for DKK1 Multiarray high binding 96-well plates (MSD) were precoated with monoclonal anti-human Dkk1 (1 mg / ml in PBS) antibody (R&D) overnight at 4°C, then blocked with 1% casein TBS (BioRad) for 1 h at 450 rpm in a thermomixer, followed by 4 wash steps with 0.5xTBST (Sigma) at RT. Cell supernatants were added to appropriate wells and incubated at RT for 1 h at 450 rpm in a thermomixer, followed by 4 wash steps and biotinylated anti-human DKK1 (R&D) was added and incubated at RT for 1 h at 450 rpm in a thermomixer. After 4 wash steps, streptavidin sulfo-TAG solution (MSD) was added and incubated at RT for 30 min at 450 rpm in a thermomixer. Four additional wash steps were performed, followed by addition of 2xRead buffer (MSD) for detection of electrochemiluminescence counts using an MSD Sector S600 reader. ELISA was performed on cell culture supernatants stored at -80°C.

[0042] [Table 2]

[0043] Example 2: C-28 / 12 cell culture for detection of DKK1 secretion induced by compound 1 The human chondrocyte cell line C-28 / I2 (licensed from Dr. Mary Goldring, Massachusetts General Hospital, Boston, USA) was grown in DMEM / F12 medium containing 10% FCS (Millipore), 50 μg / ml L-ascorbic acid diphosphate (Wako Pure Chemical), 100 IU / ml penicillin, and 100 μg / ml streptomycin. To test the activity of compound 1, cells were seeded at 50'000 cells / well (96-well plate format, Costar) in 100 μl of DMEM / F12 medium containing 1% FCS (Millipore), 50 μg / ml L-ascorbic acid phosphate (Wako Pure Chemical), 100 IU / ml penicillin, 100 μg / ml streptomycin supplemented with compound 1 (Novartis) at the concentrations specified in the figure or vehicle control and incubated for 24 h. Cell culture was performed at 37°C in a humidified incubator with 5% CO2.

[0044] A dose-dependent increase in DKK1 protein of up to 4.5-fold over controls was observed in the supernatants of C-28 / I2 cells, which occurred within 24 hours of Compound 1 treatment (Figure 1).

[0045] Example 3: C-28 / 12 cell culture for detection of DKK1 secretion induced by ANGPTL mutants The human chondrocyte cell line C-28 / I2 (licensed from Dr. Mary Goldring, Massachusetts General Hospital, Boston, USA) was grown in DMEM / F12 medium containing 10% FCS (Millipore), 50 μg / ml L-ascorbic acid diphosphate (Wako Pure Chemical), 100 IU / ml penicillin, and 100 μg / ml streptomycin. To test the activity of compound 1 and ANGPTL variants, cells were seeded at 50'000 cells / well (96-well plate format, Costar) in 100 μl of DMEM / F12 medium containing 1% FCS (Millipore), 50 μg / ml L-ascorbic acid phosphate (Wako Pure Chemical), 100 IU / ml penicillin, 100 μg / ml streptomycin supplemented with compound 1 (Novartis), ANGPTL2 (R&D), ANGPTL3 (Novartis), or ANGPTL4 (R&D) at the concentrations specified in the figure, or vehicle control, and incubated for 24 hours. Cell culture was performed at 37°C in a humidified incubator with 5% CO2.

[0046] ANGPTL2 and ANGPTL4, two ANGPTL family members previously reported to be expressed in cartilage and affect chondrogenesis and cartilage matrix remodeling, respectively, were approximately 20-fold more potent than compound 1 in stimulating the secretion of DKK1 (Figure 2). However, ANGPTL2, ANGPTL3, and ANGPTL4 induced strong cytotoxicity at concentrations above 3 μM and were therefore tested at lower concentrations than compound 1. No cytotoxicity was observed with compound 1 (Figure 2).

