Recombinant expression of bone marrow-derived growth factors

Abstract

The present invention relates generally to the field of recombinant gene expression in host cells. In particular, the present invention relates to recombinant human bone marrow derived growth factor (MYDGF) proteins that exhibit minimal degradation when expressed in host cells. Therefore, the recombinant protein is highly suited for medical use, in particular for the treatment of cardiac tissue damage and the prevention of cell death in cardiac muscle tissue. The invention also provides nucleic acids encoding the recombinant proteins and host cells expressing the recombinant proteins. The invention also provides a method for producing a recombinant protein in a host cell.

Description

[0001] The present invention generally relates to the field of recombinant gene expression in host cells. Specifically, the present invention relates to a recombinant human bone marrow-derived growth factor (MYDGF) protein that exhibits minimal degradation when expressed in a host cell. Therefore, the recombinant protein is highly suitable for medical use, particularly for treating cardiac tissue damage and preventing cell death in myocardial tissue. The present invention also provides nucleic acids encoding the recombinant protein and host cells 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 leading causes of morbidity and mortality worldwide. Acute MI is mediated by thrombotic occlusion of the coronary arteries, which results in progressive cell death in non-perfused tissues. This triggers an inflammatory response, which results in scar formation and loss of living tissue. Severe changes in the tissue architecture in the left ventricle can cause ventricular dilatation, systolic dysfunction, and heart failure. The protein named bone marrow-derived growth factor (MYDGF) has been shown to improve tissue repair and cardiac function in rodent models of MI. Compared with wild-type mice, MYDGF-deficient mice develop larger infarct scars and more severe systolic dysfunction. It has been found that treatment with recombinant MYDGF can protect myocardial cells from cell death and repair the heart after acute MI. The development of protein-based therapeutics would be a promising approach for cardiac repair and potentially also for ischemic repair in other tissues (Ebenhoch et al., 2019; Polten et al., 2019; Botnov et al., 2018, Korf-Klingebiel et al., 2015, WO 2014 / 111458).

[0003] Currently, small amounts of recombinant human MYDGF are produced by expression in human or mammalian expression systems such as HEK-293T or CHO cells. However, large-scale production of recombinant human MYDGF (rhMYDGF) by mammalian cell expression systems is associated with high costs, which makes production unattractive from an economic point of view. 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 9 additional amino acids, thereby having a significantly higher molecular weight. Although the authors speculate in the publication that the expression system may be used to produce rhMYDGF for clinical use, the His tag will cause significant antigenicity risks when administered to human patients.

[0004] It is also known that heterologous expression of rhMYDGF is associated with considerable degradation problems. Upon recombinant expression of the protein, one or more amino acids located at the N-terminus are degraded, which results in protein fragments with a smaller 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 a MW of 17032 Da, but also a degradation product with a MW of approximately 16900 Da. The ratio of target protein to degraded protein in Zhao et al., 2020 is approximately 10:1, as can be seen from the Zhao publication. Figure 2 The impurities by degradation products are therefore quite significant and unacceptable for a protein intended for systemic medical use.

[0005] In view of the prior art described above, an object of the present invention is to provide a recombinant protein having MYDGF activity,

[0006] (i) show no or only minimal degradation when expressed in a heterologous expression system;

[0007] (ii) can be produced with minimal potentially adverse process-derived post-translational modifications, such as carbamylation and gluconoylation, which may be detrimental to the protein's intended clinical use;

[0008] (iii) having the secondary and tertiary structures of native human MYDGF protein;

[0009] (iv) have high biological activity;

[0010] (v) associated with a low risk of antigenic epitopes derived from primary sequences other than human MYDGF, thereby generating minimal risk of anti-drug antibodies;

[0011] (vi) can be produced in a prokaryotic expression system to provide a non-glycosylated product;

[0012] (vii) can be produced at more than 0.5 g protein / 100 g cells, and can be expanded to more than 100 g, preferably more than 200 g, more preferably more than 300 g protein / batch.

[0013] Not all objectives will be achieved by all embodiments of the invention. The scope of the invention is defined by the claims. However, it is preferred that 2, 3, 4, 5 or 6 of the aforementioned objectives of the invention are met. Summary of the invention

[0014] In a first aspect, the present invention relates to a method for recombinantly expressing a MYDGF protein in a host cell.

[0015] In a second aspect, the invention relates to a composition obtainable by the method of the first aspect of the invention.

[0016] In a third aspect, the present invention relates to use of the composition of the second aspect of the present invention for preparing a pharmaceutical composition.

[0017] In a fourth aspect, the present invention relates to a pharmaceutical composition comprising the composition of the second aspect of the present invention.

[0018] In a fifth aspect, the present invention relates to a pharmaceutical composition according to the fourth aspect of the present invention for use as a medicament.

[0019] In a sixth aspect, the present invention relates to a composition of the second aspect of the invention or a pharmaceutical composition of the fourth aspect of the invention, which is used in the following methods: (i) treating or preventing a disease or condition selected from the following: damage, trauma, ischemia, reperfusion injury, trauma, mechanical overload, poisoning, surgery, primary or acquired cardiomyopathy, post-ischemic contractile dysfunction, myocardial infarction, preferably acute myocardial infarction, angina pectoris, heart failure, cardiac inflammation, heart failure, hypertrophy and fibrosis; (ii) promoting or improving cardiac tissue regeneration, cardiomyocyte proliferation, neovascularization, cardiac function or left ventricular contractile function after myocardial infarction; (iii) protecting cardiomyocytes from death, for example by apoptosis or necrosis; or (iv) reducing the infarct area after myocardial infarction, preferably acute myocardial infarction.

[0020] In a seventh aspect, the present invention relates to a protein having an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and preferably having an amino acid sequence of SEQ ID NO: 1.

[0021] In an eighth aspect, the present invention relates to a nucleic acid encoding the protein of the seventh aspect of the present invention.

[0022] In a ninth aspect, the present invention relates to a vector comprising the nucleic acid of the eighth aspect of the present invention.

[0023] In the 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.

[0024] In an eleventh aspect, the present invention relates to a pharmaceutical composition comprising the protein of the seventh aspect of the present invention.

[0025] 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.

[0026] In the thirteenth 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, which is used in the following methods: (i) treating or preventing a disease or condition selected from the following: damage, trauma, ischemia, reperfusion injury, trauma, mechanical overload, poisoning, surgery, primary or acquired cardiomyopathy, post-ischemic contractile dysfunction, myocardial infarction, preferably acute myocardial infarction, angina pectoris, heart failure, cardiac inflammation, heart failure, hypertrophy and fibrosis; (ii) promoting or improving cardiac tissue regeneration, cardiomyocyte proliferation, neovascularization, cardiac function or left ventricular contractile function after myocardial infarction; (iii) protecting cardiomyocytes from death, for example by apoptosis or necrosis; or (iv) reducing the infarct area after myocardial infarction, preferably acute myocardial infarction.

[0027] In a fourteenth aspect, the present invention relates to a method for recombinantly expressing a protein of the seventh aspect of the present invention in a host cell.

[0028] In a fifteenth aspect, the invention relates to a composition obtainable by the method of the fourteenth aspect of the invention.

[0029] Finally, in the sixteenth aspect, the present invention relates to use of the host cell of the tenth aspect of the present invention for recombinantly expressing MYDGF protein. DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention provides such proteins, which have MYDGF activity and achieve minimal degradation and process-derived post-translational modifications when recombinantly expressed in host cells. The present invention also provides methods for mass production of these proteins in cell-based expression systems. The method requires significantly reduced purification efforts for providing a homogeneous protein composition suitable for being formulated into a pharmaceutical product. If SEQ ID NO: 1 or SEQ ID NO: 2 is mentioned below, it must be understood that SEQ ID NO: 1 is a preferred alternative.

[0032] The proteins disclosed herein in the context of the present invention 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 mature human MYDGF protein. The native human MYDGF protein is expressed as a precursor protein having an N-terminal signal peptide of 31 amino acids and a C-terminal KDEL-like endoplasmic reticulum (ER) retention sequence. The sequence of the 173 amino acid MYDGF precursor protein is shown in SEQ ID NO:5 herein. After cutting the N-terminal signal peptide, mature MYDGF is released. The sequence of the mature human MYDGF protein consists of 142 amino acids and is shown in SEQ ID NO:6 herein. The protein of the present invention differs from mature MYDGF only in a single amino acid that has been added to its N-terminus. Therefore, 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 as "[+A]" or "[+A] variant" hereinafter in this article. 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".

[0033] The protein is associated with a particularly low risk of containing antigenic epitopes that are not derived from human MYDGF. Accordingly, the protein of the present invention achieves minimal risk of generating anti-drug antibodies. The difference between the protein of the present invention and the native human MYDGF protein is a single amino acid, which means that the region added to the native protein is too small to generate new epitopes. The absence of anti-drug antibodies makes the protein of the present invention highly suitable for therapeutic purposes. Preferably, administration of the protein to humans will generate only minimal levels of anti-drug antibodies or antibodies to endogenous MYDGF, and more preferably no antibodies at all.

[0034] As can be seen from the examples below, the proteins of the present invention show low levels of chemical and post-translational modifications when expressed in a cell-based expression system, i.e., an expression system using eukaryotic or prokaryotic cells for protein recombinant production. The effective reduction of chemical and post-translational modifications is of fundamental importance in the production of proteins for pharmaceutical applications. In particular, the proteins of the present invention are characterized by low levels of carbamylation and gluconylation. As used herein, carbamylation 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 of the protein from inclusion bodies and protein refolding, carbamylation preferably occurs in less than 6.0% (w / w) of the total protein of SEQ ID NO: 1, more preferably in less than 5.5% (w / w), less than 5.0% (w / w), less than 4.5% (w / w) or less than 4.0% (w / w) of the total protein having the amino acid sequence of SEQ ID NO: 1. Similarly, after expressing the protein in a cell-based expression system, isolating the protein from inclusion bodies and refolding the protein, carbamylation preferably occurs in less than 6.0% (w / w) of the total protein of SEQ ID NO: 2, more preferably less than 5.5% (w / w), less than 5.0% (w / w), less than 4.5% (w / w) or less than 4.0% (w / w) of the total protein having the amino acid sequence of SEQ ID NO: 2. In general, a carbamylation level of less than 6.0% (w / w) is acceptable and does not pose a risk for pharmaceutical applications.

[0035] In addition, the proteins disclosed herein are characterized by a low degree of gluconylation. Glucosylation of recombinantly expressed proteins is frequently observed in bacterial host cells, such as in E. coli BL21 (DE3) cells. This modification results from the formation of 6-phosphogluconolactone (6-PGLac), a compound produced by glucose 6-phosphate dehydrogenase. For the proteins of the present invention, after expression in a cell-based expression system, isolation of the protein from inclusion bodies, and protein refolding, gluconylation preferably occurs only in less than 4.0% (w / w) of the total protein of SEQ ID NO: 1, more preferably in less than 3.5% (w / w), less than 3.0% (w / w), less than 2.5% (w / w) or less than 2.0% (w / w) of the total protein of SEQ ID NO: 1. Similarly, carbamylation preferably occurs in less than 4.0% (w / w) of the total protein of SEQ ID NO: 2, more preferably less than 3.5% (w / w), less than 3.0% (w / w), less than 2.5% (w / w) or less than 2.0% (w / w) of the total protein of SEQ ID NO: 2. In general, a gluconylation level of less than 4% is acceptable and does not pose a risk for pharmaceutical applications.

