Recombinant expression of bone marrow-derived growth factors

Abstract

The present invention generally relates to the field of recombinant gene expression in host cells. In particular, the present invention relates to a recombinant human myeloid-derived growth factor (MYDGF) protein that exhibits minimal degradation upon expression in host cells. Accordingly, its recombinant protein is highly suitable for medical use, particularly for treating heart tissue damage and preventing cell death in myocardial tissue. The present invention also provides a nucleic acid encoding its recombinant protein, and a host cell expressing its recombinant protein. The present invention also provides a method for producing its recombinant protein in a host cell.

Description

Technical Field

[0001] The present invention generally relates to the field of recombinant gene expression in host cells. In particular, the present invention relates to a recombinant human myeloid-derived growth factor (MYDGF) protein that exhibits minimal degradation upon expression in host cells. Accordingly, the recombinant protein is highly suitable for medical use, particularly for treating heart tissue damage and preventing cell death in myocardial tissue. The present invention also provides a nucleic acid encoding the recombinant protein, and a host cell expressing the recombinant protein. The present invention also provides a method for producing the recombinant protein in a host cell.

Background Art

[0002] Acute myocardial infarction (MI) remains one of the major causes of morbidity and mortality worldwide. Acute MI is mediated by thrombotic occlusion of the coronary artery, which results in progressive cell death in the non-perfused tissue. This induces an inflammatory response, which leads to scar formation and loss of viable tissue. Severe changes in the tissue structure in the left ventricle can cause ventricular dilation, systolic dysfunction, and heart failure. A protein named myeloid-derived growth factor (MYDGF) has been shown to improve tissue repair and cardiac function in murine models of MI. Compared to wild-type mice, MYDGF-deficient mice develop larger infarct scars and more severe systolic dysfunction. Treatment with recombinant MYDGF has been found to protect cardiomyocytes from cell death and repair the heart after acute MI. The development of protein-based therapies is a promising approach for heart repair and also potentially for ischemia repair in other tissues (Ebenhoch et al., 2019; Polten et al., 2019; Botnov et al., 2018, Korf-Klingebiel et al., 2015; WO2014 / 111458).

[0003] Currently, small amounts of recombinant human MYDGF are being produced by expression in human or mammalian expression systems such as HEK-293T cells or CHO cells. However, the larger-scale production of recombinant human MYDGF (rhMYDGF) by mammalian cell expression systems is associated with high costs, which makes its production unattractive from an economic perspective. Attempts have been made to produce rhMYDGF in bacterial expression systems. Zhao et al., 2020 describe the expression of soluble rhMYDGF with a C-terminal His tag in Escherichia coli (E. coli). The tagged protein differs from the mature human wild-type protein by nine additional amino acids, thereby having a significantly higher molecular weight. The authors speculate in that publication that the expression system could be used to produce clinical-grade rhMYDGF, but the His tag would likely result in significant antigenicity when administered to human patients.

[0004] Heterologous expression of rhMYDGF is also known to be associated with non-negligible degradation problems. After recombinant expression of the protein, one or more amino acids located at the N-terminus are degraded, resulting in protein fragments with a lower molecular weight (MW) compared to the full-length protein. For example, Zhao et al., 2020 describe that the final expression product contains not only the full-length rhMYDGF protein with an MW of 17032 Da, but also degradation products with an MW of approximately 16900 Da. The ratio of target protein to degraded protein in Zhao et al., 2020 can be read from the high-performance liquid chromatography-mass spectrometry (HPLC-MS) data shown in Figure 2 of the Zhao publication and is approximately 10:1. Thus, impurities due to degradation products are quite significant and unacceptable for proteins intended for systemic medical use. In view of the prior art shown above, it is an object of the present invention to provide a recombinant protein having MYDGF activity as follows: (i) showing no degradation or only minimal degradation upon expression in a heterologous expression system; (ii) It can be produced to a minimum extent of post-translational modifications from potentially harmful processes such as carbamoylation and glucuronoylation that can be harmful to the intended clinical use of the protein; (iii) It has the secondary and tertiary structures of the native human MYDGF protein; (iv) It has high biological activity; (v) It has a low risk for antigenic epitopes derived from primary sequences other than human MYDGF, thereby resulting in a minimum risk for anti-drug antibodies; (vi) It can be produced in a prokaryotic expression system to provide a non-glycosylated product; (vii) It can be produced in an amount of protein greater than 0.5 g per 100 g of cells, and can be scaled up to more than 100 g per batch, preferably more than 200 g per batch, more preferably more than 300 g per batch.

[0005] All of the objectives are not realized by all embodiments of the present invention. The scope of the present invention is defined by the claims. However, it is preferable to meet two, three, four, five, or six of the aforementioned objectives of the present invention.

Summary of the Invention

[0006] In a first aspect, the present invention relates to a method for recombinant expression of MYDGF protein in a host cell. In a second aspect, the present invention relates to a composition obtained from the method of the first aspect of the present invention. In a third aspect, the present invention relates to the use of the composition of the second aspect of the present invention for the preparation of a pharmaceutical composition. In a fourth aspect, the present invention relates to a pharmaceutical composition comprising the composition of the second aspect of the present invention. In a fifth aspect, the present invention relates to the pharmaceutical composition of the fourth aspect of the present invention for use as a medicament. In a sixth aspect, the present invention relates to a composition of the second aspect of the present invention or a pharmaceutical composition of the fourth aspect of the present invention for use in a method of: (i) treating or preventing a disease or condition selected from the group consisting of injury, trauma, ischemia, reperfusion injury, surgery, primary or secondary cardiomyopathy, post-ischemic systolic dysfunction, myocardial infarction, preferably acute myocardial infarction, angina pectoris, heart failure, cardiac inflammation, heart insufficiency, hypertrophy, and fibrosis; (ii) promoting or improving heart tissue regeneration, cardiomyocyte proliferation, angiogenesis, heart function, or left ventricular systolic function after myocardial infarction; (iii) protecting cardiomyocytes from death, for example, by apoptosis or necrosis; or (iv) reducing infarct size after myocardial infarction, preferably acute myocardial infarction.

[0007] In a seventh aspect, the present invention relates to a protein having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1. In an eighth aspect, the present invention relates to a nucleic acid encoding the protein of the seventh aspect of the present invention. In a ninth aspect, the present invention relates to a vector comprising the nucleic acid of the eighth aspect of the present invention. In a tenth aspect, the present invention relates to a host cell comprising the protein of the seventh aspect of the present invention, the nucleic acid of the eighth aspect of the present invention, or the vector of the ninth aspect of the present invention. In an eleventh aspect, the present invention relates to a pharmaceutical composition comprising the protein of the seventh aspect of the present invention. In a twelfth aspect, the present invention relates to the protein of the seventh aspect of the present invention or the pharmaceutical composition of the eleventh aspect of the present invention for use as a medicament.

[0008] In a 13th aspect, the invention relates to the use of a protein of the 7th aspect of the invention or a pharmaceutical composition of the 11th aspect of the invention for treating or preventing a disease or condition selected from the group consisting of injury, trauma, ischemia, reperfusion injury, surgery, primary or secondary cardiomyopathy, post-ischemic systolic dysfunction, myocardial infarction, preferably acute myocardial infarction, angina pectoris, heart failure, cardiac inflammation, cardiac dysfunction, hypertrophy, and fibrosis; promoting or improving cardiac tissue regeneration, cardiomyocyte proliferation, angiogenesis, cardiac function, or left ventricular systolic function after myocardial infarction; protecting cardiomyocytes from death, for example by apoptosis or necrosis; or reducing infarct size after myocardial infarction, preferably acute myocardial infarction. In a 14th aspect, the invention relates to a method for recombinant expression of a protein of the 7th aspect of the invention in a host cell. In a 15th aspect, the invention relates to a composition obtained from the method of the 14th aspect of the invention. Finally, in a 16th aspect, the invention relates to the use of a host cell of the 10th aspect of the invention for recombinant expression of MYDGF protein. **DETAILED DESCRIPTION OF THE INVENTION**

[0009] The present invention provides a protein having MYDGF activity and showing minimal degradation and process-derived post-translational modifications in recombinant expression in a host cell. The present invention also provides methods for producing large amounts of these proteins in a cell-based expression system. The methods require significantly reduced purification efforts to provide a homogeneous protein composition suitable for formulation into pharmaceuticals. Hereinafter, when referring to SEQ ID NO: 1 or SEQ ID NO: 2, it should be understood that SEQ ID NO: 1 is the preferred option. In the context of the present invention, the proteins disclosed herein are depicted in SEQ ID NO: 1 and SEQ ID NO: 2. Both proteins consist of 143 amino acid building blocks and contain the complete amino acid sequence of the mature human MYDGF protein. The native human MYDGF protein is expressed as a precursor protein having a 31 - amino - acid N - terminal signal peptide and a C - terminal KDEL - like endoplasmic reticulum (ER) retention sequence. The sequence of the 173 - amino - acid MYDGF precursor protein is shown herein as SEQ ID NO: 5. Cleavage of the N - terminal signal peptide releases the mature MYDGF. The sequence of the mature human MYDGF protein consists of 142 amino acids and is shown herein as SEQ ID NO: 6. The proteins of the present invention differ from mature MYDGF only in that a single amino acid is added to their N - termini. Thus, these proteins can be regarded as recombinant variants of the native human MYDGF protein. The protein of SEQ ID NO: 1 contains an additional alanine residue at its N - terminus that is not present in the mature human MYDGF protein. This variant is referred to herein below as "[+A]" or "[+A] variant". The protein of SEQ ID NO: 2 differs from the mature human MYDGF protein by an additional serine residue at its N - terminus. This variant is referred to herein as "[+S]" or "[+S] variant".

[0010] The protein is associated with a particularly low risk of containing antigenic epitopes not derived from human MYDGF. Thus, the proteins of the present invention exhibit a minimal risk of generating anti - drug antibodies. The difference between the proteins of the present invention and the native human MYDGF protein lies in a single amino acid, meaning that the region added to the native protein is too small to give rise to new epitopes. The absence of anti - drug antibodies makes the proteins of the present invention very suitable for use for therapeutic purposes. Preferably, administration of the protein to humans will generate only minimal levels, and more preferably no levels, of anti - drug antibodies or antibodies directed against endogenous MYDGF.

[0011] As can be seen from the following examples, the protein of the present invention exhibits low levels of chemical modification and post-translational modification in a cell-based expression system, i.e., an expression system using eukaryotic or prokaryotic cells for the recombinant production of proteins. An effective reduction of chemical modification and post-translational modification during the production of proteins for pharmaceutical applications is of fundamental importance. In particular, the protein of the present invention is characterized by low levels of carbamoylation and glucosylation. As used herein, carbamoylation is a non-enzymatic reaction in which a carbamoyl moiety is added to a protein, peptide, or amino acid. After expression in a cell-based expression system, isolation from protein inclusion bodies, and protein refolding, carbamoylation preferably occurs at less than 6.0% (w / w) of the total protein of SEQ ID NO: 1, more preferably less than 5.5% (w / w), 5.0% (w / w), 4.5% (w / w), or 4.0% (w / w) of the total protein having the amino acid sequence of SEQ ID NO: 1. Similarly, carbamoylation preferably occurs at less than 6.0% (w / w) of the total protein of SEQ ID NO: 2, more preferably less than 5.5% (w / w), 5.0% (w / w), 4.5% (w / w), or 4.0% (w / w) of the total protein having the amino acid sequence of SEQ ID NO: 2 after expression of the protein in a cell-based expression system, isolation from protein inclusion bodies, and protein refolding. Generally, for pharmaceutical applications, a carbamoylation level of less than 6.0% (w / w) is acceptable and does not pose a risk.

[0012] In addition, the proteins disclosed herein are characterized by low levels of glucosylation. Glucosylation of recombinantly expressed proteins is typically observed in host bacterial cells such as cells of Escherichia coli BL21(DE3). This modification results from the formation of 6-phosphogluconolactone (6-PGLac), a compound produced by the enzyme glucose-6-phosphate dehydrogenase. With respect to the proteins of the present invention, glucosylation preferably occurs at less than 4.0% (w / w) of the total protein of SEQ ID NO: 1, more preferably less than 3.5% (w / w), 3.0% (w / w), 2.5% (w / w), or 2.0% (w / w) of the total protein of SEQ ID NO: 1 after expression in a cell-based expression system, isolation from protein inclusion bodies, and protein refolding. Similarly, carbamoylation preferably occurs at less than 4.0% (w / w) of the total protein of SEQ ID NO: 2, more preferably less than 3.5% (w / w), 3.0% (w / w), 2.5% (w / w), or 2.0% (w / w) of the total protein of SEQ ID NO: 2. Generally, for pharmaceutical applications, glucosylation levels of less than 4% are acceptable and pose no risk.