[0047] Example 4: C-28 / 12 cell culture for detection of DKK1 secretion induced by truncated ANGPTL mutants The human chondrocyte cell line C-28 / I2 (licensed from Dr. Mary Goldring, Massachusetts General Hospital, Boston, USA) was grown in DMEM / F12 medium containing 10% FCS (Millipore), 50 μg / ml L-ascorbic acid diphosphate (Wako Pure Chemical), 100 IU / ml penicillin, and 100 μg / ml streptomycin. To test the activity of compound 1 and truncated ANGPTL mutants, cells were seeded at 50'000 cells / well (96-well plate format, Costar) in 100 μl of DMEM / F12 medium containing 1% FCS (Millipore), 50 μg / ml L-ascorbic acid phosphate (Wako Pure Chemical), 100 IU / ml penicillin, 100 μg / ml streptomycin, supplemented with compound 1 (Novartis) or C-terminal ANGPTL2 (R&D) at the concentrations specified in the figure, or vehicle control, and incubated for 24 h. Cell culture was performed at 37°C in a humidified incubator with 5% CO2.

[0048] C-terminal ANGPTL2 (260-493; SEQ ID NO: 4), a truncated form of an ANGPTL family member previously reported to be expressed in cartilage and to affect chondrogenesis, was as potent as compound 1 in stimulating the secretion of DKK1 (Figure 3). However, the truncated form of ANGPTL2 induced strong cytotoxicity at concentrations above 3 μM. No cytotoxicity was observed with compound 1 (Figure 3).

[0049] Example 5: hMSC cell culture for detection of the expression of chondrogenic biomarkers induced by Compound 1 For the 3D pellet culture assay, bone marrow-derived human mesenchymal stem cells (hMSCs) from four different donors (Lonza Verviers, Belgium) were first expanded for two passages in Lonza medium MSCGM-BulletKitTM and stored in liquid nitrogen. Cells were further expanded in DMEM with 1 g / L glucose, 10% FBS, 6 mM L-glutamine, 10 mM HEPES, 50 IU / ml penicillin, 50 μg / ml streptomycin, and 1 ng / ml human basic FGF (R&D Systems). For 3D culture, passage 6 cells were seeded at 3.5x105 cells / well in 96-well V-bottom plates (Costar), sedimented by centrifugation (5 min, 250g) and incubated in 20-well plates supplemented with LNA043 (recombinantly expressed in CHO cells, Novartis) or vehicle control as indicated, high glucose, 0.125% BSA (Sigma), ITS (6.25 μg / ml human insulin, 6.25 μg / ml human transferrin, 6.25 ng / ml sodium selenite (Roche), 5.3 μg / ml linoleic acid (Sigma), 50 μg / ml L-ascorbic acid phosphate (AA, Wako Pure Chemical Industries, Ltd.), 10 μg / ml ethanol (100 μg / ml ethanol ... The cells were cultured in DMEM supplemented with 100 ng / ml dexamethasone (Sigma), 40 μg / ml proline (Sigma), 100 IU / ml penicillin, and 100 μg / ml streptomycin for 4 weeks. The medium was changed three times a week. Cell culture was performed at 37°C in a humidified incubator containing 5% CO2.

[0050] Treatment with compound 1 (SEQ ID NO:2) during 28 days of chondrogenic differentiation induced a dose-dependent upregulation of the secretion of the WNT inhibitor protein DKK1 in the supernatant (Figure 4). In addition, treatment with compound 1 during 28 days of chondrogenic differentiation induced a dose-dependent increase in the expression of the glycoprotein lubricin / proteoglycan 4 (PRG4) in the cartilage superficial zone up to 5.7-fold, as detected by immunohistochemical staining of 3D pellets (Figure 5). At the gene expression level, compound 1 reduced the cartilage hypertrophy marker alkaline phosphatase and the pro-inflammatory adipokine leptin (Figure 6). Additional genes shown to be downregulated by compound 1 include FBN2, CORIN, and CLEC3b, while the COMP gene was shown to be upregulated (data not shown).

[0051] Example 6: Chondrogenic biomarkers regulated by Compound 1

[0052] [Table 3]

[0053] Having thus described several aspects of several embodiments, it should be recognized that various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of this disclosure. Accordingly, the foregoing description and drawings are by way of example only.

[0054] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments specifically described herein which equivalents are intended to be encompassed by the claims.

