TECHNICAL FIELD

The present disclosure is directed to methods and reagents for treatment or prevention of myocarditis and/or cardiomyopathy. For example, the method and reagents of the disclosure may be useful for treating or preventing cardiac hypertrophy and/or preventing cardiac fibrosis and/or improving cardiac function in a subject suffering from cardiomyopathy. In particular, the present disclosure relates to the use of an isolated or recombinant antibody or antigen binding fragment thereof, which binds to the N-terminal domain of midkine protein (hereinafter, referred to as "MK"), and inhibits or reduces the function of MK in the treatment or prevention of myocarditis and/or cardiomyopathy.

BACKGROUND

Myocarditis is an acute or chronic disease of heart muscle involving focal or diffuse inflammatory infiltrate in interstitial myocardium and degeneration, necrosis or lysis of cardiac muscle fiber. The disease can lead to myocardial damage, heart dysfunction and arrhythmia, and in more extreme circumstances involving prolonged cardiac hypertrophy and permanent cardiac muscle injury, it can progress to dilated cardiomyopathy (weakening of the heart muscle), a major cause of heart failure and death.

The aetiology of myocarditis can be due to a wide variety of injuries to the myocardium, including injury caused by toxins and drugs ( e.g ., cocaine and interleukin 2) or infectious agents, most commonly including viral (e.g., coxsackievirus, adenovirus, HIV and hepatitis C virus), bacterial (e.g., diphtheria, meningococcus, psittacosis and streptococcus), rickettsial (e.g., typhus and Rocky Mountain spotted fever), fungal (e.g., aspergillosis and candidiasis), and parasitic (Chagas disease, toxoplasmosis), as well as giant cell myocarditis, and hypersensitivity reactions to drugs such as antibiotics, sulfonamides, anticonvulsants, and anti-inflammatories. More recently, myocarditis has been associated with injury to the myocardium caused by immunotherapeutic drugs e.g. immune checkpoint inhibitors. Myocarditis induced by, or associated with, an immune checkpoint inhibitor has been referred to as“immune checkpoint inhibitor-associated myocarditis” or“ICI-associated myocarditis”.

It has also been reported that in autoimmune-prone mice and patients with type 1 diabetes, chronic myocarditis can be the result of“sterile” cardiac injury (not due to infection or toxin as discussed), in particular myocardial infarction (MI).

Treatment options for myocarditis and cardiomyopathy depend upon the underlying cause and upon the individual patient. For example, existing treatments for viral myocarditis include antibiotics, heart protective agents, and antioxidant (such as high dose vitamin C, vitamin E and coenzyme Q10). In other circumstances, more aggressive therapy may be necessary, such as drugs to reduce the heart’s workload (e.g., ACE inhibitors, Angiotensin III receptor blockers (ARBs) or beta blockers), drugs to eliminate excess fluid ( e.g ., diuretics), intravenous medication to improve the heart pumping function, placement of a pump in the aorta (intra-aortic balloon pump), use of a temporary artificial heart (e.g., ventricular assist device (VAD)), and consideration of urgent heart transplantation. Some patients may have chronic and irreversible damage to the heart muscle requiring lifelong medications, while other people need medications for just a few months and then recover completely. Variability in the disease makes it difficult to treat and existing treatments to improve heart function, repair cardiac damage, and prevent heart failure are not very effective. There is therefore a need for further and improved treatment options for myocarditis and cardiomyopathy.

Midkine (hereinafter, referred to as "MK") is a heparin-binding growth/differentiation factor originally found as a product of a gene transiently expressed in the process of retinoic acid-induced differentiation of embryonal carcinoma (EC) cells and is a polypeptide of 13 kDa in molecular weight, rich in basic amino acids and cysteine (Kadomatsu. l al. (1988) Biochem. Biophys. Res. Commun., 1511312-1318; Tomokura et al. (1999) J. Biol. Chem, 265: 10765-10770).

MK is known to have various biological activities. For example, MK expression is known to be increased in a number of different human cancer cells (Muramatsu (2002) J. Biochem. 132:359-371), and its expression has been found to promote the survival and migration of cancer cells, promote angiogenesis, and contribute to cancer progression.

MK is also known to play a central role in inflammatory processes. For example, it is known that neointimal formation after vascular injury and nephritis onset during ischemic injury in the kidney are suppressed in knockout mice deficient in MK genes. Moreover, it is also known that rheumatic injury models and post-operative adhesions are significantly suppressed in such knockout mice (W02000/10608; W02004/078210).