[0036] A further embodiment of the present invention relates to a protein having an amino acid sequence of SEQ ID NO: 1 and further having a spectrum in two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) substantially the same as shown in Table 1 below. In particular, when analyzing an 8.5 mg / ml sample of the respective MYDGF protein in 50 mM sodium phosphate buffer containing 50 mM sodium chloride and 9% (v / v) D2O pH 7.4, the protein has an NMR spectrum comprising at least 2 of the amino acids in Table 1. 1 H and / or 15 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, 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 1 H and / or 15 N peak.

[0037] Most preferably, when an 8.5 mg / ml sample of the respective MYDGF protein is analyzed in 50 mM sodium phosphate buffer, pH 7.4, containing 50 mM sodium chloride and 9% (v / v) D2O, the protein has an NMR spectrum comprising all 136 of the MYDGF proteins shown in Table 1. 1 H and / or 15 N peak or consists of it. Therefore, the protein has the secondary and tertiary structures of native human MYDGF protein.

[0038] In a further embodiment, the invention relates to a protein which folds so that more than 70% and preferably more than 80%, more than 90% or more than 95% of the 1 H and / or 15 The N peak resulted in a combined chemical shift deviation (CCSD) value of less than 0.01 ppm when compared to the corresponding peaks in Table 1. CCSD was calculated according to the following formula (Brinson et al. 2019):

[0039]

[0040] where δ H and δ N Respectively represent the given cross peak 1 H and 15 N chemical shift, and δ Href and δ Nref The same cross peak 1 H and 15 N refers to the chemical shift. If more than 70%, and preferably more than 80%, more than 90% or more than 95% of the 1 H and / or 15 The N peak results in a CCSD value below 0.01 ppm, then the protein fold is very similar to that observed for the [+A] variant in Table 1. Specifically, if more than 90% or more than 95% of the N peaks in the 2D-NMR pattern are 1 H and / or 15 The N peak achieves CCSD values ​​below 0.01 ppm, and the protein folding is almost identical to that observed for the [+A] variant in Table 1.

[0041] Table 1 :NMR spectrum [+A]

[0042]

[0043]

[0044]

[0045]

[0046] A further embodiment of the invention is a composition comprising a protein as described above, preferably a composition obtainable from recombinant expression in a bacterial expression system, such as the method described in more detail below.

[0047] The composition of the invention may comprise a protein having the amino acid sequence of SEQ ID NO: 1 together with variants thereof which are shorter in length and achieve 100% sequence identity with the amino acid sequence of SEQ ID NO: 1 over their entire length, wherein the length is at least 100 amino acids, without allowing gaps in the alignment. In said composition, the ratio of the signal obtained from the protein according to SEQ ID NO: 1 to the sum of the signals obtained from said shorter variants is higher than 20, and preferably higher than 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, and more preferably higher than 450, as determined by liquid chromatography mass spectrometry (LCMS) according to Tolonen et al. (2011). 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, as determined by LCMS according to Tolonen et al. (2011), was found to be 466. When calculating the ratio of the signal obtained from the protein according to SEQ ID NO: 1 to the sum of the signals obtained from the shorter variants, any carbamylated or gluconylated proteins were excluded. The percentage used to calculate the ratio is based on the sum of the peak intensities of the unmodified MYDGF protein and the annotated MYDGF post-translational modification (PTM) species in the deconvoluted full mass spectrum of the MYDGF protein in the composition. The minimum requirements for mass spectrometry instrumentation and data processing are outlined in Example 4 below.

[0048] The composition of the invention may also comprise a protein having the amino acid sequence of SEQ ID NO: 2 together with variants thereof which are shorter in length and achieve 100% sequence identity with the amino acid sequence of SEQ ID NO: 2 over their entire length, wherein the length is at least 100 amino acids, without allowing gaps in the alignment. Within the composition, 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, and preferably higher than 50, 75, 100, 125, 150, and more preferably higher than 175 or 180, as determined by LCMS according to Tolonen et al. (2011). 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 variants, as determined by LCMS according to Tolonen et al. (2011), was found to be 186. 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 carbamylated or gluconylated protein was excluded. The percentage used to calculate the ratio is based on the sum of peak intensities of unmodified MYDGF protein and annotated MYDGF post-translational modification (PTM) species in the deconvoluted intact mass spectrum of MYDGF protein in the composition.The minimum requirements for mass spectrometry instrumentation and data processing are outlined in Example 4 below.

[0049] In one embodiment, less than 8%, and preferably less than 7%, less than 6% or less than 5% of the MYGDF protein in the composition of the invention is carbamylated, wherein the percentage is based on the sum of the peak intensities of the unmodified MYDGF protein and the annotated MYDGF post-translational modification (PTM) species in the deconvoluted full mass spectrum of the MYDGF protein in the composition. The minimum requirements for mass spectrometry instrumentation and data processing are outlined in Example 4 below.

[0050] In another embodiment, less than 6%, and preferably less than 5%, less than 4% or less than 3% of the MYDGF protein in the composition of the invention is gluconylated, wherein the percentage is based on the sum of the peak intensities of the unmodified MYDGF protein and the annotated MYDGF PTM species in the deconvoluted intact mass spectrum of the MYDGF protein in the composition. The minimum requirements for mass spectrometry instrumentation and data processing are outlined in Example 4 below.

[0051] According to another object of the present invention, the composition of the present invention preferably comprises a small amount of DNA derived from a host cell, the host cell being used to produce the MYDGF protein. Preferably, the composition of the present invention comprises less than 20pg / mg, preferably less than 15pg / mg, more preferably less than 10pg / mg and most preferably less than 5pg host cell DNA / mg of the composition, for example less than 3pg, less than 2pg or less than 1pg host cell DNA / mg of the composition. Preferably, the presence of host cell DNA in the composition is determined by quantitative polymerase chain reaction (qPCR), for example real-time qPCR.

[0052] According to another object of the present invention, the composition of the present invention preferably also includes only a small amount of bacterial endotoxin, which is caused by the production of MYDGF protein in bacterial host cells. Specifically, it is preferred that the composition includes less than 0.2EU / mg of the composition, and preferably less than 0.1EU, less than 0.09EU or 0.08EU bacterial endotoxin / mg of the composition. Suitable methods for detecting the presence of bacterial endotoxins include the current United States Pharmacopoeia (United States Pharmacopoeia) (USP-NF 2021, Issue 2, Chapter 85), European Pharmacopoeia (European Pharmacopoeia) (2021, 10th Edition, 10.5, Chapter 2.6.14) and Japanese Pharmacopoeia (Japanese Pharmacopoeia), Supplement II, JP 17th Edition, 4.01) described in the dynamic color development method.

[0053] According to another object of the present invention, it is preferred that less than 8% (w / w), and 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 carbamylated. Similarly, it is preferred that less than 6% (w / w), and 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 gluconylated. It is particularly preferred that the composition of the present invention comprises a detectable amount of carbamylated protein, however, wherein the amount of carbamylated protein in the composition is less than 5% (w / w). Similarly, it is particularly preferred that the composition of the present invention comprises a detectable amount of gluconylated protein, however, wherein the amount of gluconylated protein in the composition is less than 5% (w / w).

[0054] The compositions of the invention may comprise urea resulting from the inclusion body solubilization and / or refolding steps.

[0055] The composition of the present invention preferably contains no or only a small amount of protein aggregates that may be caused by aggregation of protein molecules, such as dimers, trimers or oligomers of MYDGF protein variants. Preferably, the composition contains the above-mentioned MYDGF, i.e., SEQ ID NO: 1 or SEQ ID NO: 2, which is predominantly a monomer. More preferably, the monomer content of the protein in the composition is 95% (w / w) or higher, and even more preferably 96% (w / w) or higher, 97% (w / w) or higher, 98% (w / w) or higher or 99% (w / w) or more. In other words, the amount of protein aggregates in the composition is about 5% (w / w) or less, and 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 amount of protein monomers and aggregates is preferably measured by size exclusion chromatography (SEC) and more preferably by size exclusion high performance liquid chromatography (SEC HPLC).

[0056] The composition of the invention preferably comprises a protein of 143 amino acids having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The composition preferably comprises less than 5% (w / w), less than 4% (w / w), less than 3% (w / w), less than 2% (w / w) and preferably less than 1% (w / w) of protein molecules shorter than 143 amino acids as measured by LCMS.

[0057] Based on the total weight of all non-gluconylated and non-carbamylated proteins in the composition comprising at least 100 contiguous amino acids of the sequence of SEQ ID NO: 1 and 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 less than 5% (w / w), less than 4% (w / w), less than 3% (w / w), less than 2% (w / w) and more preferably less than 1% (w / w) of protein molecules that differ from the amino acid sequence of SEQ ID NO: 1 by (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. The composition comprising the +A variant (SEQ ID NO: 1) shown in Table 15 shows 0.2% of protein according to (i) or (ii).

[0058] Based on the total weight of all non-gluconylated and non-carbamylated proteins in the composition comprising at least 100 contiguous amino acids of the sequence of SEQ ID NO: 1 and 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 less than 5% (w / w), less than 4% (w / w), less than 3% (w / w), less than 2% (w / w) and more preferably less than 1% (w / w) of protein molecules that differ from the amino acid sequence of SEQ ID NO: 1 by the deletion of 1-4 amino acids at the N-terminus of SEQ ID NO: 1. The composition comprising the +A variant (SEQ ID NO: 1) shown in Table 15 shows 0.2% (w / w) of such protein deletion.

[0059] Based on the total weight of all non-gluconylated and non-carbamylated proteins in the composition comprising at least 100 contiguous amino acids of the sequence of SEQ ID NO: 2 and 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 less than 5% (w / w), less than 4% (w / w), less than 3% (w / w), less than 2% (w / w) and more preferably less than 1% (w / w) of protein molecules that differ from the amino acid sequence of SEQ ID NO: 2 by (i) a deletion of 1-4 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. The composition comprising the +S variant (SEQ ID NO: 2) shown in Table 15 shows 3.1% (w / w) of protein according to (i) or (ii).

[0060] Based on the total weight of all non-gluconylated and non-carbamylated proteins in the composition comprising at least 100 contiguous amino acids of the sequence of SEQ ID NO: 2 and 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 less than 5% (w / w), less than 4% (w / w), less than 3% (w / w), less than 2% (w / w) and more preferably less than 1% (w / w) of protein molecules that differ from the amino acid sequence of SEQ ID NO: 2 by the following: 1-4 amino acid deletions at the N-terminus of SEQ ID NO: 2. The composition comprising the +S variant (SEQ ID NO: 2) shown in Table 15 shows 0.5% (w / w) of such protein deletions.

[0061] Based on the total weight of all non-gluconylated and non-carbamylated proteins in the composition comprising at least 100 adjacent amino acids of the sequence of SEQ ID NO: 1 and determined by LCMS after reductive dimethylation (stable isotope dimethyl labeling, SIDL) according to Tolonen et al. (2011), the composition of the present invention preferably contains more than 95% (w / w), more than 96% (w / w), more than 97% (w / w), more than 98% (w / w) and 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.

[0062] Based on the total weight of all non-gluconylated and non-carbamylated proteins of at least 100 adjacent amino acids of the sequence of SEQ ID NO: 2 in the composition and determined by LCMS after reductive dimethylation (stable isotope dimethyl labeling, SIDL) according to Tolonen et al. (2011), the composition of the present invention preferably contains more than 95% (w / w), more than 96% (w / w), more than 97% (w / w), more than 98% (w / w) and 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.