[0013] A further aspect of the present invention relates to a protein having the amino acid sequence of SEQ ID NO: 1 and further having a spectrum in two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) that is essentially the same as that shown in Table 1 below. In particular, when samples of each MYDGF protein at 8.5 mg / ml in 50 mM sodium phosphate buffer at pH 7.4 containing 50 mM sodium chloride and 9% (v / v) D 2 O were analyzed, at least two of the 1 H and / or 15 N peaks in Table 1, preferably 1 H and / or 15Have an NMR spectrum containing at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, at least 30, at least 32, at least 34, at least 36, at least 38, at least 40, at least 42, at least 44, at least 46, at least 48, at least 50, at least 52, at least 54, at least 56, at least 58, at least 60, at least 62, at least 64, at least 66, at least 68, at least 70, at least 72, at least 74, at least 76, at least 78, at least 80, at least 82, at least 84, at least 86, at least 88, at least 90, at least 92, at least 94, at least 96, at least 98, at least 100, at least 102, at least 104, at least 106, at least 108, at least 110, at least 112, at least 114, at least 116, at least 118, at least 120, at least 122, at least 124, at least 126, at least 128, at least 130, at least 132, or at least 134 peaks of N.

[0014] More preferably, the protein is 50 mM sodium chloride and 9% (v / v) D 2 When analyzing a sample of each MYDGF protein at 8.5 mg / ml in 50 mM sodium phosphate buffer at pH 7.4 containing O, shown in Table 1 1 H and / or 15 Have an NMR spectrum containing all 136 of H and / or N, or consisting of the same. Therefore, the protein has the secondary and tertiary structures of the native human MYDGF protein. In yet another embodiment, the present invention relates to 1 H and / or 15More than 70% of the N peak, preferably more than 80%, more than 90%, or more than 95%, results in a combined chemical shift deviation (CCSD) value of less than 0.01 ppm compared to the corresponding peak in Table 1, for the folded protein. CCSD is calculated according to the following formula (Brinson et al. 2019):

[0015] [Number] where δ H and δ N are, respectively, the 1 H and 15 N chemical shifts of the indicated cross-peak, and δ Href and δ Nref are the 1 H and 15 N reference chemical shifts for the same cross-peak. When more than 70% of the 1 H and / or 15 N peaks in the 2D-NMR map result in a CCSD value of less than 0.01 ppm, preferably more than 80%, more than 90%, or more than 95%, the protein folding is almost identical to that observed for the [+A] variant in Table 1. Specifically, when more than 90% or more than 95% of the 1 H and / or 15 N peaks in the 2D-NMR map show a CCSD value of less than 0.01 ppm, the protein folding is almost identical to that observed for the [+A] variant in Table 1.

[0016] [Table 1] TIFF2025518285000004.tif228153 TIFF2025518285000005.tif238153 TIFF2025518285000006.tif37153 A further embodiment of the present invention is a composition comprising a protein as described above, preferably a composition obtained from recombinant expression in a bacterial expression system such as the method described in more detail below.

[0017] The composition of the present invention may contain a protein having the amino acid sequence of SEQ ID NO: 1, together with its variants that are shorter in length and show 100% sequence identity with the amino acid sequence of SEQ ID NO: 1 over their entire length, and the length is at least 100 amino acids in a situation where gaps are not allowed in the alignment. In said composition, when determined by liquid chromatography mass spectrometry (LCMS) according to Tolonen et al (2011), the ratio of the sum of the signals obtained from the protein according to SEQ ID NO: 1 to the signal obtained from said shorter variant is higher than 20, preferably higher than 50, higher than 75, higher than 100, higher than 125, higher than 150, higher than 175, higher than 200, higher than 225, higher than 250, higher than 275, higher than 300, higher than 325, higher than 350, higher than 375, higher than 400, higher than 425, more preferably higher than 450. As can be seen from Table 15 below, the ratio of the signal obtained from the protein according to SEQ ID NO: 1 to the signal obtained from the shorter variant was found to be 466 when determined by LCMS according to Tolonen et al (2011). When calculating the ratio of the sum of the signal obtained from the protein according to SEQ ID NO: 1 to the signal obtained from said shorter variant, any carbamoylated protein or glucosonylated protein is excluded. The percentage for the calculation of the ratio is based on the sum of the peak intensities of the unmodified MYDGF protein, plus the annotated post-translational modification (PTM) species of MYDGF, in the deconvoluted intact mass spectrum of the MYDGF protein in the composition. The minimum requirements for the mass spectrometer and data processing are outlined in Example 4 below.

[0018] The composition of the present invention may also contain a protein having the amino acid sequence of SEQ ID NO: 2, together with variants thereof that are shorter in length and show 100% sequence identity with the amino acid sequence of SEQ ID NO: 2 over their entire length, and the length is at least 100 amino acids in a situation where gaps are not allowed in the alignment. In the said composition, when determined by LCMS according to Tolonen et al (2011), the ratio of the signal obtained from the protein according to SEQ ID NO: 2 to the sum of the signals obtained from the shorter variants is higher than 20, preferably higher than 50, higher than 75, higher than 100, higher than 125, higher than 150, more preferably higher than 175 or higher than 180. As can be seen from Table 15 below, the ratio of the signal obtained from the protein according to SEQ ID NO: 2 to the signal obtained from the shorter variant was found to be 186 when determined by LCMS according to Tolonen et al (2011). When calculating the ratio of the signal obtained from the protein according to SEQ ID NO: 2 to the sum of the signals obtained from the shorter variants, any carbamoylated proteins and glucosaminylated proteins are excluded. The percentage for the calculation of the ratio is based on the sum of the peak intensities of the unmodified MYDGF protein, plus the annotated post-translational modification (PTM) species of MYDGF, in the deconvoluted intact mass spectrum of the MYDGF protein in the composition. The minimum requirements for the mass spectrometer and data processing are outlined in Example 4 below.

[0019] In one embodiment, less than 8%, preferably less than 7%, less than 6%, or less than 5% of the MYDGF protein in the composition of the present invention is carbamoylated, and the percentage is based on the sum of the peak intensities of the unmodified MYDGF protein, plus the annotated post-translational modification (PTM) species of MYDGF, in the deconvoluted intact mass spectrum of the MYDGF protein in the composition. The minimum requirements for the mass spectrometer and data processing are outlined in Example 4 below. In another embodiment, less than 6%, preferably less than 5%, less than 4%, or less than 3% of the MYDGF protein in the composition of the invention is glucosylated, and the percentage is based on the sum of the peak intensities of the unmodified MYDGF protein, plus the annotated PTM species of MYDGF, in the deconvoluted intact mass spectrum of the MYDGF protein in the composition. The minimum requirements for the mass spectrometer and data processing are outlined in Example 4 below.

[0020] According to another object of the invention, the composition of the invention preferably contains a low amount of DNA derived from the host cell used for the production of the MYDGF protein. Preferably, the composition of the invention contains less than 20 pg / mg, preferably less than 15 pg / mg, more preferably less than 10 pg / mg, and most preferably 5 pg of host cell DNA per mg of the composition, for example, less than 3 pg, less than 2 pg, or less than 1 pg of host cell DNA per mg of the composition. Preferably, the presence of host cell DNA in the composition is determined by quantitative polymerase chain reaction (qPCR), such as real-time qPCR. According to another object of the invention, the composition of the invention also preferably contains only a low amount of bacterial endotoxin resulting from the production of the MYDGF protein in host bacterial cells. Specifically, it is preferred that the composition contains less than 0.2 EU per mg of the composition, preferably less than 0.1 EU per mg of the composition, less than 0.09 EU, or less than 0.08 EU of bacterial endotoxin. Suitable methods for detecting the presence of bacterial endotoxin include the kinetic colorimetric methods described in the latest United States Pharmacopeia (USP-NF 2021, 2nd, Chapter 85), the European Pharmacopeia (10th Edition 2021, 10.5, Chapter 2.6.14), and the Japanese Pharmacopeia, Supplement II, JP 17th Edition, 4.01).

[0021] According to another object of the present invention, less than 8% (w / w), preferably less than 7% (w / w), less than 6% (w / w), or less than 5% (w / w) of the protein in the composition of the present invention is carbamoylated. Similarly, less than 6% (w / w), preferably less than 5% (w / w), less than 4% (w / w), or less than 3% (w / w) of the protein in the composition of the present invention is glucosonoylated. The composition of the present invention contains a detectable amount of carbamoylated protein, however, it is particularly preferred that the amount of carbamoylated protein in the composition is less than 5% (w / w). Similarly, the composition of the present invention contains a detectable amount of glucosonoylated protein, however, it is particularly preferred that the amount of glucosonoylated protein in the composition is less than 5% (w / w). The composition of the present invention may contain urea resulting from the inclusion body solubilization and / or refolding step. The composition of the present invention preferably does not contain protein aggregates that may result from the aggregation of protein molecules, such as dimers, trimers, or oligomers of the MYDGF protein variant, or contains only a low amount of protein aggregates. Preferably, the composition contains the above-mentioned MYDGF, that is, the protein mainly as a monomer of SEQ ID NO: 1 or SEQ ID NO: 2. More preferably, the monomer content of the protein in the composition is 95% (w / w) or more, even more preferably 96% (w / w) or more, 97% (w / w) or more, 98% (w / w) or more, or 99% (w / w) or more. In other words, the amount of protein aggregates in the composition is about 5% (w / w) or less, even more preferably 4% (w / w) or less, 3% (w / w) or less, 2% (w / w) or less, or 1% (w / w) or less, or not detectable at all. The amounts of protein monomer and aggregate are preferably measured by size exclusion chromatography (SEC), more preferably by size exclusion high performance liquid chromatography (SEC HPLC).

[0022] The composition of the present invention preferably contains a 143 - amino - acid protein having the amino - acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The composition preferably contains protein molecules shorter than 143 amino acids, less than 5% (w / w), less than 4% (w / w), less than 3% (w / w), less than 2% (w / w), preferably less than 1% (w / w) as measured by LCMS. The composition of the present invention preferably contains, based on the total mass of all non - glucosylated and non - carbamoylated proteins in the composition containing at least 100 consecutive amino acids of the sequence of SEQ ID NO: 1, protein molecules that differ from the amino - acid sequence of SEQ ID NO: 1 by either (i) a deletion of 1 - 4 amino acids at the N - terminus of SEQ ID NO: 1 or (ii) an addition of a single amino acid at the N - terminus of SEQ ID NO: 1, as determined by LCMS after reductive dimethylation (stable isotope dimethyl labeling, SIDL) according to Tolonen et al (2011), less than 5% (w / w), less than 4% (w / w), less than 3% (w / w), less than 2% (w / w), more preferably less than 1% (w / w). The composition shown in Table 15 containing the +A variant (SEQ ID NO: 1) shows 0.2% of the protein according to (i) or (ii).

[0023] The composition of the present invention preferably contains, based on the total mass of all non - glucosylated and non - carbamoylated proteins in the composition containing at least 100 consecutive amino acids of the sequence of SEQ ID NO: 1, protein molecules that differ from the amino - acid sequence of SEQ ID NO: 1 by a deletion of 1 - 4 amino acids at the N - terminus of SEQ ID NO: 1, as determined by LCMS after reductive dimethylation (stable isotope dimethyl labeling, SIDL) according to Tolonen et al (2011), less than 5% (w / w), less than 4% (w / w), less than 3% (w / w), less than 2% (w / w), more preferably less than 1% (w / w). The composition shown in Table 15 containing the +A variant (SEQ ID NO: 1) shows 0.2% (w / w) of such protein deletion. The composition of the present invention preferably contains, based on the total mass of all non-gluconoylated and non-carbamoylated proteins in the composition containing at least 100 consecutive amino acids of the sequence of SEQ ID NO: 2, less than 5% (w / w), less than 4% (w / w), less than 3% (w / w), less than 2% (w / w), more preferably less than 1% (w / w) of protein molecules that differ from the amino acid sequence of SEQ ID NO: 2 by either (i) a deletion of 1 to 4 amino acids at the N-terminus of SEQ ID NO: 2 or (ii) an addition of a single amino acid at the N-terminus of SEQ ID NO: 2, as determined by LCMS after reductive dimethylation (stable isotope dimethyl labeling, SIDL) according to Tolonen et al (2011). The composition shown in Table 15 containing the +S variant (SEQ ID NO: 2) exhibits 3.1% (w / w) of the protein according to (i) or (ii).