The three-dimensional structure of MK has been determined by NMR and reported (Iwasaki et al. (1997) EMBO J. 16, p. 6936-6946). MK is composed of: an N-terminal fragment (hereinafter, referred to as an "N-fragment") consisting of amino acid residues 1 to 52; a C-terminal fragment (hereinafter, referred to as a "C-fragment") consisting of amino acid residues 62 to 121; and a loop region (amino acid residues 53 to 61) (hereinafter, referred to as a "loop") that links these fragments.

Each of the N- and C-fragments is mainly composed of: a portion having a three- dimensional structure consisting of three antiparallel [betaj-sheets (hereinafter, referred to as a "domain"; the domain (consisting of amino acid residues 15 to 52) in the N-fragment is referred to as an "N-domain", and the domain (consisting of amino acid residues 62 to 104) in the C-fragment is referred to as a "C-domain"); and a terminally located portion devoid of the domain that does not assume a particular three-dimensional structure (hereinafter, referred to as a "tail"; the tail (consisting of amino acid residues 1 to 14) in the N-fragment is referred to as an "N-tail", and the tail (consisting of amino acid residues 105 to 121) in the C-fragment is referred to as a "C-tail").

Anti-MK antibodies against the C-domain and the N-domain are known e.g., as disclosed in W02008/059616, WO2012/122590 and W02016/058047. Based on previous findings that MK has a number of biological activities and is implicated in a range of diseases and conditions, these and other anti-MK antibodies may be therapeutically effective for a number of diseases/conditions.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each of the appended claims.

SUMMARY

The present disclosure is based on the inventors’ finding that treatment of an animal model of myocarditis with an anti-MK antibody which binds specifically to the N-domain of MK, significantly reduced cardiac hypertrophy, fibrosis and heart failure relative to animals which received vehicle control, IgG isotype control or an anti-MK antibody which binds specifically to the C-domain of MK.

The present disclosure thus provides a method of treating and/or preventing myocarditis or cardiomyopathy in a subject, said method comprising administering to the subject an isolated or recombinant protein comprising an antigen binding domain of an antibody which binds specifically to an epitope located within the N-domain of MK protein and inhibits or reduces a function of MK.

In one example, administering the isolated or recombinant protein to the subject prevents cardiac hypertrophy and/or prevents cardiac fibrosis and/or improves heart function in a subject.

In one example, the subject is suffering from myocarditis and administering the isolated or recombinant protein to the subject prevents development of cardiomyopathy.

In one example, the myocarditis is the result of injury to the myocardium caused by toxins and/or drugs e.g., including therapeutic drugs. Therapeutic drugs may include, for example, immunotherapeutic drug e.g. immune checkpoint inhibitors. In some examples, myocarditis induced by an immune checkpoint inhibitor is referred to as immune checkpoint inhibitor (ICI)-associated myocarditis. In one example, the myocarditis is the result of injury to the myocardium caused by infectious agents, such as e.g., viral, bacterial, rickettsial, fungal, and parasitic agents. In one example, the myocarditis is the result of injury to the myocardium caused by an autoimmune disease. In one example, the myocarditis is giant cell myocarditis. In one example, the myocarditis is the result of a hypersensitivity reaction to a drug, such as an antibiotics, sulfonamides, anticonvulsants, and anti-inflammatories.

In one example, the myocarditis is acute. In one example, the myocarditis is subacute. In one example, the myocarditis is chronic.

In accordance with an example in which the method treats or prevents cardiomyopathy, the cardiomyopathy may be selected from the group consisting of hypertrophic cardiomyopathy, dilated cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular dysplasia, metabolic cardiomyopathy and unclassified cardiomyopathy. In one example, the cardiomyopathy is hypertrophic cardiomyopathy. In one example, the cardiomyopathy is dilated cardiomyopathy. In one example, the cardiomyopathy is restrictive cardiomyopathy. In one example, the cardiomyopathy is arrhythmogenic right ventricular dysplasia. In one example, the cardiomyopathy is metabolic cardiomyopathy ( e.g diabetic cardiomyopathy, alcoholic cardiomyopathy, or drug-induced cardiomyopathy). In one example, the cardiomyopathy is unclassified cardiomyopathy.

In one example, the epitope to which the isolated or recombinant protein binds is located within the N-domain of MK as defined by amino acid residues 1-61 of the sequence set forth in SEQ ID NO: 1.