[0063] The composition of the present invention preferably comprises 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) and 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, based on the total weight of all proteins in the composition comprising at least 100 consecutive amino acids of the sequence of SEQ ID NO: 1 and determined by liquid chromatography mass spectrometry (LCMS), wherein gluconylated proteins, carbamylated proteins, anhydrous proteins and Na+ adducts are ignored for the calculation of the percentages.

[0064] The composition of the present invention preferably comprises 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) and 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, based on the total weight of all proteins in the composition comprising at least 100 consecutive amino acids of the sequence of SEQ ID NO: 2 and determined by liquid chromatography mass spectrometry (LCMS), wherein gluconylated proteins, carbamylated proteins, anhydrous proteins and Na+ adducts are ignored for the calculation of the percentages.

[0065] The proteins of the invention share at least one biological activity of the naturally occurring human mature MYDGF protein, which makes them useful for therapeutic applications. Therefore, the present invention also relates to a composition as described above for use as a medicament, in particular for the known uses of MYDGF, see for example WO 2014 / 111458 and WO 2021 / 148411.

[0066] Preferably, the protein is active in: (i) treating or preventing a disease or condition selected from the group consisting of: injury, trauma, ischemia, reperfusion injury, trauma, mechanical overload, poisoning, surgery, primary or acquired cardiomyopathy, post-ischemic systolic dysfunction, myocardial infarction, preferably acute myocardial infarction, angina pectoris, heart failure, cardiac inflammation, cardiac insufficiency, hypertrophy and fibrosis; (ii) promoting or improving cardiac tissue regeneration, cardiomyocyte proliferation, neovascularization, cardiac function or left ventricular systolic function after myocardial infarction; (iii) protecting cardiomyocytes from death, for example, by apoptosis or necrosis; or (iv) reducing the infarct size after myocardial infarction, preferably acute myocardial infarction. See WO 2014 / 111458.

[0067] The cardiomyopathy to be treated may be a hereditary cardiomyopathy or a cardiomyopathy caused by a spontaneous mutation. The cardiomyopathy to be treated may also be an acquired cardiomyopathy, preferably an ischemic cardiomyopathy caused by atherosclerosis or other coronary artery diseases, a cardiomyopathy caused by myocardial infection or poisoning, a hypertensive heart disease caused by pulmonary hypertension and / or arterial hypertension or a valvular heart 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 noncompacting mitochondrial myopathy, dilated cardiomyopathy (DCM), restrictive cardiomyopathy (RCM), Takotsubo cardiomyopathy, Loeffler endocarditis, diabetic cardiomyopathy, alcoholic cardiomyopathy or obesity-related cardiomyopathy.

[0068] The heart failure to be treated is preferably chronic heart failure. 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 intermediate ejection fraction (HFmrEF). See WO 2021 / 148411.

[0069] It is particularly preferred that the above-mentioned protein has at least part of the activity of naturally occurring human mature MYDGF protein in enhancing coronary endothelial cells or coronary endothelial cell proliferation. In particular, it is preferred that the protein has at least 50% of the activity of naturally occurring human mature MYDGF protein in enhancing coronary endothelial cell proliferation, and preferably at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% of the activity. The protein can also play an activity in enhancing coronary endothelial cell proliferation that is higher than the activity of 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% activity relative to human mature MYDGF protein. Preferably, the activity in enhancing the proliferation of coronary endothelial cells is determined as described in the potency assay of Example 6 below. The activity of the protein is calculated by the following formula: Activity [%] = EC of mature MYDGF 50 / EC of the test protein 50 )x 100.

[0070] In another embodiment, it is preferred that the protein has at least 50% of the activity of the [+G]-HEK variant in enhancing the proliferation of coronary endothelial cells, wherein the [+G]-HEK variant has been 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 the activity. The protein may also exert an activity in enhancing the proliferation of coronary endothelial cells that is greater 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 the proliferation of coronary endothelial cells is determined as described in the potency assay of Example 6 below. The activity of the protein is calculated by the following formula: Activity [%] = [+G] - EC of HEK 50 ) / test protein EC 50 )x 100.

[0071] In yet another embodiment, it is preferred that the above protein has at least 50% of the activity of naturally occurring human mature MYDGF protein in enhancing cardiomyocyte proliferation, and preferably at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% of the activity. The protein can also exert an activity in enhancing cardiomyocyte proliferation that is higher than the activity of 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% activity relative to 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 following formula: Activity [%] = EC of mature MYDGF 50 / EC of the test protein 50 )x 100.

[0072] In yet another embodiment, it is preferred that the protein has at least 50% of the activity of the [+G]-HEK variant in enhancing cardiomyocyte proliferation, wherein the [+G]-HEK variant has been 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 the activity. The protein may also exert an activity in enhancing cardiomyocyte proliferation that is greater 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 following formula: Activity [%] = [+G] - EC of HEK 50 ) / test protein EC 50 )x 100.

[0073] In yet another embodiment, it is preferred that the above protein enhances the proliferation of coronary endothelial cells, EC, when measured in the potency assay of Example 6 described below. 50 Preferably, the protein enhances coronary endothelial cell proliferation, EC50 Less than 95ng / ml, less than 90ng / ml, less than 85ng / ml, less than 80ng / ml, less than 75ng / ml, less than 70ng / ml, less than 65ng / ml or less than 60ng / ml.

[0074] In yet another embodiment, it is preferred that the protein enhances cardiomyocyte proliferation, EC 50 Preferably, the protein enhances coronary endothelial cell proliferation, EC, when measured in the potency assay of Example 7. 50 Less than 95ng / ml, less than 90ng / ml, less than 85ng / ml, less than 80ng / ml, less than 75ng / ml, less than 70ng / ml, less than 65ng / ml, less than 60ng / ml, less than 55ng / ml, less than 50ng / ml, less than 45ng / ml, less than 40ng / ml, less than 35ng / ml, less than 30ng / ml or less than 25ng / ml.

[0075] The invention also relates to nucleic acids encoding proteins which, when matured, result in 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 may be DNA or RNA. However, it is preferred that the nucleic acid is a DNA molecule.

[0076] The present invention also relates to a vector or plasmid comprising a nucleic acid encoding a protein that, when matured, results in one of the MYDGF proteins of the present invention. Preferably, the vector will be an expression vector that allows expression of a protein that matures into a protein of SEQ ID NO: 1 or SEQ ID NO: 2 in a prokaryotic or eukaryotic cell. It is particularly preferred that the vector is a prokaryotic expression vector, i.e., a vector that allows expression of a recombinant protein in a prokaryotic cell environment. Even more preferably, the vector is a bacterial expression vector, i.e., a vector that allows expression of a recombinant protein in a bacterial cell. The vector preferably comprises an origin of replication, a promoter, a polylinker for cloning, a transcription terminator, and a gene that allows selection, such as a gene encoding a protein that confers antibiotic resistance. A large number of expression vectors for E. coli and other bacterial hosts have been described. Examples of vectors suitable for protein expression in E. coli cells include, for example, vectors of the pBluescript series, the pUC series, the pQE series, or the pET series. The vector preferably comprises an inducible promoter system that is capable of initiating expression after the addition of an inducer compound.

[0077] In a preferred embodiment, the vector carrying the nucleic acid encoding the above-mentioned MYDGF protein is a pET type vector. These vectors generally include a replication origin, a T7 promoter specific for T7 RNA polymerase, a lac operator for binding to 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 combination of IPTG or lactose with the lac repressor protein causes a conformational change, which causes the dissociation of the protein from the operator and induces expression from the T7 promoter. Suitable pET vectors for use in the methods 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).

[0078] The expression vector can be transformed into a eukaryotic or prokaryotic host cell by any suitable method. For example, an expression vector for E. coli can be introduced into a host cell, for example, by electroporation or chemical methods, such as calcium phosphate-mediated transformation, as described in Maniatis et al. 1982, Molecular Cloning, A laboratory Manual, Cold Spring Harbor Laboratory.

[0079] The present invention also relates to a host cell comprising a protein, nucleic acid or vector as described above. The host cell can be a eukaryotic cell or a prokaryotic cell, but it is particularly preferred that it is a prokaryotic host cell, such as a bacterial cell. Although the type of bacterial cell is not particularly limited, it is preferred that the host cell is an Escherichia coli cell, such as an Escherichia coli cell BL21 cell.

[0080] The present invention also relates to the above composition for use in the preparation of a pharmaceutical composition, ie a composition comprising the above protein, preferably a composition obtainable by a recombinant expression method in a cell-based expression system, such as the method described in more detail below.

[0081] The present invention also relates to a pharmaceutical composition comprising the above-mentioned protein or the above-mentioned composition. In addition to the protein, the composition may also contain a pharmaceutically acceptable carrier and other excipients commonly used in the preparation of pharmaceutical compositions. Generally, the pharmaceutical composition can be formulated for different routes of administration. It is preferred that the composition of the present invention is formulated for parenteral administration, such as intravenous, intra-arterial, intracoronary or intravenous administration. Compositions suitable for injection or infusion may include solutions or dispersions and powders for the extemporaneous preparation of such injectable solutions or dispersions. The composition for injection must be sterile and should be stable under the conditions of manufacture and storage. Preferably, the composition for injection or infusion also includes a preservative, such as chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. For intravenous administration, suitable carriers may include physiological saline, antibacterial water, Cremophor EL TM (BASF) or phosphate buffered saline (PBS). Sterile solutions for injection or infusion can be prepared by incorporating the required amount of MYDGF protein into an appropriate solvent, followed by filter sterilization.

[0082] In addition to the MYDGF protein, the pharmaceutical composition of the present invention may further comprise an additional active agent that is effective in: (i) treating or preventing a disease selected from the following: injury, trauma, ischemia, reperfusion injury, trauma, mechanical overload, poisoning, surgery, primary or acquired cardiomyopathy, post-ischemic systolic dysfunction, myocardial infarction, preferably acute myocardial infarction, angina pectoris, heart failure, cardiac inflammation, cardiac insufficiency, hypertrophy and fibrosis; (ii) promoting or improving cardiac tissue regeneration, cardiomyocyte proliferation, neovascularization, cardiac function or left ventricular systolic function after myocardial infarction; (iii) protecting cardiomyocytes from death, for example, by apoptosis or necrosis; or (iv) reducing the infarct size after myocardial infarction, preferably acute myocardial infarction, in a subject in need thereof.

[0083] For example, the pharmaceutical composition may include one or more angiotensin converting enzyme (ACE) inhibitors, such as benazepril, zofenopril, perindopril, trandolapril, captopril, enalapril, lisinopril and ramipril. The pharmaceutical composition may also include one or more diuretics, including chlorothiazide, hydrochlorothiazide, bendroflumethiazide, spironolactone, chlorthalidone, methylchlorothiazide, polythiazide, triamterene, furosemide, ethacrynic acid, metolazone, bumetanide, indapamide, amiloride, acetazolamide, torsemide and eplerenone. The pharmaceutical composition may also include one or more beta blockers, including acebutolol, atenolol, betaxolol, bisoprolol, carvedilol, celiprolol, esmolol, metoprolol, nebivolol, propranolol, sotalol and / or timolol.

[0084] If the protein is used in combination with any of the above additional active agents, such as ACE inhibitors, diuretics and / or beta blockers, the two active agents 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 agent. The separate compositions may be administered simultaneously, i.e., at two different administration sites at the same time, or they may be administered sequentially (in either order) to the same administration site or to different administration sites.