[0024] The composition of the present invention preferably contains, based on the total mass of all non-gluconoylated and non-carbamoylated proteins in the composition containing at least 100 consecutive amino acids of the sequence of SEQ ID NO: 2, less than 5% (w / w), less than 4% (w / w), less than 3% (w / w), less than 2% (w / w), more preferably less than 1% (w / w) of protein molecules that differ from the amino acid sequence of SEQ ID NO: 2 by a deletion of 1 to 4 amino acids at the N-terminus of SEQ ID NO: 2, as determined by LCMS after reductive dimethylation (stable isotope dimethyl labeling, SIDL) according to Tolonen et al (2011). The composition shown in Table 15 containing the +S variant (SEQ ID NO: 2) exhibits 0.5% (w / w) of such protein deletion. The composition of the present invention preferably contains, based on the total mass of all non-gluconoylated and non-carbamoylated proteins in the composition containing at least 100 consecutive amino acids of the sequence of SEQ ID NO: 1, more than 95% (w / w), more than 96% (w / w), more than 97% (w / w), more than 98% (w / w), more preferably more than 99% (w / w) of protein molecules having a length of 143 amino acids and consisting of the amino acid sequence of SEQ ID NO: 1, as determined by LCMS after reductive dimethylation (stable isotope dimethyl labeling, SIDL) according to Tolonen et al (2011).

[0025] The composition of the present invention preferably comprises, based on the total mass of all non-gluconoylated and non-carbamoylated proteins in the composition containing at least 100 consecutive amino acids of the sequence of SEQ ID NO: 2, more than 95% (w / w), more than 96% (w / w), more than 97% (w / w), more than 98% (w / w), more preferably more than 99% (w / w) of protein molecules having a length of 143 amino acids and consisting of the amino acid sequence of SEQ ID NO: 2, as determined by LCMS after reductive dimethylation (stable isotope dimethyl labeling, SIDL) according to Tolonen et al (2011). The composition of the present invention preferably comprises, based on the total mass of all proteins in the composition containing at least 100 consecutive amino acids of the sequence of SEQ ID NO: 1, more than 90% (w / w), more than 91% (w / w), more than 92% (w / w), more than 93% (w / w), more than 94% (w / w), more preferably more than 95% (w / w) of protein molecules having a length of 143 amino acids and consisting of the amino acid sequence of SEQ ID NO: 1, as measured by liquid chromatography mass spectrometry (LCMS), and gluconoylated proteins, carbamoylated proteins, dehydrated proteins, and Na+ adducts are ignored for percentage calculations.

[0026] The composition of the present invention preferably comprises, based on the total mass of all proteins in the composition containing at least 100 consecutive amino acids of the sequence of SEQ ID NO: 2, more than 90% (w / w), more than 91% (w / w), more than 92% (w / w), more than 93% (w / w), more than 94% (w / w), more preferably more than 95% (w / w) of protein molecules having a length of 143 amino acids and consisting of the amino acid sequence of SEQ ID NO: 2, as measured by liquid chromatography mass spectrometry (LCMS), and gluconoylated proteins, carbamoylated proteins, dehydrated proteins, and Na+ adducts are ignored for percentage calculations. The proteins of the present invention share at least one biological activity of the naturally occurring human mature MYDGF protein that makes them useful for therapeutic application. Accordingly, the present invention also relates to such compositions for use as medicaments, in particular for uses known for MYDGF, see for example WO2014 / 111458 and WO2021 / 148411.

[0027] Preferably, the protein is (i) active in treating or preventing a disease or condition selected from the group consisting of injury, wound, ischemia, reperfusion injury, trauma, mechanical overload, poisoning, surgery, primary or secondary cardiomyopathy, post-ischemic systolic dysfunction, myocardial infarction, preferably acute myocardial infarction, angina, heart failure, cardiac inflammation, cardiac dysfunction, hypertrophy, and fibrosis; (ii) promoting or improving cardiac tissue regeneration, cardiomyocyte proliferation, angiogenesis, cardiac function, or left ventricular systolic function after myocardial infarction; (iii) protecting cardiomyocytes from death, such as by apoptosis or necrosis; or (iv) reducing infarct size after myocardial infarction, preferably acute myocardial infarction. See WO2014 / 111458. The cardiomyopathy that can be treated can be a genetic cardiomyopathy or a cardiomyopathy caused by a spontaneous mutation. The cardiomyopathy that can be treated can also be a secondary cardiomyopathy, preferably an ischemic cardiomyopathy caused by atherosclerotic or other coronary artery disease, a cardiomyopathy caused by myocardial infection or intoxication, a hypertensive heart disease caused by pulmonary hypertension and / or arterial hypertension, or a cardiomyopathy caused by a heart valve disease. The cardiomyopathy is preferably a cardiomyopathy selected from the group consisting of hypertrophic cardiomyopathy (HCM or HOCM), arrhythmogenic right ventricular cardiomyopathy (ARVC), isolated ventricular noncompaction mitochondrial myopathy, dilated cardiomyopathy (DCM), restrictive cardiomyopathy (RCM), takotsubo cardiomyopathy, Reiffel endocarditis, diabetic cardiomyopathy, alcoholic cardiomyopathy, or a cardiomyopathy associated with obesity.

[0028] The heart failure that can be treated is preferably chronic heart failure. The heart failure or chronic heart failure can be heart failure with preserved ejection fraction (HFpEF), heart failure with reduced ejection fraction (HFrEF), or heart failure with mildly reduced ejection fraction (HFmrEF). See WO2021 / 148411. It is particularly preferred that the above protein has at least a part of the activity of the naturally occurring human mature MYDGF protein in enhancing coronary endothelial cell proliferation or coronary endothelial cell growth. In particular, it is preferred that the protein has at least 50%, preferably at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the activity of the naturally occurring human mature MYDGF protein in enhancing coronary endothelial cell proliferation. The protein may also have an activity in enhancing coronary endothelial cell proliferation that is higher than the activity of the naturally occurring human mature MYDGF protein, for example, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 180%, at least 190%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, or at least 500% of the activity with respect to the human mature MYDGF protein. Preferably, the activity in enhancing coronary endothelial cell proliferation is determined as described in the potency assay of Example 6 below. The activity of the protein is calculated by the formula: Activity [%] = EC of mature MYDGF 50 / EC of test protein 50 ) × 100.

[0029] In another embodiment, it is preferred that the protein has at least 50% of the activity of the [+G]-HEK variant in enhancing coronary endothelial cell proliferation, and the [+G]-HEK variant is produced as described in Polten et al. (2019) and Ebenhoch et al. (2019). It is particularly preferred that the protein has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of said activity. The protein may also have an activity in enhancing coronary endothelial cell proliferation that is higher than the activity of the [+G]-HEK variant, for example, at least 110%, at least 120%, at least 130%, at least 90%, at least 140%, at least 150%, at least 160%, at least 180%, at least 190%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, or at least 500% of the activity relative to the [+G]-HEK variant. Preferably, the activity in enhancing coronary endothelial cell proliferation is determined as described in the potency assay of Example 6 below. The activity of the protein is calculated by the formula: Activity [%]=[+G]-HEK's EC 50 / Test protein's EC 50 )×100.

[0030] In yet another embodiment, it is preferred that the protein has at least 50%, preferably at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the activity of the naturally occurring human mature MYDGF protein in enhancing cardiomyocyte proliferation. The protein may also have an activity in enhancing cardiomyocyte proliferation that is higher than the activity of the naturally occurring human mature MYDGF protein, for example, at least 110%, at least 120%, at least 130%, at least 90%, at least 140%, at least 150%, at least 160%, at least 180%, at least 190%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, or at least 500% of the activity relative to the human mature MYDGF protein. Preferably, the activity in enhancing cardiomyocyte proliferation is determined as described in the potency assay of Example 7 below. The activity of the protein is calculated by the formula: Activity [%] = EC of mature MYDGF 50 / EC of test protein 50 ) × 100.

[0031] In yet another embodiment, the protein preferably has at least 50% of the activity of the [+G]-HEK variant in enhancing cardiomyocyte proliferation, and the [+G]-HEK variant is produced as described in Polten et al. (2019) and Ebenhoch et al. (2019). It is particularly preferred that the protein has at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of said activity. The protein may also have an activity in enhancing cardiomyocyte proliferation that is higher than the activity of the [+G]-HEK variant, for example, at least 110%, at least 120%, at least 130%, at least 90%, at least 140%, at least 150%, at least 160%, at least 180%, at least 190%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, or at least 500% of the activity relative to the [+G]-HEK variant. Preferably, the activity in enhancing cardiomyocyte proliferation is determined as described in the potency assay of Example 7 below. The activity of the protein is calculated by the formula: Activity [%]=[+G]-HEK's EC 50 ) / EC of test protein 50 )×100.

[0032] In yet another embodiment, when the above protein is measured in the potency assay of Example 6 below, it preferably enhances coronary artery endothelial cell proliferation with an EC 50 of less than 100 ng / ml. Preferably, when the protein is measured in the potency assay of Example 6, it enhances coronary artery endothelial cell proliferation with an EC 50 of less than 95 ng / ml, less than 90 ng / ml, less than 85 ng / ml, less than 80 ng / ml, less than 75 ng / ml, less than 70 ng / ml, less than 65 ng / ml, or less than 60 ng / ml. In yet another embodiment, when the protein is measured in the potency assay of Example 7 below, it has an EC 50It is preferable to enhance cardiomyocyte proliferation. Preferably, when measured in the potency assay of Example 7, the protein has an EC of less than 95 ng / ml, less than 90 ng / ml, less than 85 ng / ml, less than 80 ng / ml, less than 75 ng / ml, less than 70 ng / ml, less than 65 ng / ml, or less than 60 ng / ml, less than 55 ng / ml, less than 50 ng / ml, less than 45 ng / ml, less than 40 ng / ml, less than 35 ng / ml, less than 30 ng / ml, or less than 25 ng / ml 50 enhances coronary artery endothelial cell proliferation.

[0033] The present invention also relates to a nucleic acid encoding a protein that is mature and gives rise to a protein as described above, i.e., a protein having the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The nucleic acid can be DNA or RNA. However, the nucleic acid is preferably a DNA molecule. The present invention also relates to a vector or plasmid containing a nucleic acid encoding a protein that is mature and gives rise to one of the MYDGF proteins of the present invention. Preferably, the vector is an expression vector that enables the expression of a protein that matures into the protein of SEQ ID NO: 1 or SEQ ID NO: 2 in prokaryotic or eukaryotic cells. It is particularly preferred that the vector is a prokaryotic expression vector, i.e., a vector that enables the expression of recombinant proteins in a prokaryotic cell environment. Even more preferably, the vector is a bacterial expression vector, i.e., a vector that enables the expression of recombinant proteins in bacterial cells. The vector preferably contains an origin of replication, a promoter, a polylinker for cloning, a transcription terminator, and a gene that enables selection, for example, a gene encoding a protein that confers antibiotic resistance. An enormous number of expression vectors have been described for E. coli and other bacterial hosts. Examples of vectors suitable for protein expression in E. coli cells include, for example, vectors of the pBluescript series, pUC series, pQE series, or pET series. The vector preferably contains an inducible promoter system that can initiate expression upon the addition of an inducer compound.

[0034] In a preferred embodiment, the vector having the nucleic acid encoding the above MYDGF protein is a pET type vector. These vectors typically include an origin of replication, a T7 promoter specific for T7 RNA polymerase, a lac operator for binding the lacI repressor protein, a polylinker for cloning the nucleic acid sequence encoding the protein to be expressed, a transcription termination sequence, an ampicillin or kanamycin resistance gene, and a lacI gene encoding the lac repressor protein. In the absence of isopropyl-β-D-thiogalactopyranoside (IPTG) or lactose, the repressor protein binds to the lac operator, thereby inhibiting the T7 promoter and blocking the expression of the target protein. The binding of IPTG or lactose to the lac repressor protein causes a conformational change that results in the detachment of the protein from the operator and the induction of expression from the T7 promoter. Suitable pET vectors for use in the method of the present invention include, but are not limited to, pET21a(+), pET24a(+), pET28a(+), pET29a(+), pET30a(+), pET41a(+), pET44a(+), pET21b(+), pET24b(+), pET26b(+), pET28b(+), pET29b(+), pET30b(+), pET42b(+), and pET44b(+). Vectors based on the pET-26b(+) backbone are particularly preferred. Further examples of suitable vectors are described, for example, in "Cloning Vectors" (Pouwels et al. (eds.) Elsevier, Amsterdam New York Oxford, 1985).