Claims

1. A method for determining the chondrogenic induction activity of an ANGPTL polypeptide, comprising exposing a cell culture to the ANGPTL polypeptide, measuring the expression and / or secretion levels of a chondrogenic biomarker, and comparing the expression and / or secretion levels with a cell culture not exposed to the ANGPTL polypeptide.

2. 2. The method of claim 1, wherein the ANGPTL polypeptide is SEQ ID NO:

2.

3. The method of claim 1 , wherein the cell culture is comprised of chondrocytes.

4. The method of claim 1 , wherein the cell culture is comprised of mesenchymal stem cells.

5. The method of claim 4 , wherein the mesenchymal stem cells are human.

6. The method of claim 1 , wherein the expression and / or secretion levels of the chondrogenic biomarkers are measured by immunosorbent assay.

7. The method of claim 1 , wherein the immunosorbent assay is an ELISA or a Western blot.

8. 2. The method of claim 1, wherein the biomarker is annexin A6, CD44, CD151, ITM2A, FAM20B, FoxC1, FoxC2, SOX5, SOX6, SOX9, ACAN, cathepsin B, CHAD, CHADL, chondroadherin, collagen II, collagen IV, collagen IX, CRTAC1, DSPG3, decorin, IBSP / sialoprotein II, matrilin-1, matrilin-3, matrilin-4, MIA, otraprin / OTOR, URB, DKK1, FBN2, LEP, ALPL, CORIN, CLEC3b, or COMP.

9. The method of claim 8, wherein the biomarker is DKK1.

10. 10. The method of any one of claims 1 to 9, wherein the expression and / or secretion levels of the biomarkers DKK1, SOX9, ACAN or COMP are increased compared to a cell culture not exposed to the ANGPTL polypeptide.

11. 10. The method of any one of claims 1 to 9, wherein the expression and / or secretion levels of the biomarkers LEP, ALPL, CLEC3b, CORIN or FBN2 are reduced compared to a cell culture not exposed to the ANGPTL polypeptide.

12. A method for determining the biological activity of an ANGPTL polypeptide that induces chondrogenesis, comprising adding a certain amount of the polypeptide to a cell culture and measuring the amount of DKK1 secretion, wherein the increase in DKK1 secretion after exposure to the polypeptide is increased compared to the secretion of DKK1 in a cell culture to which the polypeptide has not been added.

13. 13. The method of claim 12, wherein the ANGPTL polypeptide is ANGPTL2, ANGPTL3, or ANGPTL4.

14. The method of claim 12 , wherein the compound is an ANGPTL3 derivative.

15. 15. The method of claim 14, wherein the compound is SEQ ID NO:

2.

16. The method of claim 12 , wherein the compound is an ANGPTL2 derivative.

17. 17. The method of claim 16, wherein the compound is SEQ ID NO:

4.

18. The method of claim 12 , wherein the compound is an ANGPTL4 derivative.

19. A method for identifying a substance having a chondrogenesis-inducing effect, comprising: a. Culturing cells capable of expressing chondrogenic biomarkers; b. adding the substance to the cell culture; c. Measuring the secretion of chondrogenic biomarkers after addition of the substance; Including, A method in which the substance has a chondrogenic inducing effect if the level of a chondrogenic biomarker after addition of the substance is altered compared to the level of the biomarker in a cell culture not exposed to the substance.

20. 20. The method of claim 19, wherein the level of the chondrogenic biomarker is elevated.

21. 20. The method of claim 19, wherein the level of the chondrogenic biomarker is reduced.

22. 22. The method of claim 20 or 21, wherein the chondrogenic biomarker is annexin A6, CD44, CD151, ITM2A, FAM20B, FoxC1, FoxC2, SOX5, SOX6, SOX9, ACAN, cathepsin B, CHAD, CHADL, chondroadherin, collagen II, collagen IV, collagen IX, CRTAC1, DSPG3, decorin, IBSP / sialoprotein II, matrilin-1, matrilin-3, matrilin-4, MIA, otraprin / OTOR, URB, DKK1, FBN2, LEP, ALPL, CORIN, CLEC3b, or COMP.

Images