[0085] The present invention also relates to a protein or pharmaceutical composition as described above, which is used as a medicament. More specifically, the protein or pharmaceutical composition is suitable for use as a medicament for (i) treating or preventing a disease or condition selected from the group consisting of: injury, trauma, ischemia, reperfusion injury, trauma, mechanical overload, poisoning, surgery, primary or acquired cardiomyopathy, post-ischemic systolic dysfunction, myocardial infarction, preferably acute myocardial infarction, angina pectoris, heart failure, cardiac inflammation, cardiac insufficiency, hypertrophy and fibrosis; (ii) promoting or improving cardiac tissue regeneration, cardiomyocyte proliferation, neovascularization, cardiac function or left ventricular systolic function after myocardial infarction; (iii) protecting cardiomyocytes from death, for example, by apoptosis or necrosis; or (iv) reducing the infarct size after myocardial infarction, preferably acute myocardial infarction.

[0086] The present invention also relates to a method for: (i) treating or preventing a disease or condition selected from the group consisting of: injury, trauma, ischemia, reperfusion injury, trauma, mechanical overload, poisoning, surgery, primary or acquired cardiomyopathy, post-ischemic contractile dysfunction, myocardial infarction, preferably acute myocardial infarction, angina pectoris, heart failure, cardiac inflammation, cardiac insufficiency, hypertrophy and fibrosis; (ii) promoting or improving cardiac tissue regeneration, cardiomyocyte proliferation, neovascularization, cardiac function or left ventricular contractile function after myocardial infarction; (iii) protecting cardiomyocytes from death, for example, by apoptosis or necrosis; or (iv) reducing the infarct size after myocardial infarction, preferably acute myocardial infarction, in a subject in need thereof, wherein the method comprises administering an effective amount of a pharmaceutical composition as described above comprising a protein of SEQ ID NO: 1 or SEQ ID NO: 2.

[0087] The present invention also provides a method for recombinantly expressing MYDGF protein in a host cell, the method comprising the following steps:

[0088] (a) providing a host cell as described above, preferably a host cell comprising a nucleic acid encoding a protein which, after maturation, consists of 143 amino acids having an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2;

[0089] (b) culturing the host cell under conditions allowing expression of the MYDGF protein;

[0090] (c) isolating inclusion bodies containing the MYDGF protein from the host cell; and

[0091] (d) solubilizing the inclusion bodies and refolding the MYDGF protein.

[0092] The above method of the present invention relates to the production of the proteins shown in SEQ ID NO:1 and SEQ ID NO:2. The method utilizes a host cell as described above, preferably a prokaryotic host cell comprising a nucleic acid encoding a protein, which leads to one of the proteins of the present invention when mature. The bacterial host cell used in the method of the present invention preferably contains a nucleic acid encoding a protein that leads to the protein of SEQ ID NO:1 when mature, or a nucleic acid encoding a protein that leads to the protein of SEQ ID NO:2 when mature, and the nucleic acid is inserted in an expression vector that allows the recombinant protein to be expressed in the host cell. The host cell can be any type of eukaryotic cell or prokaryotic cell suitable for expressing recombinant exogenous proteins (i.e., proteins that are not naturally produced by host cells). 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 of Escherichia coli. The use of Escherichia coli strain BL21 or its derivative strain is most preferred.

[0093] Preferably, step (a) of the above method comprises (i) providing a host cell comprising a nucleic acid comprising an open reading frame of a sequence according to SEQ ID NO: 11 or SEQ ID NO: 12 operably linked to a promoter, flanked by a start codon and a stop codon; or

[0094] (ii) providing a host cell comprising a nucleic acid encoding a protein which before maturation consists of 144 amino acids having an amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16.

[0095] The amino acid sequence of SEQ ID NO:15 or SEQ:16 is a MYDGF protein including an N-terminal methionine residue derived from a start codon. These proteins undergo a maturation process that removes the N-terminal methionine. Maturation is preferably accomplished by one or more host cell derived aminopeptidases, more preferably one or more host cell derived methionine aminopeptidases. For example, one or more aminopeptidases may be produced by a bacterial host cell such as Escherichia 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, the nucleic acid being an expression vector that is preferably circular, coiled or supercoiled.

[0096] In step (b) of the method, the bacterial host cell is cultured under conditions that allow expression of the protein in the bacterial host cell. The conditions for providing expression of the MYDGF protein will depend on the prokaryotic host cell and the expression vector used in the process. Suitable conditions can be easily selected and applied by those skilled in the art. For example, if an inducible bacterial expression system, such as a pET vector, is used, the conditions that allow expression of the target protein typically include a culture temperature of 20-42°C, preferably 30-40°C, and more preferably 35-38°C. The culture medium typically has a pH of 6.5 to 9.0, more typically 7.0 to 8.0, and preferably 7.5. The fermentation of the culture can last for a period of time ranging from a few hours to a few days. The cells can be cultured in a batch or fed batch process. For example, if the culture is performed as a batch process, the culture time typically ranges from about 12 hours to about 36 hours. When a continuous process is used, the fermentation time may be as long as 21 days or more. Suitable methods for culturing bacterial host cells are described in Encyclopaedia of Bioprocess Technology: Fermentation, Biocatalysis, and Bioseparation, Vols. 1-5, Flickinger, MC, Drew, SW (eds.), 1999 John Wiley & Sons. Preferably, the protein is expressed in the host cell using an inducible expression system, e.g., a system that allows initiation of protein expression by addition of an inducer compound such as IPTG or lactose to the culture medium.

[0097] Proteins expressed in this way will accumulate in the host cell in an insoluble form in so-called inclusion bodies. This means that the expressed protein accumulates within the cell and is deposited in the form of insoluble aggregates of inactive, misfolded protein.

[0098] In step (c) of the method of the invention, inclusion bodies containing insoluble MYDGF protein are isolated from host cells. To this end, bacterial host cells are harvested after cultivation and disrupted, for example, by high-pressure homogenization or other generally known cell lysis procedures. Inclusion bodies can be isolated from the lysate by different methods, for example by tubular centrifugation, such as high-speed tubular centrifugation. Methods for isolating inclusion bodies from bacterial cells are generally known and are described, for example, in Peternel & Komel (2010) and Eggenreich et al. (2020).

[0099] In step (d) of the method of the present invention, the MYDGF protein in the isolated inclusion bodies obtained from step (c) is dissolved and refolded. Methods for dissolving proteins are known and include, for example, incubating the inclusion bodies in the presence of urea, guanidine hydrochloride (GuHCl) and / or DTT, followed by filtration. It is preferred in this article that the refolding of the protein is performed in the presence of urea. Filtration may include one or more of depth filtration, ultrafiltration and / or diafiltration. Methods for refolding proteins are also known and include, for example, incubating the protein dissolved from the inclusion bodies in the presence of urea, CaCl2 and / or cystamine. Kits for dissolving and refolding proteins from inclusion bodies are marketed by different manufacturers.

[0100] The method may further comprise step (e), wherein a refolded MYDGF protein of 143 amino acids having an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is obtained. This step is after step (d) of the above method.

[0101] Preferably, the method of the present invention comprises a further step (f), wherein the solubilized and refolded MYDGF protein obtained from step (e) is further purified. Protein purification can be carried out according to conventional methods, and may include one or more of ultrafiltration, diafiltration, hydrophobic interaction chromatography and / or anion exchange chromatography.

[0102] Preferably, the purification in step (f) is accomplished by anion exchange chromatography or hydrophobic interaction chromatography. These types of chromatography can be performed by contacting the MYDGF protein with a chromatography resin material under conditions that allow the MYDGF protein to adsorb to the resin. The resin material is then optionally washed to remove impurities, such as non-proteinaceous materials or proteins other than MYDGF. In the final step, the MYDGF protein is eluted from the resin.

[0103] More preferably, the purification in step (f) is accomplished by anion exchange chromatography. If anion exchange chromatography is used, the adsorption of the MYDGF protein to the anion exchange chromatography resin is preferably performed under conditions of 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 lowering the pH of the liquid phase (i.e., the mobile phase).

[0104] The method of the present invention allows the production of particularly high amounts of the protein of SEQ ID NO: 1 or SEQ ID NO: 2. As shown in Examples 2 and 3, the protein can be produced with the method of the present invention with a productivity of more than 0.4 g protein / 100 g cells, and preferably more than 0.5 g protein / 100 g cells, more than 0.6 g protein / 100 g cells, more than 0.7 g protein / 100 g cells, more than 0.8 g protein / 100 g cells, more than 0.9 g protein / 100 g cells, and more preferably more than 1.0 g protein / 100 g cells. In other words, the process of the present invention allows the production of the protein of SEQ ID NO: 1 or SEQ ID NO: 2 in amounts of more than 100 g protein / batch, and preferably more than 150 g protein / batch, more than 200 g protein / batch, more than 250 g protein / batch, more than 300 g protein / batch, and more preferably more than 350 g or 400 g protein / batch (see Example 3).

[0105] The present invention also relates to the use of bacterial host cells such as those described elsewhere herein for recombinant expression of MYDGF protein.The host cell is a prokaryotic or eukaryotic cell comprising a nucleic acid, plasmid or vector encoding a protein as described herein above.

[0106] A further embodiment of the present invention is a protein according to one of the embodiments mentioned above, which can be obtained by heterologous expression in bacteria, and preferably obtained by the method described above. A further embodiment of the present invention is a protein according to one of the embodiments mentioned above, which can be obtained by production in the form of inclusion bodies and refolding. A further embodiment of the present invention is a composition comprising MYDGF protein, wherein the composition can be obtained by heterologous expression in bacteria, and preferably obtained by the method described above, wherein the composition comprises less than 1% (w / w) of protein molecules shorter than 143 amino acids.

[0107] In yet another aspect, the present invention provides a method for producing MYDGF protein in a cell-based expression system, comprising

[0108] (a) providing a host cell, preferably a host cell comprising a nucleic acid encoding a protein which, after maturation, consists of SEQ ID NO: 1 or SEQ ID NO: 2;

[0109] (b) culturing the host cell under conditions that allow expression of the protein;

[0110] (c) isolating inclusion bodies containing the MYDGF protein from the host cell; and

[0111] (d) solubilizing the inclusion bodies and refolding the MYDGF protein.

[0112] The different method steps of the method have been described above. It is again preferred that the refolding of the protein is performed in the presence of urea. Accordingly, the protein refolding in step (d) preferably comprises incubating the protein in the presence of urea.

[0113] The method may further comprise step (e), wherein a refolded MYDGF protein of 143 amino acids having an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is obtained. This step is after step (d) of the above method.

[0114] The above method may further comprise an additional step (f), wherein the MYDGF protein expressed by the host cell and separated from the inclusion body is purified. This step may comprise a method commonly used in the field of protein purification, such as ultrafiltration, diafiltration and / or anion exchange chromatography.

[0115] As described elsewhere herein, the host cell can be any host cell suitable for recombinant protein production. The host cell can be eukaryotic or prokaryotic. The use of prokaryotic host cells is preferred. Even more preferred is that the host cell is a bacterial cell, such as an E. coli cell. The use of the E. coli cells of strain BL21 or its derivative strains is particularly preferred.

[0116] The nucleic acid encoding the protein contained by the host cell can be a DNA or RNA molecule, and preferably a DNA molecule, which upon maturation results in the protein of SEQ ID NO: 1 or SEQ ID NO: 2. 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 herein.

[0117] The present invention also relates to a composition obtainable by the above method, wherein the composition comprises a protein of 143 amino acids having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The composition preferably comprises less than 1% (w / w) of protein molecules shorter than 143 amino acids, as measured by LCMS.