[0035] The expression vector can be transformed into a host eukaryotic or host prokaryotic cell by any suitable method. For example, the expression vector used for E. coli can be introduced into the host cell, as described by Maniatis et al. 1982, Molecular Cloning, A laboratory Manual, Cold Spring Harbor Laboratory, for example, by electroporation or by a chemical method such as calcium phosphate-mediated transformation. The present invention also relates to a host cell containing the protein, nucleic acid, or vector as described above. The host cell can be a eukaryotic or prokaryotic cell, but it is particularly preferred that it is a host prokaryotic cell such as a bacterial cell. The type of bacterial cell is not particularly limited, but it is preferred that the host cell is an E. coli cell such as an Escherichia coli BL21 cell.

[0036] The present invention also relates to the above composition for the preparation of a pharmaceutical composition, i.e., a composition containing the above protein, preferably a composition obtained by a method of recombinant expression in a cell-based expression system, for example, a method described in detail below. The present invention also relates to a pharmaceutical composition comprising the above protein or the above composition. The pharmaceutical composition may contain, in addition to the protein, pharmaceutically acceptable carriers and other excipients commonly used for the formulation of pharmaceutical compositions. Generally, the pharmaceutical composition can be formulated for various routes of administration. Preferably, the pharmaceutical composition of the present invention is formulated for parenteral administration, such as by intravenous, intraarterial, intracoronary, or intramuscular injection. Examples of pharmaceutical compositions suitable for injection or infusion include solutions or dispersions, and powders for the immediate preparation of such injectable solutions or dispersions. The pharmaceutical composition for injection must be sterile and should be stable under the conditions of manufacture and storage. Preferably, the pharmaceutical composition for injection or infusion also contains preservatives such as chlorobutanol, phenol, ascorbic acid, thimerosal, and other similar substances. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (trademark) (BASF), or phosphate buffered saline (PBS). The sterile solution for injection can be prepared by incorporating the required amount of MYDGF protein into a suitable solvent and then filter sterilizing it.

[0037] In addition to the MYDGF protein, the pharmaceutical composition of the present invention may further contain, in a subject in need thereof, (i) an additional active substance effective to treat or prevent a disease selected from the group consisting of injury, wound, ischemia, reperfusion injury, trauma, mechanical overload, poisoning, surgery, primary or secondary cardiomyopathy, post-ischemic systolic dysfunction, myocardial infarction, preferably acute myocardial infarction, angina pectoris, heart failure, cardiac inflammation, cardiac dysfunction, hypertrophy, and fibrosis; (ii) to promote or improve cardiac tissue regeneration, cardiomyocyte proliferation, angiogenesis, cardiac function, or left ventricular systolic function after myocardial infarction; (iii) to protect cardiomyocytes from death, such as by apoptosis or necrosis; or (iv) to reduce infarct size after myocardial infarction, preferably acute myocardial infarction. For example, the pharmaceutical composition may comprise one or more angiotensin-converting enzyme (ACE) inhibitors such as benazepril, zofenopril, perindopril, trandolapril, captopril, enalapril, lisinopril, and ramipril. The pharmaceutical composition may also comprise one or more diuretics such as chlorothiazide, hydrochlorothiazide, bendroflumethiazide, spironolactone, chlorthalidone, methyclothiazide, polythiazide, triamterene, furosemide, ethacrynic acid, metolazone, bumetanide, indapamide, amiloride, acetazolamide, torsemide, and eplerenone. The pharmaceutical composition may also comprise one or more beta blockers such as acebutolol, atenolol, betaxolol, bisoprolol, carvedilol, celiprolol, esmolol, metoprolol, nebivolol, propranolol, sotalol, and / or timolol.

[0038] When the protein is used in combination with any of the above additional active substances, such as an ACE inhibitor, a diuretic, and / or a beta blocker, the two active substances may also be administered separately from each other, i.e., in the form of separate pharmaceutical compositions, one containing the MYDGF protein and the other containing the additional active substance. The separate pharmaceutical compositions can be administered simultaneously, i.e., at the same time at two separate administration sites, or they can be administered sequentially (in either order) at the same administration site or at different administration sites. The present invention also relates to a protein or pharmaceutical composition as described above for use as a medicament. More specifically, the protein or pharmaceutical composition (i) treats or prevents a disease or condition selected from the group consisting of injury, wound, ischemia, reperfusion injury, trauma, mechanical overload, poisoning, surgery, primary or secondary cardiomyopathy, post-ischemic systolic dysfunction, myocardial infarction, preferably acute myocardial infarction, angina pectoris, heart failure, cardiac inflammation, cardiac dysfunction, hypertrophy, and fibrosis; (ii) promotes or improves cardiac tissue regeneration, cardiomyocyte proliferation, angiogenesis, cardiac function, or left ventricular systolic function after myocardial infarction; (iii) protects cardiomyocytes from death, for example by apoptosis or necrosis; or (iv) is suitable for use as a medicament for reducing infarct size after myocardial infarction, preferably acute myocardial infarction.

[0039] The present invention also relates to a method for (i) treating or preventing a disease or condition selected from the group consisting of injury, wound, ischemia, reperfusion injury, trauma, mechanical overload, poisoning, surgery, primary or secondary cardiomyopathy, post-ischemic systolic dysfunction, myocardial infarction, preferably acute myocardial infarction, angina pectoris, heart failure, cardiac inflammation, cardiac dysfunction, hypertrophy, and fibrosis; (ii) promoting or improving cardiac tissue regeneration, cardiomyocyte proliferation, angiogenesis, cardiac function, or left ventricular systolic function after myocardial infarction; (iii) protecting cardiomyocytes from death, for example by apoptosis or necrosis; or (iv) reducing infarct size after myocardial infarction, preferably acute myocardial infarction, in a subject in need thereof, the method comprising administering an effective amount of a pharmaceutical composition as described above comprising the protein of SEQ ID NO: 1 or SEQ ID NO: 2. The present invention also relates to a method for recombinant expression of MYDGF protein in a host cell, (a) supplying a host cell as described above, preferably a host cell comprising a nucleic acid encoding a 143-amino acid protein having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 after maturation; (b) culturing the host cell under conditions that allow expression of the MYDGF protein; (c) Isolating inclusion bodies containing the MYDGF protein from the host cell; and (d) Solubilizing the inclusion bodies and refolding the MYDGF protein A method is provided that includes the above.

[0040] The method of the present invention described above is directed to the production of the proteins shown in SEQ ID NO: 1 and SEQ ID NO: 2. The method uses a host cell as described above, preferably a host prokaryotic cell containing a nucleic acid encoding a protein that results in one of the mature proteins of the present invention. The host bacterial cell used in the method of the present invention preferably contains a nucleic acid encoding a protein that results in the mature protein of SEQ ID NO: 1 or a nucleic acid encoding a protein that results in the mature protein of SEQ ID NO: 2, which is inserted into an expression vector that enables the expression of the recombinant protein in the host cell. The host cell can be any type of eukaryotic or prokaryotic cell suitable for use in the expression of a recombinant exogenous protein, i.e., a protein not naturally produced by the host cell. Preferably, the host cell is a prokaryotic cell such as a bacterial cell. More preferably, the cell is a bacterial cell belonging to the genus Escherichia, and even more preferably to the species E. coli. The use of the E. coli BL21 strain or its derivative is most preferred.

[0041] Preferably, step (a) of the above method is (i) Supplying a host cell containing a nucleic acid having an open reading frame flanked by start and stop codons, according to the sequence of SEQ ID NO: 11 or SEQ ID NO: 12, operably linked to a promoter; or (ii) Supplying a host cell containing a nucleic acid encoding a 144 - amino - acid protein having the amino - acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16 before maturation including. The amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16 is a MYDGF protein containing an N-terminal methionine residue derived from the start codon. These proteins are exposed to a maturation process that removes the N-terminal methionine. Maturation is preferably effected by one or more host cell-derived aminopeptidases, more preferably one or more host cell-derived methionine aminopeptidases. For example, one or more aminopeptidases can be produced by host bacterial cells, such as E. coli, used for recombinant expression. It is particularly preferred that step (a) comprises providing a host cell comprising a nucleic acid of SEQ ID NO: 7 or SEQ ID NO: 8 which is an expression vector, preferably a circular, coiled or supercoiled expression vector.

[0042] In step (b) of the method, the host bacterial cells are cultured under conditions that allow for the expression of the protein in the host bacterial cells. The conditions for imparting the expression of the MYDGF protein depend on the host prokaryotic cells and the expression vector used in the process. Appropriate conditions can be readily selected and applied by those skilled in the art. For example, when an inducible bacterial expression system such as a pET vector is used, the conditions for enabling the expression of the target protein typically include a culture temperature of 20 to 42 °C, preferably 30 to 40 °C, more preferably 35 to 38 °C. The culture medium typically has a pH of 6.5 to 9.0, more typically 7.0 to 8.0, preferably 7.5. The fermentation of the culture can be continued for a period ranging from several hours to several days. The cells can be cultured in a batch or fed-batch process. For example, when the culture is carried out as a batch process, the culture time is usually in the range of about 12 hours to about 36 hours. When a continuous process is used, the fermentation time can be up to 21 days or more. Suitable methods for culturing host bacterial cells are described in Encyclopaedia of Bioprocess Technology: Fermentation, Biocatalysis, and Bioseparation, Volumes 1-5, Flickinger, M.C., Drew, S.W. (eds.), 1999 John Wiley & Sons. Preferably, the protein is expressed in the host cells using an inducible expression system, such as a system that allows for the initiation of protein expression by the addition of an inducer compound such as IPTG or lactose to the culture medium.

[0043] The protein expressed in this way accumulates in the host cells in an insoluble form in so-called inclusion bodies. This means that the expressed protein accumulates intracellularly and deposits in the form of insoluble aggregates of inactive misfolded proteins. In step (c) of the method of the present invention, the inclusion bodies containing insoluble MYDGF protein are isolated from the host cells. For that purpose, the host bacterial cells are collected after cultivation and disrupted, for example, by high-pressure homogenization or other generally known cell lysis procedures. The inclusion bodies can be isolated from their solubilized solution by various methods, for example, by cylindrical centrifugation such as high speed tubular centrifugation. Methods for the isolation of inclusion bodies from bacterial cells are generally known and are described, for example, in Peternel & Komel (2010) and Eggenreich et al. (2020).

[0044] In step (d) of the method of the present invention, the MYDGF protein in the isolated inclusion bodies obtained from step (c) is solubilized and refolded. Methods for solubilizing proteins are known and include, for example, incubation of the inclusion bodies in the presence of urea, guanidine hydrochloride (GuHCl), and / or DTT, followed by filtration. It is preferred herein that the refolding of the protein is carried out in the presence of urea. Filtration can include one or more of depth filtration, ultrafiltration, and / or diafiltration. Methods for refolding proteins are likewise known and include, for example, incubation of the protein solubilized from the inclusion bodies in the presence of urea, CaCl 2 , and / or cystamine. Kits for solubilizing and refolding proteins from inclusion bodies are commercially available from various manufacturing companies. The method can also include step (e) in which a 143-amino acid refolded MYDGF protein having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is obtained. This step follows step (d) of the above method.

[0045] Preferably, the method of the present invention includes an additional step (f) in which the solubilized and refolded MYDGF protein obtained from step (e) is further purified. Protein purification can be carried out according to routine methods and may include one or more of ultrafiltration, diafiltration, hydrophobic interaction chromatography, and / or anion exchange chromatography. Preferably, the purification in step (f) is effected by anion exchange chromatography or hydrophobic interaction chromatography. These types of chromatography can be carried out by contacting the MYDGF protein with a chromatographic resin material under conditions that allow adsorption of the MYDGF protein to the resin. Thereafter, the resin material may be washed to remove impurities such as non-proteinaceous materials or proteins other than MYDGF. In a final step, the MYDGF protein is eluted from the resin. More preferably, the purification in step (f) is effected by anion exchange chromatography. When anion exchange chromatography is used, the adsorption of the MYDGF protein to the anion exchange chromatography resin is preferably carried out at a low ionic strength, for example, at a conductivity of less than 3 mS / cm, less than 2 mS / cm, less than 1.5 mS / cm, or less than 1 mS / cm. Elution can be achieved by increasing the salt concentration and / or decreasing the pH of the liquid phase, i.e., the mobile phase.

[0046] The method of the present invention enables the production of the protein of SEQ ID NO: 1 or SEQ ID NO: 2, particularly in large amounts. As shown in Examples 2 and 3, the protein can be produced by the method of the present invention with a productivity of more than 0.4 g of protein per 100 g of cells, preferably more than 0.5 g of protein per 100 g of cells, more than 0.6 g of protein per 100 g of cells, more than 0.7 g of protein per 100 g of cells, more than 0.8 g of protein per 100 g of cells, more than 0.9 g of protein per 100 g of cells, and more preferably more than 1.0 g of protein per 100 g of cells. In other words, the method of the present invention enables the production of the protein of SEQ ID NO: 1 or SEQ ID NO: 2 in an amount of more than 100 g of protein per batch, preferably more than 150 g of protein per batch, more than 200 g of protein per batch, more than 250 g of protein per batch, more than 300 g of protein per batch, and more preferably more than 350 g or more than 400 g of protein per batch (see Example 3). The present invention also relates to the use of a host cell for the recombinant expression of the MYDGF protein. The host cell is a prokaryotic or eukaryotic cell containing a nucleic acid, plasmid, or vector encoding the protein as described above in this specification.