In one example, the isolated or recombinant protein useful in the method of the disclosure recognizes at least a portion of a high electrostatic potential cluster located at amino acid residues 1-61 of the sequence set forth in SEQ ID NO: 1.

In one example, the isolated or recombinant protein useful in the method of the disclosure binds specifically to a conformational epitope formed by the amino acid sequence set forth in SEQ ID NO: 1, wherein the epitope includes at least two residues selected from the group consisting of 18W, 20W, 34F, 35R, 36E, 38T, 43T, 45R, 47R and 49R. In one example, the isolated or recombinant protein binds an epitope defined by residues 18W, 20W, 35R and 49R. In one example, the isolated or recombinant protein binds an epitope defined by residues 18W, 20W, 36E, 38T, 43T and 45R. In one example, the isolated or recombinant protein binds an epitope defined by residues 18W, 20W, 34F, 36E, 45R and 47R.

In one example, an isolated or recombinant protein useful in the method of the disclosure comprises:

(i) a heavy chain variable domain (VH) comprising the sequence set forth in SEQ ID NO:

2 or 33 or a sequence exhibiting 95% or greater identity thereto;

(ii) a light chain variable domain (VL) comprising the sequence set forth in SEQ ID NO: 3 or a sequence exhibiting 95% or greater identity thereto;

(iii) a VH comprising the sequence set forth in SEQ ID NO: 2 or 33 or a sequence exhibiting 95% or greater identity thereto and a VL comprising the sequence set forth in SEQ ID NO: 3 or a sequence exhibiting 95% or greater identity thereto; (iv) a VH comprising CDR1, CDR2 and CDR3 comprising the sequences set forth in SEQ ID NO: 4, 5 and 6 respectively, and a VL comprising CDR1, CDR2 and CDR3 comprising the sequences set forth in SEQ ID NO: 7, 8 and 9 respectively; or

(v) three CDRs comprised within the sequence set forth in SEQ ID NO: 2 or 33 and three CDRs comprised within the sequence set forth in SEQ ID NO: 3.

In one example, the isolated or recombinant protein comprises a VH comprising the sequence set forth in SEQ ID NO: 2 or a sequence exhibiting 95% or greater identity thereto. In one example, the isolated or recombinant protein comprises a VH comprising the sequence set forth in SEQ ID NO: 33 or a sequence exhibiting 95% or greater identity thereto. In one example, the isolated or recombinant protein comprises a VL comprising the sequence set forth in SEQ ID NO: 3 or a sequence exhibiting 95% or greater identity thereto. In one example, the isolated or recombinant protein comprises a VH comprising the sequence set forth in SEQ ID NO: 2 or a sequence exhibiting 95% or greater identity thereto and a VL comprising the sequence set forth in SEQ ID NO: 3 or a sequence exhibiting 95% or greater identity thereto. In one example, the isolated or recombinant protein comprises a VH comprising the sequence set forth in SEQ ID NO: 33 or a sequence exhibiting 95% or greater identity thereto and a VL comprising the sequence set forth in SEQ ID NO: 3 or a sequence exhibiting 95% or greater identity thereto. In one example, the isolated or recombinant protein comprises a VH comprising CDR1, CDR2 and CDR3 comprising the sequences set forth in SEQ ID NO: 4, 5 and 6 respectively, and a VL comprising CDR1, CDR2 and CDR3 comprising the sequences set forth in SEQ ID NO: 7, 8 and 9 respectively. In one example, the isolated or recombinant protein comprises three CDRs comprised within the sequence set forth in SEQ ID NO: 2 or 33 and three CDRs comprised within the sequence set forth in SEQ ID NO: 3.

In one example, an isolated or recombinant protein useful in the method of the disclosure comprises:

(i) a heavy chain variable domain (VH) comprising the sequence set forth in SEQ ID NO:

10 or a sequence exhibiting 95% or greater identity thereto;

(ii) a light chain variable domain (VL) comprising the sequence set forth in SEQ ID NO:

11 or a sequence exhibiting 95% or greater identity thereto;

(iii) a VH comprising the sequence set forth in SEQ ID NO: 10 or a sequence exhibiting 95% or greater identity thereto and a VL comprising the sequence set forth in SEQ ID NO: 11 or a sequence exhibiting 95% or greater identity thereto;

(iv) a VH comprising CDR1, CDR2 and CDR3 comprising the sequences set forth in SEQ ID NO: 12, 13 and 14 respectively, and a VL comprising CDR1, CDR2 and CDR3 comprising the sequences set forth in SEQ ID NO: 15, 16 and 17 respectively; or (v) three CDRs comprised within the sequence set forth in SEQ ID NO: 10 and three CDRs comprised within the sequence set forth in SEQ ID NO: 11.