[0118] The composition will contain no or only a small amount of carbamylated protein. In a preferred embodiment, the composition contains a detectable amount of carbamylated protein. For example, in embodiments, the amount of carbamylated 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 carbamylated protein will be limited to less than 8% (w / w) in the composition, and preferably less than 7% (w / w), less than 6% (w / w) or less than 5% (w / w) protein. In another preferred embodiment, the composition does not contain a detectable amount of carbamylated protein.

[0119] Similarly, the composition will contain no or only a small amount of gluconylated protein. In a preferred embodiment, the composition contains a detectable amount of gluconylated protein. For example, in one embodiment, the amount of gluconylated 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 gluconylated protein will be limited to less than 6% (w / w), less than 5% (w / w), less than 4% (w / w) or less than 3% (w / w) of protein in the composition. In another preferred embodiment, the composition does not contain a detectable amount of gluconylated protein.

[0120] In particularly preferred embodiments, the composition comprises a detectable amount of carbamylated protein, however, wherein less than 7% (w / w) or less than 5% (w / w) of the protein in the composition is carbamylated. For example, the composition may comprise at least 0.05% (w / w) or at least 0.1% (w / w) carbamylated protein, however, wherein less than 7% (w / w) of the protein in the composition is carbamylated. In yet another particularly preferred embodiment, the composition may comprise at least 0.05% (w / w) or at least 0.1% (w / w) carbamylated protein, however, wherein less than 5% (w / w) of the protein in the composition is carbamylated.

[0121] Similarly, in another particularly preferred embodiment, the composition comprises a detectable amount of gluconylated protein, however, wherein less than 5% (w / w) or less than 3% (w / w) of the protein in the composition is gluconylated. For example, the composition may comprise at least 0.05% (w / w) or at least 0.1% (w / w) gluconylated protein, however, wherein less than 5% (w / w) of the protein in the composition is gluconylated. In yet another particularly preferred embodiment, the composition may comprise at least 0.05% (w / w) or at least 0.1% (w / w) gluconylated protein, however, wherein less than 3% (w / w) of the protein in the composition is gluconylated.

[0122] In one embodiment, the composition comprises a protein having the amino acid sequence of SEQ ID NO: 1 together with variants thereof which are shorter in length and achieve 100% sequence identity with the amino acid sequence of SEQ ID NO: 1 over their entire length, wherein the length is at least 100 amino acids, without allowing gaps in the alignment. Within the composition, the ratio of the signal obtained from the protein according to SEQ ID NO: 1 to the sum of the signals obtained from the shorter variants is higher than 20, and preferably higher than 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, and more preferably higher than 450 as determined by LCMS after reductive dimethylation (stable isotope dimethyl labeling, SIDL) according to Tolonen et al. (2011). 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, as determined by LCMS according to Tolonen et al. (2011), was found to be 466.

[0123] In one embodiment, the composition comprises a protein having the amino acid sequence of SEQ ID NO: 2 together with variants thereof, the variants being shorter in length and achieving 100% sequence identity with the amino acid sequence of SEQ ID NO: 2 over its entire length, wherein the length is at least 100 amino acids, without allowing gaps in the alignment. Within the composition, 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, and preferably higher than 50, 75, 100, 125, 150, and more preferably higher than 175 or 180, as determined by LCMS after reductive dimethylation (stable isotope dimethyl labeling, SIDL) according to Tolonen et al. (2011). 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 variants, as determined by LCMS according to Tolonen et al. (2011), was found to be 186.

[0124] Preferably, the composition comprises a protein that is folded such that more than 70%, and preferably more than 80%, more than 90% or more than 95% of the protein in the two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) pattern is 1 H and / or 15 The N peak resulted in a combined chemical shift deviation (CCSD) value of less than 0.01 ppm when compared to the corresponding peak in Table 1.

[0125] The above compositions contain minimal impurities or post-translational modifications and can therefore be used to prepare pharmaceutical compositions. The pharmaceutical compositions can be prepared as described elsewhere herein.

[0126] The above composition or pharmaceutical composition is preferably used as a medicament, and it can be used as described above, for example, for treating or preventing cardiac insufficiency in a subject in need thereof, treating cardiomyopathy, promoting cardiac tissue regeneration, promoting cardiomyocyte proliferation, promoting neovascularization, promoting cardiac function, reducing infarct size, treating or preventing fibrosis, treating or preventing hypertrophy, or treating or preventing heart failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0127] Figure 1 The NMR spectrum of the MYDGF[+A] variant described in Example 5 is shown.

[0128] Figure 2 The NMR spectrum of the MYDGF[+G-HEK] variant described in Example 5 is shown.

[0129] Figure 3 Shows the Figure 1 and Figure 2 The superposition of two spectra.

[0130] Figure 4 Dose response curves of different MYDGF variants in HCAEC migration assay are shown. Recovery data are derived from cells treated with vehicle (control), VEGFA (50 ng / mL), different concentrations of [+G]-HEK (reference) or different concentrations of MYDGF variant [+A] batch V301. 4-PL curve fits of [+G]-HEK and respective [+A] batches are shown.

[0131] Figure 5 Dose response curves of different MYDGF variants in HCAEC migration assay are shown. Recovery data are derived from cells treated with vehicle (control), VEGFA (50 ng / mL), different concentrations of [+G]-HEK (reference) or different concentrations of MYDGF variant [+A] batch V302. 4-PL curve fits of [+G]-HEK and respective batches [+A] are shown.

[0132] Figure 6 Dose response curves of different MYDGF variants in HCAEC migration assay are shown. Recovery data are derived from cells treated with vehicle (control), VEGFA (50 ng / mL), different concentrations of [+G]-HEK (reference) or different concentrations of MYDGF variant [+A] batch V303. 4-PL curve fits of [+G]-HEK and respective [+A] batches are shown.

[0133] Figure 7 Comparison of different MYDGF variants and batches in ischemia / reperfusion assays is shown. Metabolic activity was determined for cells stimulated with vehicle (control), IGF-1 (50 ng / mL), different concentrations of [+G]-HEK (reference) or different concentrations of MYDGF (batches V301, V302 and V303), respectively. 4-PL curve fits for [+G]-HEK and respective batches [+A] are shown.

[0134] Figure 8 Shown is the cardiac function in FVB / N mice as evaluated by echocardiography. Left ventricular end-diastolic area (LVEDA), left ventricular end-systolic area (LVESA) and fractional area change (FAC) as evaluated by transthoracic echocardiography at the 6th day (A) and the 28th day (B) after sham operation or I / R operation in FVB / N mice. Treatment and animal number (in column) are indicated. MYDGF indicates human protein, and Mydgf indicates mouse protein.

[0135] Fig. 9 (A) Infarct scar size at day 28 post-MI in FVB / N mice is shown. Exemplary images and summary data are shown. Scar size was assessed by Masson's trichrome staining. Treatment and number of animals are indicated (within columns). MYDGF indicates human protein, Mydgf indicates mouse protein. (B) Capillary density at day 28 post-MI in FVB / N mice. Exemplary images and summary of data are shown. Capillary density was assessed by fluorescent IB4 / WGA staining. Treatment is indicated. Six mice / group were used. MYDGF indicates human protein, Mydgf indicates mouse protein. Example

[0136] The invention will be illustrated by the following examples, which are given by way of example only. Specifically, the examples describe the generation of production strains and heterologous expression and purification of MYDGF variants.

[0137] As described in more detail in the Examples below, the production process for human MYDGF was first developed at a 5L scale using a Research Cell Bank (RCB), and then validated by a pooled run at a 20L scale using a GMP-compliant working cell bank (WCB). Finally, expression was transferred to a current good manufacturing practice (cGMP) facility at a 200L scale, which resulted in a batch yield of 16-18kg of wet inclusion bodies (IB). The downstream process for purifying MYDGF protein from inclusion bodies was first developed at a laboratory scale, then validated by a pooled run at a pilot scale using inclusion bodies from a 10L fermentation aliquot, and finally transferred to a cGMP facility where one of the downstream batches started with 10kg of wet IB (representing a fermentation aliquot of approximately 110-125L).

[0138] Non-clinical batches and clinical batches of MYDGF are manufactured at a 200L scale. Several batches are performed in a GMP facility. Batching results in a high yield of typically 330-355g MYDGF from a 125L fermentation aliquot. This reflects a yield of up to 2.84g / L fermentation. The MYDGF produced by this process meets all quality requirements required for use in toxicology and clinical studies. The monomer content measured by HP size exclusion chromatography is routinely higher than 99%, high molecular weight impurities (aggregates) are less than 1% and low molecular weight impurities (fragments) are less than 0.1%. The endotoxin content is less than 0.03EU / mg protein. The host cell DNA content is ≤3pg / mg protein.

[0139] Example 1 : Vector preparation and transfection

[0140] For the production of cell banks, a derivative of the E. coli strain BL21 (DE3) was used, which had been modified so that it did not produce phage. This strain was transformed with one of the vectors shown in SEQ ID NO: 7-10, carrying the gene encoding the respective MYDGF variant. The gene codons of the respective variant were optimized for high expression rates in E. coli and synthesized by ATUM (Newark, California, USA). Plasmids encoding the following MYDGF variants were generated:

[0141] ●[+A] variant, in which the N-terminal V residue in position +1 of mature human MYDGF is preceded by an A residue (aa sequence shown in SEQ ID NO: 1: AVSEPTTVAFDVRPGGVVHSFSHNVGPGDKYTCMFTYASQGGTNEQWQMSLGTSEDHQHFTCTIWRPQGKSYLYFTQFKAEVRGAEIEYAMAYSKAAFERESDVPLKTEEFEVTKTAVAHRPGAFKAELSKLVIVAKASRTEL). The expression vector encoding this variant is shown in SEQ ID NO: 7.

[0142] [+S] variant, in which the N-terminal V residue in position +1 of mature human MYDGF is preceded by an S residue (aa sequence shown in SEQ ID NO: 2: SVSEPTTVAFDVRPGGVVHSFSHNVGPGDKYTCMFTYASQGGTNEQWQMSLGTSEDHQHFTCTIWRPQGKSYLYFTQFKAEVRGAEIEYAMAYSKAAFERESDVPLKTEEFEVTKTAVAHRPGAFKAELSKLVIVAKASRTEL). The expression vector encoding this variant is shown in SEQ ID NO: 8.

[0143] The [+G] variant, in which the N-terminal V residue in position +1 of mature human MYDGF is preceded by a G residue (aa sequence shown in SEQ ID NO: 3: GVSEPTTVAFDVRPGGVVHSFSHNVGPGDKYTCMFTYASQGGTNEQWQMSLGTSEDHQHFTCTIWRPQGKSYLYFTQFKAEVRGAEIEYAMAYSKAAFERESDVPLKTEEFEVTKTAVAHRPGAFKAELSKLVIVAKASRTEL). The expression vector encoding this variant is shown in SEQ ID NO: 9.

[0144] -[-V] variant, in which the N-terminal V residue in position +1 of mature human MYDGF is deleted (aa sequence shown in SEQ ID NO:4: SEPTTVAFDVRPGGVVHSFSHNVGPGDKYTCMFTYASQGGTNEQWQMSLGTSEDHQHFTCTIWRPQGKSYLYFTQFKAEVRGAEIEYAMAYSKAAFERESDVPLKTEEFEVTKTAVAHRPGAFKAELSKLVIVAKASRTEL). The expression vector encoding this variant is shown in SEQ ID NO:10.

[0145] In the event of inconsistencies between any sequence listed above and the sequence shown in the accompanying sequence listing, the above paragraph controls. Hyphens that eventually appear within the sequence are the result of truncation due to text processing and must be ignored.