[0047] A further embodiment of the present invention is a protein according to one embodiment described above, obtained by heterologous expression in bacteria, preferably by the method described above. A further embodiment of the present invention is a protein according to one embodiment described above, obtained by production and refolding in the form of inclusion bodies. A further embodiment of the present invention is a composition comprising the MYDGF protein, wherein the composition is obtained by heterologous expression in bacteria, preferably by the method described above, and the composition contains less than 1% (w / w) of protein molecules shorter than 143 amino acids. In yet another aspect, the present invention provides a method for producing the MYDGF protein in a cell-based expression system, comprising the following steps: (a) Supplying a host cell, preferably a host cell containing a nucleic acid encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 after maturation; (b) Culturing the host cell under conditions that allow expression of the protein; (c) Isolating inclusion bodies containing the MYDGF protein from the host cell; and (d) Solubilizing the inclusion bodies and refolding the MYDGF protein.

[0048] Separate method steps of the method are described above. It is also preferably the case here that the refolding of the protein is carried out in the presence of urea. Accordingly, the refolding of the protein in step (d) preferably includes incubation of the protein in the presence of urea. The method may also include step (e) in which a refolded MYDGF protein of 143 amino acids having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is obtained. This step follows step (d) of the above method. The above method may also include an additional step (f) in which the MYDGF protein expressed by the host cell and isolated from the inclusion bodies is purified. This step may include methods commonly used in the field of protein purification such as ultrafiltration, diafiltration, and / or anion exchange chromatography. As described elsewhere herein, the host cell can be any host cell suitable for use in the production of recombinant proteins. The host cell can be eukaryotic or prokaryotic. The use of host prokaryotic cells is preferred. It is even more preferred that the host cell is a bacterial cell such as an E. coli cell. The use of E. coli cells of the BL21 strain or its derivatives is particularly preferred.

[0049] The nucleic acid contained by a host cell encoding a protein that yields a mature protein of SEQ ID NO:1 or SEQ ID NO:2 can be a DNA or RNA molecule, preferably a DNA molecule. The nucleic acid can be contained in a vector such as a eukaryotic or prokaryotic expression vector. Preferably, the vector is a prokaryotic expression vector as described elsewhere in this specification. The present invention also refers to a composition obtained from the above method, comprising a 143 - amino - acid protein having the amino - acid sequence of SEQ ID NO:1 or SEQ ID NO:2. The composition preferably contains less than 1% (w / w) of protein molecules shorter than 143 amino acids when measured by LCMS. The composition does not contain or contains only a small amount of carbamoylated protein. In a preferred embodiment, the composition contains a detectable amount of carbamoylated protein. For example, in one embodiment, the amount of carbamoylated protein is 0.01% (w / w), 0.02% (w / w), 0.05% (w / w), or 0.1% (w / w). At the same time, the total amount of carbamoylated protein is limited to less than 8% (w / w), preferably less than 7% (w / w), 6% (w / w), or 5% (w / w) of the protein in the said composition. In another preferred embodiment, the composition does not contain a detectable amount of carbamoylated protein.

[0050] Similarly, the composition does not contain or contains only a small amount of glucosaminoylated protein. In a preferred embodiment, the composition contains a detectable amount of glucosaminoylated protein. For example, in one embodiment, the amount of glucosaminoylated protein is 0.01% (w / w), 0.02% (w / w), 0.05% (w / w), or 0.1% (w / w). At the same time, the total amount of glucosaminoylated protein is limited to less than 6% (w / w), preferably less than 5% (w / w), 4% (w / w), or 3% (w / w) of the protein in the said composition. In another preferred embodiment, the composition does not contain a detectable amount of glucosaminoylated protein. In a particularly preferred embodiment, the composition comprises a detectable amount of carbamoylated protein, however, less than 7% (w / w) or less than 5% (w / w) of the protein in the composition is carbamoylated. For example, the composition may comprise at least 0.05% (w / w) or at least 0.1% (w / w) of carbamoylated protein, however, less than 7% (w / w) of the protein in the composition is carbamoylated. In yet another particularly preferred embodiment, the composition may comprise at least 0.05% (w / w) or at least 0.1% (w / w) of carbamoylated protein, however, less than 5% (w / w) of the protein in the composition is carbamoylated.

[0051] Similarly, in another particularly preferred embodiment, the composition comprises a detectable amount of glucosaminoylated protein, however, less than 5% (w / w) or less than 3% (w / w) of the protein in the composition is glucosaminoylated. For example, the composition may comprise at least 0.05% (w / w) or at least 0.1% (w / w) of glucosaminoylated protein, however, less than 5% (w / w) of the protein in the composition is glucosaminoylated. In yet another particularly preferred embodiment, the composition may comprise at least 0.05% (w / w) or at least 0.1% (w / w) of glucosaminoylated protein, however, less than 3% (w / w) of the protein in the composition is glucosaminoylated. In one embodiment, the composition comprises a protein having the amino acid sequence of SEQ ID NO: 1, together with variants thereof that are shorter in length and exhibit 100% sequence identity with the amino acid sequence of SEQ ID NO: 1 over their entire length, and the length is at least 100 amino acids in a situation where gaps are not allowed in the alignment. In the composition, when determined by LCMS after reductive dimethylation (stable isotope dimethyl labeling, SIDL) according to Tolonen et al (2011), the ratio of the sum of the signals obtained from the protein according to SEQ ID NO: 1 to the signal obtained from the shorter variant is higher than 20, preferably higher than 30, higher than 40, higher than 50, higher than 60, higher than 70, higher than 75, higher than 80, higher than 85, higher than 90, higher than 100, higher than 125, higher than 150, higher than 175, higher than 200, higher than 225, higher than 250, higher than 275, higher than 300, higher than 325, higher than 350, higher than 375, higher than 400, higher than 425, and more preferably higher than 450. As can be seen from Table 15 below, the ratio of the signal obtained from the protein according to SEQ ID NO: 1 to the signal obtained from the shorter variant was found to be 466 when determined by LCMS according to Tolonen et al (2011).

[0052] In one embodiment, the composition comprises a protein having the amino acid sequence of SEQ ID NO: 2, together with its variants that are shorter in length and show 100% sequence identity with the amino acid sequence of SEQ ID NO: 2 over their entire length, and the length is at least 100 amino acids in a situation where gaps are not allowed in the alignment. In the said composition, when determined by LCMS after reductive dimethylation (stable isotope dimethyl labeling, SIDL) according to Tolonen et al (2011), the ratio of the sum of the signals obtained from the protein by SEQ ID NO: 2 to the signal obtained from the shorter variant is higher than 20, preferably higher than 50, higher than 75, higher than 100, higher than 125, higher than 150, more preferably higher than 175 or higher than 180. As can be seen from Table 15 below, the ratio of the signal obtained from the protein by SEQ ID NO: 2 to the signal obtained from the shorter variant was found to be 186 when determined by LCMS according to Tolonen et al (2011). Preferably, the composition is in a two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) map 1 H and / or 15 more than 70%, preferably more than 80%, more than 90%, or more than 95% of the peaks of N produce a composite chemical shift deviation (CCSD) value of less than 0.01 ppm compared to the corresponding peaks in Table 1, and contains a folded protein.

[0053] The above composition contains a minimal amount of impurities or post-translational modifications and can thus be used in the preparation of pharmaceutical compositions. The pharmaceutical compositions can be prepared as described elsewhere in this specification. The above composition or pharmaceutical composition is preferably used as a medicament and, as described above, for example, in a subject in need thereof, to treat or prevent heart failure, to treat cardiomyopathy, to promote heart tissue regeneration, to promote cardiomyocyte proliferation, to promote angiogenesis, to promote heart function, to reduce infarct size, to treat or prevent fibrosis, to treat or prevent hypertrophy, or to treat or prevent heart failure.

Brief Description of the Drawings

[0054]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Example

[0055] The present invention is illustrated by the following examples, which are shown by way of example only. Specifically, the examples describe the preparation of production stocks and the heterologous expression and purification of MYDGF variants. As described in more detail in the following examples, the production process for human MYDGF was first developed at 5 L scale using a research cell bank (RCB), and then verified by scale-up at 20 L scale using a good manufacturing practice (GMP)-compliant working cell bank (WCB). Finally, the expression was transferred to a current Good Manufacturing Practice (cGMP) facility at 200 L scale, which yielded a batch harvest of 16 - 18 kg of wet inclusion bodies (IBs). The downstream process for purification of MYDGF protein from inclusion bodies was first developed at laboratory scale and then verified by scale-up at pilot scale using inclusion bodies from 10 L of fermentation aliquots, and finally transferred to the cGMP facility, where one downstream batch was initiated from 10 kg of wet IBs representing approximately 110 - 125 L of fermentation aliquots.

[0056] Non-clinical and clinical batches of MYDGF were manufactured at 200 L scale. Some batches were conducted in a GMP facility. The batches yielded typically 330 - 355 g of MYDGF from one 125 L fermentation aliquot, which reflects a yield of up to 2.84 g / L fermentation. The MYDGF produced by this process met all quality requirements necessary for use in toxicological and clinical studies. The monomer content, measured by HP size exclusion chromatography, was above 99% as specified, with less than 1% high molecular weight impurities (aggregates) and less than 0.1% low molecular weight impurities (fragments). The endotoxin content was 0.03 EU / mg protein. The host cell DNA content was ≤3 pg / mg protein.

[0057] (Example 1): Preparation and transfection of vectors For the production of cell banks, a derivative strain of Escherichia coli BL21(DE3) that has been modified not to produce phage was used. This strain was transformed with one of the vectors shown in SEQ ID NOs: 7-10, each of which has the gene encoding each MYDGF variant. The genes for each variant were codon-optimized for high expression in Escherichia coli and synthesized by ATUM (Newark, California, USA). Plasmids encoding the following MYDGF variants were prepared: · [+A] variant in which the N-terminal V residue at position +1 of mature human MYDGF is preceded by an A residue (amino acid sequence shown in SEQ ID NO: 1: AVSEPTTVAFDVRPGGVVHSFSHNVGPGDKYTCMFTYASQGGTNEQWQMSLGTSEDHQHFTCTIWRPQGKSYLYFTQFKAEVRGAEIEYAMAYSKAAFERESDVPLKTEEFEVTKTAVAHRPGAFKAELSKLVIVAKASRTEL). The expression vector encoding this variant is shown in SEQ ID NO: 7. · [+S] variant in which the N-terminal V residue at position +1 of mature human MYDGF is preceded by an S residue (amino acid sequence shown in SEQ ID NO: 2: SVSEPTTVAFDVRPGGVVHSFSHNVGPGDKYTCMFTYASQGGTNEQWQMSLGTSEDHQHFTCTIWRPQGKSYLYFTQFKAEVRGAEIEYAMAYSKAAFERESDVPLKTEEFEVTKTAVAHRPGAFKAELSKLVIVAKASRTEL). The expression vector encoding this variant is shown in SEQ ID NO: 8. · [+G] variant in which the N-terminal V residue at position +1 of mature human MYDGF is preceded by a G residue (amino acid sequence shown in SEQ ID NO: 3: GVSEPTTVAFDVRPGGVVHSFSHNVGPGDKYTCMFTYASQGGTNEQWQMSLGTSEDHQHFTCTIWRPQGKSYLYFTQFKAEVRGAEIEYAMAYSKAAFERESDVPLKTEEFEVTKTAVAHRPGAFKAELSKLVIVAKASRTEL). The expression vector encoding this variant is shown in SEQ ID NO: 9. · [-V] variant of mature human MYDGF lacking the +1 N-terminal V residue (amino acid sequence shown in SEQ ID NO: 4: SEPTTVAFDVRPGGVVHSFSHNVGPGDKYTCMFTYASQGGTNEQWQMSLGTSEDHQHFTCTIWRPQGKSYLYFTQFKAEVRGAEIEYAMAYSKAAFERESDVPLKTEEFEVTKTAVAHRPGAFKAELSKLVIVAKASRTEL). The expression vector encoding this variant is shown in SEQ ID NO: 10.