In one example, the isolated or recombinant protein comprises a VH comprising the sequence set forth in SEQ ID NO: 10 or a sequence exhibiting 95% or greater identity thereto. In one example, the isolated or recombinant protein comprises a VL comprising the sequence set forth in SEQ ID NO: 11 or a sequence exhibiting 95% or greater identity thereto. In one example, the isolated or recombinant protein comprises a VH comprising the sequence set forth in SEQ ID NO: 10 or a sequence exhibiting 95% or greater identity thereto and a VL comprising the sequence set forth in SEQ ID NO: 11 or a sequence exhibiting 95% or greater identity thereto. In one example, the isolated or recombinant protein comprises a VH comprising CDR1, CDR2 and CDR3 comprising the sequences set forth in SEQ ID NO: 12, 13 and 14 respectively, and a VL comprising CDR1, CDR2 and CDR3 comprising the sequences set forth in SEQ ID NO: 15, 16 and 17 respectively. In one example, the isolated or recombinant protein comprises three CDRs comprised within the sequence set forth in SEQ ID NO: 10 and three CDRs comprised within the sequence set forth in SEQ ID NO: 11.

In one example, an isolated or recombinant protein useful in the method of the disclosure comprises:

(i) a heavy chain variable domain (VH) comprising the sequence set forth in SEQ ID NO:

18 or a sequence exhibiting 95% or greater identity thereto;

(ii) a light chain variable domain (VL) comprising the sequence set forth in SEQ ID NO:

19 or a sequence exhibiting 95% or greater identity thereto;

(iii) a VH comprising the sequence set forth in SEQ ID NO: 18 or a sequence exhibiting 95% or greater identity thereto and a VL comprising the sequence set forth in SEQ ID NO: 19 or a sequence exhibiting 95% or greater identity thereto;

(iv) a VH comprising CDR1, CDR2 and CDR3 comprising the sequences set forth in SEQ ID NO: 20, 21and 22 respectively, and a VL comprising CDR1, CDR2 and CDR3 comprising the sequences set forth in SEQ ID NO: 23, 24 and 25 respectively; or

(v) three CDRs comprised within the sequence set forth in SEQ ID NO: 18 and three CDRs comprised within the sequence set forth in SEQ ID NO: 19.

In one example, the isolated or recombinant protein comprises a VH comprising the sequence set forth in SEQ ID NO: 18 or a sequence exhibiting 95% or greater identity thereto. In one example, the isolated or recombinant protein comprises a VL comprising the sequence set forth in SEQ ID NO: 19 or a sequence exhibiting 95% or greater identity thereto. In one example, the isolated or recombinant protein comprises a VH comprising the sequence set forth in SEQ ID NO: 18 or a sequence exhibiting 95% or greater identity thereto and a VL comprising the sequence set forth in SEQ ID NO: 19 or a sequence exhibiting 95% or greater identity thereto. In one example, the isolated or recombinant protein comprises a VH comprising CDR1, CDR2 and CDR3 comprising the sequences set forth in SEQ ID NO: 20, 2 land 22 respectively, and a VL comprising CDR1, CDR2 and CDR3 comprising the sequences set forth in SEQ ID NO: 23, 24 and 25 respectively. In one example, the isolated or recombinant protein comprises three CDRs comprised within the sequence set forth in SEQ ID NO: 18 and three CDRs comprised within the sequence set forth in SEQ ID NO: 19.

According to any example herein, the isolated or recombinant protein comprises a heavy chain variable domain (VH) and a light chain variable domain (VL).

In one example, the VH and the VL are in a single polypeptide chain. In accordance with this example, the isolated or recombinant protein may be:

(i) a single chain Fv fragment (scFv);

(ii) a dimeric scFv (di-scFv); or

(iii) at least one of (i) and/or (ii) linked to an Fc or a heavy chain constant domain (CH) 2 and/or CH3.

In another example, the VL and VH are provided in separate polypeptide chains. In accordance with this example, the isolated or recombinant protein may be:

(i) a diabody;

(ii) a triabody;

(iii) a tetrabody;

(iv) a Fab;

(v) a F(ab’)2;

(vi) a Fv; or

(iv) one of (i) to (iii) linked to a Fc or a heavy chain constant domain (CH) 2 and/or

CH3.