[0146] To prepare the expression strain, the Gene Pulser Xcell TM The above vector plasmids were used to transform E. coli cells by electroporation using the BioRad Electroporation System. The proteins were expressed in the form of inclusion bodies (IB) accumulated in the cytoplasm of E. coli cells, as further described in the Examples below.

[0147] Example 2 : Expression of MYDGF variants

[0148] Expression and purification of MYDGF variants were performed by the following general protocol:

[0149] 1. Fermentation

[0150] 2. IB Preparation

[0151] 3. Dissolution / Refolding

[0152] 4. Ultrafiltration / diafiltration

[0153] 5. Anion Exchange Chromatography

[0154] 6. Preferably: Hydrophobic Interaction Chromatography

[0155] 7. Preferably: Concentrated and formulated

[0156] Example 2.1 : Fermentation

[0157] A cell bank vial of the production strain obtained from Example 1 was thawed at room temperature. The pre-culture (PC) consisted of two 1L flasks with 300mL seed medium / flask. The composition of the seed medium is depicted in Table 2 below. All buffers and media were prepared with reverse osmosis (RO) water and sterilized using a nanofiltration device or heat sterilization before use.

[0158] Table 2 :Seed culture medium

[0159]

[0160]

[0161] Defoamers and antibiotics were added as needed. Kanamycin was added as an antibiotic (to a final concentration of 50 μg / mL). Each flask was inoculated with 100 μL of the production strain. The culture was grown for approximately 9.4 hours with a target OD (optical density at 550 nm) of 1.75 ± 0.5.

[0162] The main culture (MC) was performed in a 20 L bulk stainless steel bioreactor containing 10 L of batch medium. The composition of the batch medium is depicted in Table 3 below.

[0163] Table 3 : Batch culture medium

[0164]

[0165] 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 discharge pressure and 20% DO set point. After the carbon source consumption was observed via the dissolved oxygen (DO) peak, the exponential feeding (concentration 600 g / L glucose; μ = 0.25 h -1 ). The composition of the feed medium is depicted in Table 4 below.

[0166] Table 4 : Feed medium

[0167]

[0168] After 9 hours of exponential feed rate (60.48 to 573.78 g / hour), the feed rate remained constant at a rate of 573.78 g / hour for the rest of the fermentation time (11.5 hours). 11.5 hours after the start of exponential feed, a 60-minute temperature ramp (from 33.5°C to 30.0°C) was started, and the ramp was completed immediately before the induction with IPTG. 12.5 hours after the start of feed, the culture was induced via a bolus injection of IPTG. MC was terminated 20.5 hours after the start of feed. At the end of the culturing process, the culture broth was immediately cooled to <12°C, diluted to 15% of the target cell wet weight (WCW) with reverse osmosis (RO) water, and the bacterial cell mass was separated from the supernatant via centrifugation with a CEPA centrifuge. The biomass was harvested and transferred to downstream processing together with the supernatant.

[0169] By using LabChip Product quantification was performed using a PCR system (Perkin Elmer), which provides an automated high-throughput alternative to traditional SDS-PAGE and protein quantification. Sample preparation was performed using a liquid handling system (Tecan FreedomEVO 150). For product quantification from fermentation samples, analytical cell disruption of samples from fermentation was facilitated via enzymatic cell lysis. 90 μL of fermentation suspension was washed with cell disruption buffer (FastBreak 100). TM Lysonase in cell lysis reagent (Promega) TM (Merck), with 32 μL Lysonase / 1 mL FastBreak TM The samples were diluted with PBS (reagent) in a ratio of 9:10 (v / v). For total product determination (soluble fraction and insoluble fraction), the samples were mixed before each pipetting step. Finally, the samples were diluted into the system's specific sample buffer. In order to minimize the amount of sample buffer required, all dilution steps were performed in PBS or another formulation buffer.

[0170] For final dilution, 8 μ L samples (from PBS dilution) or standard curve samples are diluted in 28 μ L non-reducing sample buffer in 96 orifice plates (Eppendorftwin.tec PCR plate 95100401).For reducing conditions, 28 μ L reducing sample buffer (with 35mM DTT) are used. Plate foil (Eppendorf PCR foil 0030127790) is sealed, centrifuged briefly (30 seconds at 25g) and denatured 10 minutes at 70 ℃.After denaturation, plate is centrifuged 5 minutes at 2200g, to spin down all evaporated liquids.After centrifugation, take out foil and dilute it via 140 μ L DI water.96 orifice plates are sealed with foil (Eppendorf PCR foil 95100401), and plate is centrifuged 10 minutes at 2200g, to precipitate any potential aggregation that will cause LabChip to analyze failure. After centrifugation, the plates were analyzed in a LabChip GXII with the setting "HT Protein Express 100 High Sensitivity". LabChip preparation was performed according to the manufacturer's instructions. Standard curves were prepared by diluting reference materials. As reference material, the [+G] variant was used, which had been produced in HEK 293-6E cells, as described in Polten (2019), see page 1303, column 1 and Figure S1; and Ebenhoch (2019), see page 8, column 1.

[0171] Quantitation was performed via linear fitting in the range of 1 mg / mL to 0.1 mg / mL. Reducing and non-reducing conditions did not change the integrated area used for quantitation, however, a shift in run time was observed that did not affect quantitation.

[0172] Example 2.2 : Purification and analysis of inclusion bodies

[0173] The frozen E. coli biomass obtained from Example 2.1 was resuspended 1:5 (w / v) with IB preparation buffer 1 (1M urea, 50mM Tris, 0.1% (v / v) polysorbate 20, pH 7.5). After 15 minutes of resuspension with ultraturrax, the E. coli cells were broken by high pressure homogenization using 3 passes at 650-700 bar. The dense and heavy inclusion bodies (IB) and large cell fragments were separated by high-speed tubular centrifugation using a GLE rotor (CEPA). The feed flow rate was 55mL / min and the centrifugation speed was 24,500g. The tube had an inner diameter of 3.2mm. The recovered pellets were washed twice with HQ water. In all cases, the pellets were diluted 1:5 (w / v) and resuspended using ultraturrax. After the HQ water step, the pellets contained mainly IB.

[0174] Example 2.3 : Protein solubilization, refolding and purification

[0175] The frozen IB obtained from Example 2.3 was dissolved in a dissolution buffer (8 M urea, 0.14 M GuHCl, 6 mM DTT, 50 mM Tris, pH 8) at room temperature. The mixture was first stirred with an ultraturrax for 10 minutes and then with a propeller mixer for 180 minutes. The target concentration during the dissolution process was 5 mg / mL with a target volume of 100 mL. Subsequently, the dissolution pool was filtered on 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 dissolution buffer. Subsequently, the dissolution pool was loaded directly. Use The system collects the filtrate by UV monitoring. The inclusion body lysate is diluted with 1:5 refolding buffer (4M urea, 0.3125M Tris, 12.5mM CaCl2, 3.75mM cystamine, pH 7). The refolded collection of recovery is stirred overnight. On the second day, it is filtered on a CUNO depth filter (filter E16E01A60ZB05A, 3M Deutschland GmbH, Neuss, Germany).

[0176] After filtration with a depth filter, the filtrate was subjected to ultrafiltration / diafiltration (UFDF) to perform buffer exchange. 2 UFDF was performed using a diafiltration buffer (20 mM Tris, pH 9) using an Ultracell 3 kDa, C-size. A concentration factor of 2 and a diafiltration factor of 5 were used.

[0177] After UFDF, the filtrate was subjected to ion exchange chromatography (IEX). YMC Biopro IEX 75 μm posts were used, with a column diameter of 1 cm, a bed height of 9 cm and a column volume of 7.5 ml. The post was first balanced with 3 column volumes (CV) of equilibration buffer 1 (20 mM Hepes, 1 M NaCl pH 7) for 5 minutes, and then balanced with 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. Protein was eluted with 5 CV of elution buffer 1 (20 mM Hepes, 1 M NaCl, pH 7) for 5 minutes, and then eluted with 10 CV of elution buffer 2 (20 mM Hepes, pH 7) for 5 minutes using a gradient (0% to 100% 20 mM Hepes, 1 M NaCl pH 7). Finally, the post was stripped with 1 M HCl for 5 minutes with 5 CV.

[0178] Example 2.4 : Protein yield and product homogeneity

[0179] At least one purification was performed for all 4 variants. In addition, a second purification experiment was performed for the [+A] and [+S] variants. Purification resulted in high yield and high purity for each of the four variants. In particular, the overall process yield of the [+A] variant after purification and refolding was found to be 2.4 g / L for the first batch and 5.3 g / L for the second batch.

[0180] Analytical high performance size exclusion chromatography was performed to test purity. The purified [+A] variant showed a high purity of 99.75% main peak, 0.25% low molecular weight impurities and 0.0% aggregation level. Similarly, high purity levels were also achieved for [-V] variant (99.64% main peak, 0.0% low molecular weight impurities, 0.04% aggregation) and [+S] variant (99.73% main peak, 0.2% low molecular weight impurities, 0.05% aggregation). In contrast, when checked with high performance size exclusion chromatography, the homogeneity of the [+G] variant product was poor, showing 60.36% main peak purity and 39.64% aggregation.

[0181] The process yields from bench-scale purification runs for all N-terminal variants are summarized in the following table:

[0182] Table 5a : Summary of laboratory-scale production of MYDGF N-terminal variants. MYDGF amounts at different process steps are provided in mg MYDGF

[0183]

[0184]

[0185] The fed-batch fermentation process applied to all four variants resulted in very high cell densities at the end of fermentation (OD 326-348 at 550 nm; 310,42-337,58 g / L wet cell mass). Very high volumetric titers of the recombinant MYDGF variants were achieved (23.1-27.1 g / L fermentation).

[0186] Zhao et al. (2020) reported the fermentation of MYDGF-6His fusion protein in E. coli, followed by extraction and purification from E. coli cell lysate.

[0187] Table 5b : Characteristics of the method according to Zhao et al. (2020) compared with the method according to the present invention.

[0188]

[0189] Table 5c : Summary of the purification of rhMYDGF as published in Zhao et al. (2020).

[0190]

[0191]

[0192] *See Zhao et al. (2020), at page 1192, section 3.1, lines 18–20, citing Figure 1 C refers to a purity of more than 95%. See also Figure 2 A.

[0193] The above data are the average of three independent experiments. Protein was quantified by BCA method. The amount of target protein was estimated by densitometry analysis of protein bands in SDS-PAGE gel. Total protein = protein concentration (mg / mL) × volume (mL). Yield = total protein (mg) × purity (%).

[0194] Example 3 :Advanced MYDGF manufacturing process

[0195] The manufacturing process for the [+A] variant was further developed based on Example 2. The fermentation process for MYDGF was first developed at 5 L scale using a research cell bank (RCB), then validated by pooling runs at 20 L scale using a GMP working cell bank (WCB), and finally transferred to 200 L scale. A typical 200 L fermentation batch produced 16-18 kg wet IB.

[0196] The downstream process for purification of MYDGF drug substance from intracellular inclusion bodies was first developed at bench scale, then validated by pooled runs at pilot scale using inclusion bodies from a 10-L fermentation aliquot, and finally transferred to one of the downstream cGMP facilities starting with 10 kg wet IB (representing approximately 110-125 L of fermentation aliquot).

[0197] Table 6a: Description of the manufacturing process of CMC1a API (upstream process part)

[0198]

[0199]

[0200] Table 6b :Description of the CMC1a API manufacturing process (downstream part)

[0201]

[0202]

[0203] Several batches were performed under GMP conditions. Batching resulted in high yields of typically 330-355 g MYDGF from a 125 L fermentation aliquot. This reflects an overall process yield of up to 2.84 g / L fermentation. The MYDGF API produced by this process meets all quality requirements required for use in toxicology and clinical studies.