[0058] If there is a conflict between any of the sequences listed above and the sequences shown in the attached sequence listing, the above paragraph shall take precedence. Hyphens that finally appear within the sequence are the result of document processing truncation and must be ignored. To prepare the expression strain, E. coli cells were transformed by electroporation using the above vector plasmid with the Gene Pulser Xcell (TM) electroporation system (BioRad). The protein was expressed in E. coli cells in the form of inclusion bodies (IBs) that accumulate in the cytoplasm, as further described in the examples below.

[0059] (Example 2): Expression of MYDGF Variant The expression and purification of the MYDGF variant were carried out according to the following general scheme: 1. Fermentation 2. IB Preparation 3. Solubilization / Refolding 4. Ultrafiltration / Diafiltration 5. Anion Exchange Chromatography 6. Preferably: Hydrophobic Interaction Chromatography 7. Preferably: Concentration and Formulation

[0060] (Example 2.1): Fermentation One cell bank vial of the production strain obtained from Example 1 was thawed at room temperature. The preculture (PC) consisted of two 1 L shake flasks containing 300 mL of seed culture medium per flask. The composition of the seed culture is depicted in Table 2 below. All buffers and media were prepared with reverse osmosis (RO) water and sterilized prior to use using either a nanofiltration device or dry heat sterilization.

[0061]

Table 2

[0062] If necessary, antifoaming agents and antibiotics were added. Kanamycin was added as an antibiotic (to a final concentration of 50 μg / mL). Each shake flask was inoculated with 100 μL of the production strain. The cultures were grown for approximately 9.4 hours aiming for an OD (optical density at 550 nm) of 1.75 ± 0.5. The main culture (MC) was carried out in a 20 L total volume stainless steel bioreactor containing 10 L of batch medium. The composition of the batch medium is depicted in Table 3 below.

[0063]

Table 3

[0064] The batch medium was inoculated with 100 mL of cell broth from the preculture. During the batch phase and the exponential feeding phase, the fermentation process parameters were kept constant at 33.5 °C, pH 6.8, 1.0 bar top pressure, and a 20% DO set point. After carbon source depletion was observed by a dissolved oxygen (DO) peak, an exponential feed (concentration 600 g / L glucose; μ = 0.25 h -1 ) was started. The composition of the feed medium is depicted in Table 4 below.

[0065]

Table 4

[0066] After 9 hours of exponential feed rate (60.48 - 573.78 g / hour), the feed rate was kept constant at a rate of 573.78 g / hour during the remainder of the fermentation (11.5 hours). A 60-minute temperature gradient (33.5 °C - 30.0 °C) was started 11.5 hours after the start of the exponential feed and the gradient was ended immediately prior to induction with IPTG. The culture was induced with a bolus of IPTG 12.5 hours after the start of the feed. MC was ended 20.5 hours after the start of the feed. At the end of the culture process, the culture broth was immediately cooled to <12 °C, diluted to 15% of the target wet cell weight (WCW) with reverse osmosis (RO) water, and the bacterial cell population was separated from the supernatant by centrifugation in a CEPA centrifuge. The biomass was collected together with the supernatant and transferred to downstream processing.

[0067] Quantification of the product was performed by using an automated high-throughput LabChip GXII (registered trademark) system (Perkin Elmer), an alternative to traditional SDS-PAGE and protein quantification. Sample preparation was performed using a liquid handling system (Tecan Freedom EVO 150). For product quantification from fermentation samples, analytical cell disruption of samples from the fermentation was facilitated by enzymatic cell lysis. 90 μL of the fermentation suspension was diluted in a 9:10 ratio (v / v) with cell disruption buffer (FastBreak (trademark) cell lysis reagent (Promega) with Lysonase (trademark) (Merck), 32 μL of Lysonase per mL of FastBreak (trademark) reagent). For total product determination (soluble and insoluble fractions), samples were mixed prior to each pipetting step. Finally, the samples were diluted into the specific sample buffer of the system. To minimize the amount of sample buffer required, all dilution steps were performed in PBS or another formulated buffer.

[0068] For the final dilution, 8 μL of the sample or standard curve sample (from PBS dilution) was diluted into 28 μL of non-reducing sample buffer in a 96-well plate (Eppendorf twin.tec PCR plate 95100401). For the reducing condition, 28 μL of reducing sample buffer (containing 35 mM DTT) was used. The plate was sealed with foil (Eppendorf PCR foil 0030127790), centrifuged briefly (at 25 g for 30 seconds), and denatured at 70 °C for 10 minutes. After denaturation, the plate was centrifuged at 2200 g for 5 minutes to pellet any vaporized liquid. After centrifugation, the foil was removed and diluted with 140 μL of DI water. The 96-well plate was sealed with foil (Eppendorf twin.tec PCR plate 95100401), and the plate was centrifuged at 2200 g for 10 minutes to pellet any potential aggregates that would cause malfunction of the LabChip analysis. After centrifugation, the plate was analyzed on a LabChip GXII in the setting "HT Protein Express 100 High Sensitivity". The LabChip preparation was performed according to the manufacturer's guide. The standard curve was prepared by diluting the reference material. As the reference material, the [+G] variant produced in HEK 293-6E cells was used as described in Polten (2019) (see page 1303, first paragraph and Figure S1) and Ebenhoch (2019) (see page 8, first paragraph). Quantification was performed in the range of 1 mg / mL to 0.1 mg / mL by linear fit. The reducing and non-reducing conditions did not change the integrated area for quantification, but a shift in the run time that did not affect quantification was observed.

[0069] (Example 2.2): Purification and Analysis of Inclusion Bodies The frozen E. coli biomass obtained from Example 2.1 was resuspended at a ratio of 1:5 (w / v) using IB prep buffer 1 (1 M urea, 50 mM Tris, 0.1% (v / v) Polysorbat 20, pH 7.5). After resuspension with an ultraturrax for 15 minutes, the E. coli cells were disrupted using 3 passes at 650 - 700 bar by high-pressure homogenization. High-density and heavy inclusion bodies (IBs), as well as large cell fragments, were separated by high-speed cylindrical centrifugation using a GLE rotor (CEPA). The feed flow rate was 55 mL / min and the centrifugation speed was 24,500 g. The tube had an inner diameter of 3.2 mm. The recovered pellet was washed twice with HQ water. In all cases, the pellet was diluted at a ratio of 1:5 (w / v) and resuspended using an ultraturrax. After the HQ water step, the pellet mostly contained IBs.

[0070] (Example 2.3): Protein Solubilization, Refolding, and Purification The frozen IBs obtained from Example 2.3 were solubilized at room temperature in solubilization buffer (8 M urea, 0.14 M GuHCl, 6 mM DTT, 50 mM Tris, pH 8). The mixture was first stirred with an ultraturrax for 10 minutes and then with a propeller mixer for 180 minutes. The target concentration during solubilization was 5 mg / mL in a 100 mL target volume. Subsequently, the solubilization pool was filtered through a CUNO depth filter (filter E16E01A90ZB08A, 0.1 - 0.6 μm, 3M Deutschland GmbH, Neuss, Germany). The filter was pre-equilibrated with water for injection (WFI) and solubilization buffer. Subsequently, the solubilization pool was directly loaded. The filtrate was collected by UV monitoring using an AKTA system. The inclusion body solubilizate was refolded in refolding buffer (4 M urea, 0.3125 M Tris, 12.5 mM CaCl 2It was diluted 1:5 with 3.75 mM cystamine, pH 7). The refolded pool that had recovered was stirred overnight. The next day, it was filtered through a CU-NO depth filter (Filter E16E01A60ZB05A, 3M Deutschland GmbH, Neuss, Germany).

[0071] After filtration through the depth filter, the filtrate was subjected to ultrafiltration / diafiltration (UFDF) to perform buffer exchange. UFDF was performed using a Pellicon 3 membrane (88 cm 2 , Ultracell 3 kDa, screen type C) with a 2-fold concentration factor and a 5-fold diafiltration factor using a diafiltration buffer (20 mM Tris, pH 9). After UFDF, the filtrate was subjected to ion exchange chromatography (IEX). A YMC Biopro IEX 75 μm column with a column diameter of 1 cm, a bed height of 9 cm, and a column volume of 7.5 ml was used. The column was first equilibrated with 3 column volumes (CV) of equilibration buffer 1 (20 mM Hepes, 1 M NaCl pH 7) for 5 minutes, followed by 5 CV of equilibration buffer 2 (20 mM Tris pH 9) for 5 minutes. After loading the filtrate, the filtrate was washed with 5 CV of 20 mM Tris pH 9 for 5 minutes. The protein was eluted with 5 CV of elution buffer 1 (20 mM Hepes, 1 M NaCl, pH 7) for 5 minutes, followed by 10 CV of elution buffer 2 (20 mM Hepes, pH 7, 1 M NaCl pH 7 using a gradient (0% - 100% 20 mM Hepes)) for 5 minutes. Finally, the column was stripped with 5 CV of 1 M HCl for 5 minutes.

[0072] (Example 2.4): Protein Yield and Product Homogeneity Purification was carried out at least once for all four variants. In addition, a second purification experiment was carried out for the [+A] and [+S] variants. The purification resulted in high yields and high purity for each of the four variants. In particular, the overall process yields after purification and refolding for the [+A] variant were found to be 2.4 g / L for the first batch and 5.3 g / L for the second batch. Analytical high-performance size-exclusion chromatography was carried out to test for purity. The purified [+A] variant showed a high purity of 99.75% main peak, 0.25% low molecular weight impurities, and 0.0% aggregate level. Similarly, high purity levels were achieved for the [-V] variant (99.64% main peak, 0.0% low molecular weight impurities, 0.04% aggregates) and the [+S] variant (99.73% main peak, 0.2% low molecular weight impurities, 0.05% aggregates). In contrast, when experimented with high-performance size-exclusion chromatography, the [+G] variant product was less homogeneous, showing a main peak purity of 60.36% and 39.64% aggregates.

[0073] The process yields from the laboratory-scale purification runs for all N-terminal variants are summarized in the following table:

Table 5

[0074] Zhao et al. (2020) reported the fermentation of the MYDGF-6His fusion protein in E. coli, followed by extraction and purification from the E. coli cell lysate.

Table 6

[0075]

Table 7

[0076] (Example 3): Improved MYDGF production process Based on Example 2, the production process was further developed for the [+A] variant. The fermentation process for MYDGF was first developed at 5 L scale using a Research Cell Bank (RCB), then verified by enhanced execution at 20 L scale using a GMP Working Cell Bank (WCB), and finally transferred to 200 L scale. A typical 200 L fermentation batch yielded 16 - 18 kg of wet IBs. The downstream process for the purification of the MYDGF pro-form from intracellular inclusion bodies was first developed at laboratory scale, then verified by enhanced execution at pilot scale using inclusion bodies from a 10 L fermentation aliquot, and finally transferred to a cGMP facility where one downstream batch was started from 10 kg of wet IBs representing approximately 110 - 125 L of fermentation aliquot.

[0077]

Table 8

[0078]

Table 9

[0079] Several batches were conducted under GMP conditions. The batches typically yielded high amounts of 330 - 355 g of MYDGF from one 125 L fermentation aliquot. This reflects an overall process yield of up to 2.84 g / L of fermentation. The MYDGF bulk produced by this process met all the quality requirements necessary for use in toxicological and clinical studies. The monomer content measured by HP size exclusion chromatography was above 99% as specified, with less than 1% high molecular weight impurities (aggregates) and less than 0.1% low molecular weight impurities (fragments). The endotoxin content was less than 0.03 EU per mg of MYDGF protein. The host cell DNA content was ≤ 3 pg / mg protein.

[0080] (Example 4): Molecular weight analysis by LCMS and improved molecular weight analysis by LCMS after chemical modification by Tolonen et al. ("aLCMS") Samples of the folded and purified product obtained from Example 2 were subjected to liquid chromatography mass spectrometry (LCMS) analysis. Intact (non-reduced) molecular weight analysis was performed on the MYGDF constructs using liquid chromatography / electrospray ionization mass spectrometry (LC-ESI-MS) to (1) verify the sequences by the conformity of the observed molecular weights to the predicted values for each sequence and (2) obtain the overall profile of net post-translational modifications (PTMs) in each protein. The samples (0.5 μg / injection) were desalted using an Agilent 1290 UPLC with a 1.0 mm × 30 mm C3 POROS reversed-phase column and introduced into the mass spectrometer. Proteins were captured, desalted, and eluted from the column using a 3-minute binary gradient consisting of mobile phase A (98.9% water, 1% acetonitrile, 0.1% formic acid, and 2 mM ammonium acetate) and mobile phase B (70% isopropanol, 20% acetonitrile, 9.9% water, and 0.1% formic acid) increasing from 5% to 80% of mobile phase B at 150 μl / min. Mass spectral data of the eluted material were acquired using an Agilent 6224 time-of-flight (TOF) MS and then processed (deconvoluted) using the maximum entropy algorithm within the Mass Hunter analysis software (Agilent). The data obtained by this method are referred to herein as "intact MW LCMS data" or data "measured by liquid chromatography mass spectrometry (LCMS)".