[0204] The monomer content measured by HP size exclusion chromatography is routinely higher than 99%, with high molecular weight impurities (aggregates) lower than 1% and low molecular weight impurities (fragments) lower than 0.1%. The endotoxin content is lower than 0.03 EU / mg MYDGF protein. The host cell DNA content is ≤ 3 pg / mg protein.

[0205] Example 4 : Molecular weight analysis by LCMS and advanced molecular weight analysis by LCMS after chemical modification according to Tolonen et al. ("aLCMS")

[0206] Samples of the folded and purified products obtained from Example 2 were subjected to liquid chromatography mass spectrometry (LCMS) analysis. Liquid chromatography / electrospray ionization mass spectrometry (LC-ESI-MS) was used to perform intact (non-reduced) molecular weight analysis of the MYGDF constructs to (1) verify the sequence via consistency of observed molecular weights with predicted values ​​for each sequence, and (2) capture an overall profile of net post-translational modifications (PTMs) for each protein. An Agilent 1290 UPLC with a 1.0 mm×30 mm C3 POROS reverse phase column was used to desalt and introduce samples (0.5 μg / injection) into the mass spectrometer. A three-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 was used to capture, desalt, and elute the protein from the column. The mass spectral data of the eluted material was collected using an Agilent 6224 time-of-flight (TOF) MS, and then processed (deconvoluted) using the maximum entropy algorithm in Mass Hunter analysis software (Agilent). The data obtained by this method are referred to herein as "complete MW LCMS data" or "data measured by liquid chromatography mass spectrometry (LCMS)".

[0207] For peptide level sequence confirmation and site-specific post-transcriptional modification (PTM) analysis, trypsin and chymotrypsin were used to digest aliquots of each sample, respectively, to achieve complete sequence coverage. Each sample of 100 μg was desalted and concentrated via centrifugal pelleting of acetone precipitation and precipitation material. Each protein mass was redissolved, denatured and reduced in 10 μl denaturation / reduction buffer (5% w / v sodium deoxycholate (SDC), 10 mM dithiothreitol (DTT), 20 mM ammonium bicarbonate), and incubated at 70 ° C for 2 minutes, followed by a ten-fold dilution with 20 mM ammonium bicarbonate and 2 mM methionine. The molecules of reduction / denaturation were then split into two vials (each 50 μg), and thereafter trypsin and chymotrypsin were added to each tube with a ratio of 1:10 enzyme relative to substrate, and the samples were incubated at 37 ° C for 10 minutes. The reaction was quenched by adding 10% v / v trifluoroacetic acid, resulting in a final concentration of the reagent 1% v / v. The short (10 min) digestion step eliminates the need for an alkylation step commonly used in peptide mapping. The precipitated sodium deoxycholate is removed by centrifugation at 16,000 x g, and the supernatant containing the peptides is recovered and transferred to an autosampler vial, which is immediately stored at -80°C until analysis. The data obtained by this method is referred to herein as "peptide mapping LCMS data".

[0208] aLCMS: Additionally, since the first four N-terminal residues of various MYGDF constructs have been shown to undergo fragmentation during electrospray (at both the intact and peptide levels), reductive dimethylation (also known as stable isotope dimethyl labeling (SIDL)) was performed on aliquots of the peptide digests according to Tolonen et al. (2019) to distinguish sample-derived versus electrospray-derived N-terminal truncations. The data obtained by this method are referred to herein as "LCMS after reductive dimethylation (stable isotope dimethyl labeling, SIDL)". Briefly, 50 μg of peptides from each digest were immobilized into separate Waters Oasis SPE cartridges using a vacuum manifold. The SPE medium and peptides were adjusted to pH 5.5 with citrate buffer (90 mM citric acid, 230 mM sodium dibasic phosphate), and then 10 ml of 0.8% v / v formaldehyde (in citrate buffer) and 120 mM sodium cyanoborohydride were passed through the bound peptides for 10 minutes. The reactants were 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 retention microcentrifuge tubes and then dried in a vacuum centrifuge. The dried peptides were reconstituted in 50 μl 0.1% TFA and transferred to autosampler vials for LC-MS / MS analysis.

[0209] LC-MS / MS (tandem mass spectrometry) analysis was performed using a Vanquish UHPLC system connected to a Lumos Fusion Orbitrap (ThermoFisher), which was 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 x 150 mm C18 CSH Acquity UPLC reverse phase column (1.7 μm particles, Waters Corp.) and separated using a binary gradient as follows: 0.5% to 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 (mobile phase A = 0.1% difluoroacetic acid (DFA) in water). A top-4 data dependent acquisition (DDA) MS workflow was used to analyze the LC eluate. Full scan MS spectra were collected at 200 m / z with 120,000 resolution (FWHM), and HCD (high energy collision dissociation) and EThcD (electron transfer dissociation with supplementary HCD energy) MS / MS spectra were collected in an Orbitrap analyzer in a charge state dependent manner at 15,000 resolution. The resulting .RAW files 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.

[0210] Table 7 : MYDGF variants examined by aLCMS or LCMS:

[0211]

[0212] Table 8 : Complete LCMS MW data of the +S MYDGF variant

[0213]

[0214] (PTM = post-translational modification). Na in complete analysis + Adducts are common artifacts and not a property of the molecule. The term "N-terminus" in the peptide maps refers to the amino group at the N-terminus. Sequence coverage is 100%.

[0215] ND=PTM<lower limit of detection

[0216] Table 9 : Peptide mapping LCMS data of +S MYDGF variants

[0217]

[0218] *: Determined by complete Mw analysis. Na + Adducts added to original values

[0219] Table 10 :-V Complete LCMS MW data of MYDGF variants

[0220]

[0221] Table 11 :-V Peptide mapping LCMS data of MYDGF variants

[0222]

[0223] *Determined from full MW analysis; Na+ adducts added to original values

[0224] Table 12 : Complete MW LCMS data for the +A MYDGF variant

[0225]

[0226] Table 13 : +A Peptide mapping LCMS data of MYDGF variants

[0227]

[0228]

[0229] *Na+ adducts determined by intact mass analysis were added to the raw values

[0230] **N-terminal methionine not observed

[0231] Table 14 : Complete MW LCMS data of +G MYDGF variant

[0232]

[0233] Table 15 :a LCMS data (combined intact MW LCMS and dimethyl terminated peptide level LCMS after trypsin digestion as described*:

[0234]

[0235]

[0236] *N-terminal methionine was not observed in the +A variant; however, it was observed at 2.5% in the +S variant.

[0237] Example 5 :Structural analysis

[0238] The +G MYDGF variant (HEK) having a sequence according to SEQ ID NO: 3 was produced as described in Polten et al. (2019), and on page 1303, column 1 and Figure S1, Ebenhoch et al. (2019), page 8, column 1. The +A MYDGF variant was prepared according to Example 2.

[0239] The two-dimensional 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. 1 H / 15 N HSQC NMR spectra. Spectra were recorded using the pulse program hsqcfpf3gpphwg (Bodenhausen & Ruben 1980; Piotto et al. 1992; Sklenar et al. 1993; Mori et al. 1995) from the Bruker catalog with 48 complexity points in the indirect dimension, 1024 scans, 1 s inter-scan delay, resulting in a total experimental time of 30 h. The hsqcfpf3gpphwg pulse program describes phase-sensitive 2D H-1 / X correlation spectroscopy via double inept transmission, using the f3 channel and employing decoupling as well as flip pulses and a watergate sequence for water suppression during acquisition. NMR samples contained 8.5 mg / ml of the respective MYDGF protein in 50 mM sodium phosphate buffer at pH 7.4 containing 50 mM sodium chloride and 9% (v / v) D2O. Processing and analysis were performed with Topspin 3.5 (Bruker BioSpin)

[0240] In 2D 1 H / 15 The cross peaks observed in the N HSQC NMR spectrum 1 H and 15 The N chemical shift clearly shows that MYDGF is a folded protein in both variants +G variant (HEK) and +A variant. The dispersion of the cross peaks and their chemical shifts is significantly higher and very different from the experimentally determined random chemical shifts (Wishart et al. 1995). This publication exemplifies 2D plots comparing folded and unfolded proteins. 1 H / 15 Comparison of N HSQC NMR spectra (Dyson & Wright, 1995). The difference between random coil and observed chemical shifts is often used in NMR structure calculations as a constraint on secondary structure (α-helix or β-sheet) (Shen & Bax, 2015).

[0241] Table 16 : variants examined by NMR

[0242]

[0243] Table 17 :1H, 15N chemical shift

[0244]

[0245]

[0246]

[0247]

[0248] See Ebenhoch et al. (2019), which found that the 2D of the active +G MYDGF variant (HEK) and the +A MYDGF variant 1 H / 15 The N HSQC NMR spectra were almost identical. This confirmed that +A MYDGF had the correct tertiary structure of MYDGF.

[0249] Example 6 : Potency assay in human coronary artery endothelial cells

[0250] To determine the relative potency of the [+A] variant relative to the MYDGF+G HEK protein as a reference, potency assays were performed in human coronary artery endothelial cells (HCAEC). The +A variant was made according to Example 2. Three batches designated V301, V302, and V303 were examined. The +G variant (SEQ ID NO: 3) was produced in HEK cells as described in Polten et al. (2019), page 1303, column 1, and Figure S1, and Ebenhoch et al. (2019), page 8, column 1, and it was used as an internal activity benchmark.

[0251] Table 18 : Variants examined in the HCAEC assay

[0252]

[0253]

[0254] HCAEC is inoculated into EGM-2 medium (Lonza) containing 10% fetal calf serum (FCS) in 24-well plates with a total volume of 1ml per well at a density of 55.000-60.000 cells per well. 24 hours after inoculation (cells must be confluent), the culture medium in each well is replaced with 1mL MCDB131-Medium (Life Technologies) containing 2% FCS, and incubated for 3-4 hours. After incubation, the cell monolayer is scratched with a pipette tip (200 μl) in each well. The tip is used vertically to ensure that the scratch is large enough. Then the cells are washed once with the MCDB culture medium containing 2% FCS. Subsequently, 1mL fresh culture medium (MCDB containing 2% FCS) is added to each well. Then cells are cultured in the absence of MYDGF protein (control) or in the presence of different concentrations of MYDGF protein, wherein each well contains a starting concentration and a serial dilution. Each protein is tested at the following concentrations: 13.3, 19.7, 29.6, 44.4, 66.6 and 100 ng / mL. Human VEGFA (50 ng / mL) serves as a positive control. MYDGF is tested in duplicate in batches V301, V302, V303 and head-to-head with reference. Immediately after treatment at T = 0 hours, a microscope (Zeiss Axio Observer Z1, with 50x magnification, 5x objective lens, with phase contrast setting) is used to take photos from all holes. Photos are taken from the middle of the hole because the best contrast is visible there. The plate is then incubated at 37 ° C. After 16 hours of incubation time (T = 16 hours), photos of each hole are taken again as described above.

[0255] For determination of activity, the recovery rate in the assay was calculated by measuring the cell-free area in the 0-hour photograph and the 16-hour photograph, respectively, using axiovision software or ImageJ. Recovery rate (%) was calculated as [(cell-free area at 0 hours - cell-free area at 16 hours) / cell-free area at 0 hours] x 100. Two experiments were summarized to perform a 4-parameter logistic (4-PL) curve fit and calculate the EC 50 Values ​​(GraphPad Prism software, version 9.1.0). EC values ​​from different batches and reference were applied 50 The relative potency compared to the reference was calculated by: potency [%] = [+G] - EC of HEK 50 ) / Test batches of EC 50 )x 100.