[0081] For peptide-level sequence confirmation and site-specific post-translational modification (PTM) analysis, aliquots of each sample were digested separately with trypsin and chymotrypsin to achieve complete sequence coverage. Each 100 μg sample was desalted and concentrated by acetone precipitation and centrifugation of the precipitate material into a pellet. Each protein pellet was solubilized again, denatured, reduced in 10 μl of denaturing / reducing buffer (5% w / v sodium deoxycholate (SDC), 10 mM dithiothreitol (DTT), 20 mM ammonium bicarbonate), incubated at 70 °C for 2 minutes, and subsequently diluted 10-fold with 20 mM ammonium bicarbonate and 2 mM methionine. The reduced / denatured molecules were then split into two vials (50 μg each), and to each tube, trypsin and chymotrypsin were added separately at an enzyme-to-substrate ratio of 1:10, and the samples were incubated at 37 °C for 10 minutes. 10% v / v trifluoroacetic acid was added to quench the reaction to a final concentration of 1% v / v of the reagent. This short (10-minute) digestion step obviated the need for an alkylation step commonly used in peptide mapping. Precipitated sodium deoxycholate was removed by centrifugation at 16,000 xg, the peptide-containing supernatant was recovered, transferred to an autosampler vial, and immediately stored at -80 °C until analysis. The data obtained by this method are referred to herein as "peptide mapping LCMS data".

[0082] aLCMS: Additionally, the first four N-terminal residues of various MYDGF constructs have been shown to cause electrospray fragmentation (both at the intact level and the peptide level), so in order to delineate between sample-derived N-terminal truncations and electrospray-derived N-terminal truncations, aliquots of the peptide digest were subjected to reductive dimethylation (also known as stable isotope dimethyl labeling, SIDL) according to Tolonen et al (2019). The data obtained by this method are referred to herein as "LCMS after reductive dimethylation (stable isotope dimethyl labeling, SIDL)". Briefly, 50 μg of peptide from each digest was immobilized onto separate Waters Oasis SPE cartridges using vacuum manifold. The SPE media and peptide were adjusted to pH 5.5 with citrate buffer (90 mM citric acid, 230 mM disodium phosphate), after which 10 ml of 0.8% v / v formaldehyde (in citrate buffer) and 120 mM sodium cyanoborohydride were passed over the immobilized peptide for 10 minutes. The reaction was then removed by washing with 10 column volumes of 0.1% formic acid in water and eluted with 10 volumes of 50% acetonitrile, 0.1% formic acid. The labeled peptides were collected into low-binding microcentrifuge tubes and then dried in a vacuum centrifuge. The dried peptides were reconstituted in 50 μl of 0.1% TFA and transferred to autosampler vials for LC-MS / MS analysis.

[0083] LC-MS / MS (tandem mass spectrometry) analysis was performed using a Vanquish UHPLC system interfaced with a Lumos Fusion Orbitrap (ThermoFisher) operated under the control of Xcalibur 4.1.31.9 software (ThermoFisher). 0.5 μg of each peptide digest was loaded onto a 2.1 mm × 150 mm C18 CSH Acquity UPLC reversed-phase column (1.7 μm particles, Waters Corp.) and separated using a binary gradient as follows: mobile phase A (0.1% difluoroacetic acid (DFA) in water), 0.5% - 40% mobile phase B (99.9% acetonitrile, 0.1% DFA) at a flow rate of 200 μl / min and a column temperature of 50 °C. The LC eluate was analyzed using the top 4 data-dependent acquisition (DDA) MS workflows. Full-scan MS spectra were acquired at a resolution of 120,000 (FWHM) at 200 m / z, and MS / MS spectra of HCD (high-energy collision-induced dissociation) and EThcD (electron transfer dissociation supplemented with HCD energy) were acquired in the Orbitrap analyzer at a resolution of 15,000 in a charge state-dependent manner. The RAW files resulting from each LC-MS / MS analysis were further processed using Protein Metrics Inc. (PMI) Byonic and Byos software to identify and quantify PTMs. Manual analysis of various spectra was performed using the QualBroswer function of Xcalibur software.

[0084]

Table 10

Table 11

[0085]

Table 12

[0086]

Table 13

[0087]

Table 14

[0088]

Table 15

[0089]

Table 16

[0090]

Table 17

[0091]

Table 18

[0092] (Example 5): Structural elucidation The +G MYDGF variant (HEK) having the sequence according to SEQ ID NO: 3 was produced as described in Polten et al. (2019), page 1303, first paragraph and Figure S1, Ebenhoch et al. (2019), first paragraph on page 8. The +A MYDGF variant was prepared according to Example 2. Two-dimensional 1 H / 15The 2D H-1 / N HSQC NMR spectra were collected at 310 K on a Bruker Avance III 800 MHz spectrometer equipped with a 5 mm z-gradient TCI cryoprobe in a 2.5 mm tube. The spectra were recorded with 48 complex points in the indirect dimension, 1024 scans, a 1 s interscan delay, resulting in a total experimental time of 30 h, using the pulse program hsqcfpf3gpphwg (Bodenhausen and Ruben 1980; Piotto et al., 1992; Sklenar et al., 1993; Mori et al., 1995) from the Bruker catalog. The hsqcfpf3gpphwg pulse program describes a phase-sensitive 2D H-1 / X correlation spectrum by double inept transfer using the f3 channel, in-line decoupling during acquisition, plus flip-back pulses, and a watergate sequence for water suppression. The NMR samples contained 8.5 mg / ml of each MYDGF protein in 50 mM sodium phosphate buffer at pH 7.4 containing 50 mM sodium chloride and 9% (v / v) D 2 2O. Processing and analysis were performed with Topspin 3.5 (Bruker BioSpin).

[0093] 2D 1 H / 15 The H and 1 N chemical shifts of the cross-peaks observed in the 2D H-1 / N HSQC NMR spectra clearly show that MYDGF is a folded protein for both the +G variant (HEK) and the +A variant. The dispersion of the cross-peaks and their chemical shifts is significantly higher than the experimentally determined random chemical shifts and highly variable (Wishart et al. 1995). Comparing folded and unfolded proteins, 2D 15 H / 1 H / 15The comparison of 1H-15N HSQC NMR spectra is exemplified in this publication (Dyson & Wright, 1995). The difference between the random coil chemical shifts and the observed chemical shifts is often used as a constraint for secondary structure (α-helix or β-sheet) in NMR structure calculations (Shen & Bax, 2015).

[0094]

Table 19

[0095]

Table 20

[0096] (Example 6): Potency assay in human coronary artery endothelial cells To determine the relative potency of the [+A] variant against the MYDGF+G HEK protein as a reference, a potency assay was performed in human coronary artery endothelial cells (HCAECs). The +A variant was manufactured according to Example 2. Three batches named V301, V302, and V303 were examined. The +G variant (SEQ ID NO: 3) was generated in HEK cells as described in Polten et al. (2019), page 1303, first paragraph and Figure S1, Ebenhoch et al. (2019), first paragraph on page 8, and it was used as an internal activity benchmark.

[0097]

Table 21

[0098] HCAECs were seeded at a density of 55,000 - 60,000 cells per well in EGM-2 medium (Lonza) containing 10% fetal bovine serum (FCS) at a total volume of 1 ml per well in a 24-well plate. Twenty-four hours after seeding (cells need to be confluent), the medium in each well was replaced with 1 mL of MCDB131 medium (Life Technologies) containing 2% FCS and incubated for 3 - 4 hours. After incubation, in each well, the cell monolayer was scratched with a pipette tip (200 μl). The tip was used vertically to ensure that the scratch was large enough. Subsequently, the cells were washed once with MCDB medium containing 2% FCS. Subsequently, 1 mL of fresh medium (MCDB containing 2% FCS) was added to each well. Then, the cells were cultured in the absence of MYDGF protein (control) or in the presence of MYDGF protein at various concentrations, and each well contained the starting concentration and serial dilutions. Each protein was tested at the following concentrations: 13.3 ng / mL, 19.7 ng / mL, 29.6 ng / mL, 44.4 ng / mL, 66.6 ng / mL, and 100 ng / mL. Human VEGFA (50 ng / mL) served as a positive control. MYDGF batches V301, V302, V303 were tested in duplicates, 1:1 against the reference. Immediately after treatment at T = 0 h, pictures were taken from all wells using a microscope (Zeiss Axio Observer Z1 at 50× magnification, 5× objective lens, phase contrast setting). The pictures were taken from the center of the well such that optimal contrast was seen there. Subsequently, the plate was incubated at 37 °C. After 16 hours of incubation (T = 16 h), pictures of each well were taken again as described above.

[0099] For the determination of activity, the recovery in the assay was calculated by measuring the cell-free area using the AxioVision software or ImageJ in the photos at 0 h and 16 h, respectively. The recovery (%) was calculated as [(cell-free area at 0 h - cell-free area at 16 h) / cell-free area at 0 h] × 100. Summarizing the two experiments, a four-parameter logistic (4-PL) curve fit was performed and the EC 50 value (GraphPad Prism software, version 9.1.0) was calculated. The EC 50 values from various batches and references were applied to calculate the relative potency compared to the reference: Potency [%] = ([+G] - EC 50 of HEK / EC 50 ) × 100

[0100] [Table 22]

[0101] Results: As can be seen from Table 18, all three batches (V301, V302, V303) producing the +A MYDGF variant showed bioactivity comparable to the reference. In HCAECs, the following relative potencies were determined compared to the reference (set at 100%): V301 (177%, 95%, and 136%), V302 (114%, 110%, and 103%), V303 (99%, 111%, and 81%). The migration experiments of pooled cells resulted in EC 50 values of 37.4 ng / mL, 42.1 ng / mL, and 47.8 ng / mL for batches V301, V302, and V303, respectively. When the data from all batches (V301, V302, and V303) were pooled and a curve fit was performed, an EC 50 value of 41.1 ng / mL was calculated. The EC 50 value for the corresponding reference, calculated in the same way, was 43.0 ng / mL.

[0102] (Example 7): Potency assay in neonatal rat cardiomyocytes Neonatal rat cardiomyocytes (NRCMs) were seeded in 96-well plates and subjected to simulated ischemia / reperfusion (I / R) in the absence of the MYDGF batch (control) or in the presence of various MYDGF batches. I / R was simulated as previously described by Ebenhoch et al. (2019). Reference proteins were assayed in a 1:1 comparison. Each protein was tested at the following six concentrations: 13.3 ng / mL, 19.7 ng / mL, 29.6 ng / mL, 44.4 ng / mL, 66.6 ng / mL, and 100 ng / mL. Mouse IGF-1 (50 ng / mL) served as a positive control. Metabolic activity was evaluated by the MTS assay (Promega). MYDGF batches V301, V302, and V303 were tested in 3-4 technical replicates in 2-3 experiments. To summarize the experiment, a 4-PL curve fit was performed and EC 50 values were calculated. EC 50 values from the various batches and references were applied to calculate the relative potency against the reference (see above).

[0103] [Table 23] Results: In neonatal rat cardiomyocytes, the following relative potencies were determined compared to the reference (set at 100%): V301 (108%), V302 (431%), V303 (85%). The potency values for MYDGF batches V301 and V303 showed bioactivity comparable to the reference. The metabolic activity for batch V302 was similar to the reference (bar graph in Figure 7), but a non-ideal curve fit yielded a potency 4-fold higher. When data from all batches (V301, V302, and V302) were pooled and a curve fit was performed, an EC 50 value of 22.2 ng / mL was calculated. The EC 50 value for the corresponding reference, calculated in the same way, was 24.5 ng / mL.

[0104] (Example 8): Mouse myocardial infarction assay To compare the efficacy of human MYDGF treatment and mouse MYDGF treatment for myocardial infarction (MI) healing in mice, a mouse model of myocardial infarction was used. FVB / N mice were subjected to sham or true (ischemia / reperfusion) surgery, treated with human or mouse recombinant MYDGF, and observed for 28 days. The human and mouse MYDGF proteins used in the assay are depicted in Table 20.