[0256] Table 19 : EC of different batches of [+A] variants determined in HCAEC assay 50 and potency value

[0257]

[0258] result As can be seen from Table 18, the three +A MYDGF variant production batches (V301, V302, V303) all showed comparable bioactivity to the reference. In HCAEC, the following relative potencies compared to the reference (set to 100%) were determined: V301 (177, 95 and 136%), V302 (114, 110 and 103%), V303 (99, 111 and 81%). Pooled cell migration experiments resulted in EC values ​​of 37.4, 42.1 and 47.8 ng / mL for batches V301, V302 and V303, respectively. 50 When the data from all batches (V301 and V302, V303) were pooled and curve fitted, an EC of 41.1 ng / mL was calculated. 50 The EC values ​​calculated in the same way for the corresponding reference 50 The value is 43.0ng / mL.

[0259] Example 7 : Potency assay in neonatal rat cardiomyocytes

[0260] Neonatal rat cardiomyocytes (NRCMs) were seeded into 96-well plates and subjected to simulated ischemia / reperfusion (I / R) in the absence of MYDGF batches (control) or in the presence of different MYDGF batches. I / R was simulated as described earlier by Korf-Klingebiel et al., (2015). Reference proteins were assayed in head-to-head comparisons. Each protein was tested at the following six concentrations: 13.3, 19.7, 29.6, 44.4, 66.6 and 100 ng / mL. Mouse IGF-1 (50 ng / mL) served as a positive control. Metabolite activity was evaluated by MTS assay (Promega). MYDGF batches V301, V302 and V303 were tested in 2-3 experiments with 3-4 technical replicates. The experiments were summarized to perform 4-PL curve fitting and calculate EC 50 Apply EC values ​​from different batches and reference 50 Values ​​were used to calculate relative potency relative to a reference (see above).

[0261] Table 20 : Potency values ​​of different batches of [+A] variants determined in the neonatal rat cardiomyocyte assay

[0262]

[0263] result: In neonatal rat cardiomyocytes, the following relative potencies were determined compared to the reference (set to 100%): V301 (108%), V302 (431%), V303 (85%). The potency values ​​for batches V301 and V303 with respect to MYDGF indicated comparable biological activity to the reference. Although the metabolic activity for batch V302 was similar to the reference ( Figure 7 ), but a non-ideal curve fit resulted in a 4-fold higher potency. When data from all batches (V301 and V302 and V302) were pooled and curve fitted, an EC of 22.2 ng / mL was calculated. 50 The EC values ​​calculated in the same way for the corresponding reference 50 The value is 24.5ng / mL.

[0264] Example 8 : Myocardial infarction assay in mice

[0265] To compare the efficacy of human and mouse MYDGF treatment on the healing of myocardial infarction (MI) 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 followed up for 28 days. The human and mouse MYDGF proteins used in the assay are depicted in Table 20.

[0266] Table 21 : variants examined in the mouse myocardial infarction assay

[0267]

[0268] The +G MYDGF variant (HEK) having a sequence according to SEQ ID NO: 3 was produced essentially as described in Polten et al. (2019), and on page 1303, column 1 and Figure S1, Ebenhoch et al. (2019), page 8, column 1. The murine MYDGF-His variant was prepared according to Korf-Klingebiel et al. (2021), Supplementary Materials.

[0269] Heart failure prone FVB / N mice were subjected to sham surgery (thoracotomy without ischemia / reperfusion; no I / R) or I / R (ischemia / reperfusion) surgery. Mice were treated with human or mouse MYDGF (10 μg bolus + 10 μg / d pump for 7 days, model 1007D) or diluent only (placebo). Serial echocardiograms were performed (on days 6 and 28) and mice were followed up for 28 days. At the end of the experiment, hearts were collected and scar size was determined by Masson's trichrome staining, and capillary density was determined by fluorescent IB4 / WGA staining. Statistical significance was evaluated by one-way ANOVA with Dunnett's multiple comparison post hoc test. *P<0.05, **P<0.01, ***P<0.001 relative to sham surgery (relative to all VR groups on FAC). ##P<0.01, ###P<0.001 relative to placebo.

[0270] result : It was found that protein treatment with human MYDGF ("MYDGF") improved cardiac function (fractional area change, FAC) by 17.5% at day 6 and 16.7% at day 28 compared to placebo treatment. Mouse MYDGF ("Mydgf") increased FAC by 16.1% at day 6 and 19.0% at day 28 compared to placebo treatment (see Figure 8 ).like Fig. 9 As can be seen in Figure 2, infarct scarring was reduced by both recombinant MYDGF proteins (16.5% for human MYDGF and 12.4% for mouse MYDGF relative to placebo). Further, MYDGF protein therapy increased capillary density in the infarct border zone after MI by 21.8% for the human protein and 19.1% for the mouse protein relative to placebo (see Figure 2). Fig. 9 ). Thus, both treatments significantly improved MI healing as assessed by improved cardiac function, reduced scar size, and increased capillary density in the infarct border zone.

[0271] References

[0272] 1. Botnov, V. et al. (2018), J Biol Chem, 293(34), 13166–13175.

[0273] 2. Ebenhoch, R. et al. (2019), Nat Commun 10, 5379.

[0274] 3. Korf-Klingebiel, M. et al. (2015), Nat Med 21(2):140-9.

[0275] 4. Korf-Klingebiel, M. et al. (2021), Circulation. 144(15): 1227-40.

[0276] 5. Polten, F. et al. (2019), Anal Chem, 91, 1302-1308.

[0277] 6. Tolonen, AC et al. (2011), Mol Systems Biol 7(1).

[0278] 7. Zhao, L. et al. (2020), J Cell Mol Med, 24(2): 1189-1199.

[0279] 8. Frottin, F. et al. (2006), The Proteomics of N-terminal MethionineCleavage, Molecular & Cellular Proteomics, Volume 5, Issue 12, 2336-2349.

[0280] 9. Bodenhausen G. & Ruben DJ (1980), Chem. Phys. Lett. 69, 185.

[0281] 10. Piotto, M. et al. (1992), J Biomol NMR, 2, 661-666.

[0282] 11. Sklenar, V. et al. (1993), J Magn Reson, Series A 102, 241-245.

[0283] 12. Mori, S. et al. (1995), J Magn Reson B 108, 94-98.

[0284] 13. Wishart, DS et al. (1995), J Biomol NMR, 5, 67-81.

[0285] 14. Dyson, HJ & Wright, PE (1998), Nat Struct Biol, 5, 499-503.

[0286] 15. Shen Y. & Bax A. (2015), Methods Mol Biol, 1260:17-32.

[0287] 16.CN 111544572 A

[0288] 17.EP 2 918 676 B1

[0289] 18.US 5,744,328 B

[0290] 19.US 7,851,433 B

[0291] 20.US2015 / 0291683 A1

[0292] 21.WO 2011 / 154349 A2

[0293] 22.Peternel,S.&Komel,R.(2010),Microbial Cell Factories,9:66.

[0294] 23.Eggenreich,B.Science(2020),Journal of Biotechnology 324S,100022.

[0295] 24.Brinson,Research RG(2019),mAbs,11,94-105.

[0296] 25.WO 2021 / 148411

[0297] 26.WO 2014111458

Claims

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

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

15.

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

7.

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

5. The method of claim 4, wherein the removal of the N-terminal methionine residue is achieved 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 having 143 amino acids of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and preferably having 143 amino acids of the amino acid sequence of SEQ ID NO: 1, after step (d).

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

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

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

10. The method of claim 9, wherein the anion exchange chromatography or hydrophobic interaction chromatography step is performed by contacting the MYDGF protein with a chromatography resin material under conditions that allow the MYDGF protein to adsorb 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 performed under conditions of low ionic strength.

12. The method of claim 11, wherein the adsorption is performed 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 resin is achieved 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 prokaryotic host cell.

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

16. The method of claim 15, wherein the bacterial host cell is an E. coli cell. The method according to claim 16 , wherein the E. coli cell is an E. coli cell of strain BL21 or a derivative thereof.

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

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

12.

20. The method of any one of claims 1-19, wherein the nucleic acid is contained in a vector.

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

22. The method of 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 obtainable according to the method of any one of claims 1 to 23, wherein the composition comprises a 143 amino acid protein having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

2.

25. The composition of claim 24, wherein the composition comprises less than 1% (w / w) of protein molecules shorter than 143 amino acids as measured by liquid chromatography mass spectrometry (LCMS).

26. The composition of claim 24 or 25, wherein the composition comprises less than 20 pg, preferably less than 15 pg / mg, more preferably less than 10 pg / mg, and 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 24-26, wherein the composition comprises less than 0.2 EU / mg, and preferably less than 0.1 EU / mg or 0.08 EU / mg bacterial endotoxin.

28. A composition according to any one of claims 24-27, wherein less than 8% (w / w), and preferably less than 7% (w / w), less than 6% (w / w) or less than 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 carbamylated.

29. A composition according to any one of claims 24 to 28, wherein less than 6% (w / w), and 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 gluconylated.

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

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

32. The composition of any one of claims 24-31, wherein the composition comprises urea.

33. A composition according to any one of claims 24 to 32, wherein the composition comprises a protein of 143 amino acids having the amino acid sequence of SEQ ID NO: 1 and, after reductive dimethylation (stable isotope dimethyl labelling, SIDL), the ratio of the signal of the protein according to SEQ ID NO: 1 to the signal of a shorter variant in liquid chromatography mass spectrometry (LCMS) is at least 50, and preferably more than 100, 200, 300 or 400, wherein only the signal from non-carbamylated and non-gluconylated protein is used to calculate the ratio.

34. A composition according to any one of claims 24 to 33, wherein the composition comprises a protein of 143 amino acids having the amino acid sequence of SEQ ID NO: 2, and the ratio of the signal of the protein according to SEQ ID NO: 2 to the signal of a shorter variant in liquid chromatography mass spectrometry (LCMS) after reductive dimethylation (stable isotope dimethyl labelling, SIDL) is at least 50, and preferably more than 75, 100, 150 or 175, wherein only the signal from non-carbamylated and non-gluconylated protein is used to calculate the ratio.

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

36. The composition of any one of claims 24-35, wherein the composition comprises greater than 95%, 96%, 97%, 98% or 99% MYDGF protein in monomer content.

37. Use of a 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 24-36.

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

40. The pharmaceutical composition of claim 38 or 39, wherein the composition is formulated for parenteral administration.

41. The pharmaceutical composition of claim 40, wherein the composition is formulated for intravenous, intraarterial or intracoronary administration.

42. The pharmaceutical composition of claim 41, wherein the composition is formulated for intravenous administration.

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

44. A composition according to any one of claims 24-36 or a pharmaceutical composition according to any one of claims 38-43 for use in the following method: (i) treatment or prevention of a disease or condition selected from the group consisting of injury, trauma, ischemia, reperfusion injury, trauma, mechanical overload, poisoning, surgery, primary or acquired cardiomyopathy, post-ischemic systolic dysfunction, myocardial infarction, preferably acute myocardial infarction, angina pectoris, heart failure, cardiac inflammation, cardiac insufficiency, 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 the infarct size after myocardial infarction, preferably acute myocardial infarction.

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

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

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

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

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

50. The composition of any one of claims 24-36 or the pharmaceutical composition for use in a 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 (HFrEF) or heart failure with intermediate ejection fraction (HFmrEF).

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