[0105]

Table 24

[0106] FVB / N mice prone to heart failure were subjected to sham (thoracotomy without ischemia / reperfusion; without I / R) or I / R (ischemia / reperfusion) surgery. The mice were treated with human or mouse MYDGF (10 μg bolus + 10 μg / day pump for 7 days, Model 1007D) or diluent only (placebo). Continuous echocardiography was performed (on days 6 and 28), and the mice were observed for 28 days. At the end of the experiment, the hearts were collected, and the scar size was determined by Masson's trichrome staining and the capillary density by fluorescent IB4 / WGA staining. Statistical significance was evaluated by one-way ANOVA with Dunnett's multiple comparison post hoc test. (For all VR groups for FAC) vs. sham * P < 0.05, ** P < 0.01, *** P < 0.001. vs. placebo ##P < 0.01, P < 0.001.

[0107] Results: Protein therapy with human MYDGF ("MYDGF") was found to improve cardiac function (fractional area change, "FAC") by 17.5% on day 6 and 16.7% on day 28 compared to placebo treatment. Mouse MYDGF ("Mydgf") increased FAC by 16.1% on day 6 and 19.0% on day 28 compared to placebo treatment (see Figure 8). As can be seen from Figure 9, infarct scarring was reduced by both recombinant MYDGF proteins (16.5% for human MYDGF and 12.4% for mouse MYDGF compared to placebo). Furthermore, MYDGF protein therapy increased capillary density in the infarct border region after MI by 21.8% for the human protein and 19.1% for the mouse protein compared to placebo treatment (see Figure 9). Thus, both treatments significantly improved MI healing as evaluated by improved cardiac function, reduced scar size, and increased capillary density in the infarct border region. TIFF2025518285000034.tif144157 TIFF2025518285000035.tif50131

Claims

1. A method for recombinant expression of MYDGF protein in a host cell, comprising: (a) providing a host cell comprising a nucleic acid encoding a protein consisting of 143 amino acids having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 after maturation; (b) culturing the host cell under conditions that allow expression of the protein; (c) isolating inclusion bodies containing the MYDGF protein from the host cell; and (d) solubilizing the inclusion bodies and refolding the MYDGF protein A method comprising the steps of.

2. Step (a) is (i) providing a host cell comprising a nucleic acid containing an open reading frame flanked by start and stop codons, according to the sequence of SEQ ID NO: 11 or SEQ ID NO: 12, preferably SEQ ID NO: 11, operably linked to a promoter; or (ii) providing a host cell comprising a nucleic acid encoding a protein consisting of 144 amino acids having the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16, preferably SEQ ID NO: 15, before maturation The method according to claim 1, comprising the steps of.

3. The method according to any one of claims 1 to 3, wherein step (a) comprises providing a host cell comprising the nucleic acid of SEQ ID NO: 7 or SEQ ID NO: 8, preferably SEQ ID NO:

7.

4. The method according to any one of claims 1 to 3, wherein said maturation is removal of the N-terminal methionine residue.

5. The method according to claim 4, wherein the removal of the N-terminal methionine residue is effected by one or more host cell-derived aminopeptidases.

6. The method according to any one of claims 1 to 5, further comprising (e) obtaining a refolded MYDGF protein of 143 amino acids having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, after step (d).

7. The method according to any one of claims 1 to 6, wherein the refolding of the protein in step (d) comprises incubating the protein in the presence of urea.

8. The method according to any one of claims 1 to 7, further comprising (f) purifying the MYDGF protein.

9. The method according to claim 8, wherein step (f) comprises ultrafiltration, diafiltration, hydrophobic interaction chromatography, and / or anion exchange chromatography.

10. The method according to claim 9, wherein the step of anion exchange chromatography or hydrophobic interaction chromatography is carried out by contacting the MYDGF protein with a chromatographic resin material under conditions that allow adsorption of the MYDGF protein to the resin, optionally washing the resin, and eluting the MYDGF protein from the resin.

11. The method according to claim 10, wherein the adsorption of the MYDGF protein to the anion exchange chromatography resin is carried out under conditions of low ionic strength.

12. The method according to claim 11, wherein the adsorption is carried out at a conductivity of less than 3 mS / cm, less than 2 mS / cm, less than 1.5 mS / cm, or less than 1 mS / cm.

13. The method according to any one of claims 9 to 12, wherein the desorption of the MYDGF protein from the anion exchange chromatography resin is effected by increasing the salt concentration and / or decreasing the pH of the liquid phase.

14. The method according to any one of claims 1 to 13, wherein the host cell is a host prokaryotic cell.

15. The method according to claim 14, wherein the host prokaryotic cell is a bacterial cell.

16. The method according to claim 15, wherein the host bacterial cell is an Escherichia coli cell.

17. The method according to claim 16, wherein the Escherichia coli cell is an Escherichia coli cell of the BL21 strain or a derivative thereof.

18. The method according to any one of claims 1 to 18, wherein the nucleic acid is DNA or RNA.

19. The method according to claim 18, wherein the nucleic acid comprises the sequence of SEQ ID NO: 11 or SEQ ID NO:

12.

20. The method according to any one of claims 1 to 19, wherein the nucleic acid is contained within a vector.

21. The method according to claim 20, wherein the vector is a prokaryotic expression vector.

22. The method according to claim 20 or 21, wherein the vector comprises a T7 promoter.

23. The method according to any one of claims 20 to 22, wherein the vector comprises or consists of the sequence of SEQ ID NO: 7 or SEQ ID NO:

8.

24. A composition obtained from the method according to any one of claims 1 to 23, the composition comprising a 143 - amino acid protein having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

2.

25. The composition according to claim 24, which contains protein molecules shorter than 143 amino acids and less than 1% (w / w) when measured by liquid chromatography-mass spectrometry (LC-MS).

26. The composition according to claim 24 or 25, which contains less than 20 pg / mg, preferably less than 15 pg / mg, more preferably less than 10 pg / mg, most preferably less than 5 pg / mg, less than 3 pg / mg, less than 2 pg / mg, or less than 1 pg / mg of host cell DNA.

27. The composition according to any one of claims 24 to 26, which contains less than 0.2 EU / mg, preferably less than 0.1 EU / mg or less than 0.08 EU / mg of bacterial endotoxin.

28. The composition according to any one of claims 24 to 27, wherein less than 8% (w / w), preferably less than 7% (w / w), 6% (w / w), or 5% (w / w), less than 4% (w / w), less than 3% (w / w), or less than 2% (w / w) of the protein in the composition is carbamoylated.

29. The composition according to any one of claims 24 to 28, wherein less than 6% (w / w), preferably less than 5% (w / w), less than 4% (w / w), or less than 3% (w / w), or less than 2% (w / w) of the protein in the composition is glucosylated.

30. The composition according to any one of claims 24 to 29, wherein less than 8%, preferably less than 7%, less than 6%, or less than 5% of the MYGDF protein in the composition of the present invention is carbamoylated, and the percentage is based on the sum of the peak intensities of the unmodified MYDGF protein and the annotated post-translational modification (PTM) species of MYDGF in the deconvoluted intact mass spectrum of the MYDGF protein in the composition.

31. The composition according to any one of claims 24 to 30, wherein less than 6%, preferably less than 5%, less than 4%, or less than 3% of the MYDGF protein in the composition of the present invention is glucosylated, and the percentage is based on the sum of the peak intensities of the unmodified MYDGF protein and the annotated post-translational modification (PTM) species of MYDGF in the deconvoluted intact mass spectrum of the MYDGF protein in the composition.

32. The composition according to any one of claims 24 to 31, which contains urea.

33. The composition according to any one of claims 24 to 32, wherein the composition contains a 143 - amino - acid protein having the amino - acid sequence of SEQ ID NO: 1, and the ratio of the signal for the protein according to SEQ ID NO: 1 to the signal for a shorter variant in liquid chromatography - mass spectrometry (LCMS) after reductive dimethylation (stable - isotope dimethyl labeling, SIDL) is at least 50, preferably higher than 100, higher than 200, higher than 300, or higher than 400, where only the signals from non - carbamoylated and non - glucosonoylated proteins are used to calculate the ratio.

34. The composition according to any one of claims 24 to 33, wherein the composition contains a 143 - amino - acid protein having the amino - acid sequence of SEQ ID NO: 2, and the ratio of the signal for the protein according to SEQ ID NO: 2 to the signal for a shorter variant in liquid chromatography - mass spectrometry (LCMS) after reductive dimethylation (stable - isotope dimethyl labeling, SIDL) is at least 50, preferably higher than 75, higher than 100, higher than 150, or higher than 175, where only the signals from non - carbamoylated and non - glucosonoylated proteins are used to calculate the ratio.

35. The composition is in a two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) map 1 H and / or 15 more than 70%, preferably more than 80%, more than 90%, or more than 95% of the peaks of N result in a composite chemical shift deviation (CCSD) value of less than 0.01 ppm compared to the corresponding peaks in Table 1, and the composition according to any one of claims 24 to 34, comprising a folded protein that is folded

36. The composition according to any one of claims 24 to 35, comprising a MYDGF protein having a monomer content of more than 95%, more than 96%, more than 97%, more than 98%, or more than 99%.

37. Use of the composition according to any one of claims 24 to 36 for the preparation of a pharmaceutical composition.

38. A pharmaceutical composition comprising the composition according to any one of claims 24 to 36.

39. The pharmaceutical composition according to claim 38, further comprising a pharmaceutically acceptable carrier.

40. The pharmaceutical composition according to claim 38 or 39, formulated for parenteral administration.

41. The pharmaceutical composition according to claim 40, formulated for intravenous, intra - arterial, or intracoronary administration.

42. The pharmaceutical composition according to claim 41, formulated for intravenous administration.

43. The composition according to any one of claims 24 to 36 or the pharmaceutical composition according to any one of claims 38 to 42 for use as a medicament.

44. (i) treating or preventing a disease or condition selected from the group consisting of injury, trauma, ischemia, reperfusion injury, external trauma, mechanical overload, poisoning, surgery, primary or secondary cardiomyopathy, post-ischemic systolic dysfunction, myocardial infarction, preferably acute myocardial infarction, angina pectoris, heart failure, inflammation of the heart, cardiac dysfunction, hypertrophy, and fibrosis; (ii) promoting or improving cardiac tissue regeneration, cardiomyocyte proliferation, angiogenesis, cardiac function, or left ventricular systolic function after myocardial infarction; (iii) protecting cardiomyocytes from death, for example, by apoptosis or necrosis; or (iv) reducing infarct size after myocardial infarction, preferably acute myocardial infarction The composition according to any one of claims 24 to 36 or the pharmaceutical composition according to any one of claims 38 to 43 for use in the method.

45. (i) treating or preventing a disease or condition selected from the group consisting of injury, trauma, ischemia, reperfusion injury, external trauma, mechanical overload, poisoning, surgery, primary or secondary cardiomyopathy, post-ischemic systolic dysfunction, myocardial infarction, preferably acute myocardial infarction, angina pectoris, heart failure, and inflammation of the heart; (ii) improving left ventricular systolic function after myocardial infarction; or (iii) protecting cardiomyocytes from death, for example, by apoptosis or necrosis The composition according to any one of claims 24 to 36 or the pharmaceutical composition for use in the method according to claim 44, for use in the method.

46. The composition according to any one of claims 24 to 36 or the pharmaceutical composition for use in the method according to claim 44 or 45, wherein the cardiomyopathy is a hereditary cardiomyopathy or a cardiomyopathy caused by a spontaneous mutation.

47. The composition according to any one of claims 24 to 36 or the pharmaceutical composition for use in the method according to claim 44 or 45, wherein the cardiomyopathy is a secondary cardiomyopathy, preferably an ischemic cardiomyopathy caused by atherosclerosis or other coronary artery diseases, a cardiomyopathy caused by myocardial infection or intoxication, a hypertensive heart disease caused by pulmonary hypertension and / or arterial hypertension, and a cardiomyopathy caused by heart valve diseases.

48. The composition or pharmaceutical composition according to any one of claims 24 to 36 for use in the method according to claim 44 or 45, wherein the cardiomyopathy is selected from the group consisting of hypertrophic cardiomyopathy (HCM or HOCM), arrhythmogenic right ventricular cardiomyopathy (ARVC), isolated ventricular non-compaction mitochondrial myopathy, dilated cardiomyopathy (DCM), restrictive cardiomyopathy (RCM), takotsubo cardiomyopathy, Reiffel endocarditis, diabetic cardiomyopathy, alcoholic cardiomyopathy, or cardiomyopathy associated with obesity.

49. The composition or pharmaceutical composition according to any one of claims 24 to 36 for use in the method according to claim 44 or 45, wherein the heart failure is chronic heart failure.

50. The composition or pharmaceutical composition according to any one of claims 24 to 36 for use in the method according to claim 49, wherein the heart failure or chronic heart failure is heart failure with preserved ejection fraction (HFpEF), heart failure with reduced ejection fraction (HF rEF), or heart failure with mildly reduced ejection fraction (HFmrEF).

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