In one example, the isolated or recombinant protein is a chimeric, de-immunized, humanized or human antibody.

In one example, the isolated or recombinant protein may comprise a human or non human primate heavy chain immunoglobulin constant region selected from a group consisting of IgGl, IgG2, IgG3, IgG4, IgM, IgE and IgA. For example, the isolated or recombinant protein may comprise a human or non-human primate heavy chain immunoglobulin constant region from an IgGl. For example, the isolated or recombinant protein may comprise a human or non-human primate heavy chain immunoglobulin constant region from an IgG4. For example, the isolated or recombinant protein may comprise a human or non-human primate heavy chain immunoglobulin constant region from an IgG2.

In one example, the isolated or recombinant protein may be conjugated to a compound. For example, the isolated or recombinant protein may conjugated to a compound is selected from the group consisting of a radioisotope, a detectable label, a therapeutic compound, a colloid, a toxin, a nucleic acid, a peptide, a protein, a compound that increases the half-life of the protein in a subject and mixtures thereof.

In one example, the subject has received or will receive treatment with one or more other agents for treatment of myocarditis or cardiomyopathy. In one example, the method comprises administering the isolated or recombinant protein in combination with one or more other agents for treatment of myocarditis or cardiomyopathy.

The present disclosure also provides for use of an isolated or recombinant protein comprising an antigen binding domain of an antibody which binds specifically to an epitope located within the N-domain of midkine (MK) protein and which inhibits or reduces a function of MK in the preparation of a medicament for treating and/or preventing myocarditis or cardiomyopathy in a subject in need thereof.

Suitable isolated or recombinant proteins are as described in any example hereof.

In one example, treatment with the medicament prevents cardiac hypertrophy and/or prevents cardiac fibrosis and/or improves heart function in the subject.

In one example, the subject is suffering from myocarditis and treatment with the medicament prevents development of cardiomyopathy in the subject.

In one example, the myocarditis is the result of injury to the myocardium caused by toxins and/or drugs e.g., including therapeutic drugs. Therapeutic drugs may include, for example, immunotherapeutic drug e.g. immune checkpoint inhibitors. In some examples, myocarditis induced by an immune checkpoint inhibitor is referred to as immune checkpoint inhibitor (ICI)-associated myocarditis. In one example, the myocarditis is the result of injury to the myocardium caused by infectious agents, such as e.g., viral, bacterial, rickettsial, fungal, and parasitic agents. In one example, the myocarditis is the result of injury to the myocardium caused by an autoimmune disease. In one example, the myocarditis is giant cell myocarditis. In one example, the myocarditis is the result of a hypersensitivity reaction to a drug, such as an antibiotics, sulfonamides, anticonvulsants, and anti-inflammatories.

In one example, the myocarditis is acute. In one example, the myocarditis is subacute. In one example, the myocarditis is chronic.

In accordance with an example in which the method treats or prevents cardiomyopathy, the cardiomyopathy may be selected from the group consisting of hypertrophic cardiomyopathy, dilated cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular dysplasia, metabolic cardiomyopathy and unclassified cardiomyopathy. In one example, the cardiomyopathy is hypertrophic cardiomyopathy. In one example, the cardiomyopathy is dilated cardiomyopathy. In one example, the cardiomyopathy is restrictive cardiomyopathy. In one example, the cardiomyopathy is arrhythmogenic right ventricular dysplasia. In one example, the cardiomyopathy is metabolic cardiomyopathy ( e.g ., diabetic cardiomyopathy, alcoholic cardiomyopathy, or drug-induced cardiomyopathy). In one example, the cardiomyopathy is unclassified cardiomyopathy.

BRIEF DESCRIPTION OF FIGURES

Figure 1 shows that blocking midkine with an antibody targeting the N-domain of midkine (i.e., MK-Ab2) reduced heart/body weight ratio in EAM mice at day 21 post treatment. Single data points represent individual mice n = 10 for non-immunized group; n = 25 for immunized groups. *P < 0.05

Figure 2 shows that blocking midkine with an antibody targeting the N-domain of midkine (i.e., MK-Ab2) reduces cardiac fibrosis in an EAM mouse model of myocarditis (a) Representative cross sections of cardiac tissue of non-immunized mice (left panel), vehicle- treated immunized mice (middle panel) as well as immunized mice after treatment with the anti-N-MK antibody MK-Ab2 (right panel) on day 63 using Masson's trichrome staining. Fibrotic tissue appears blue. Scale bar (overview), 1 mm; (magnification), 100 pm. (b) Degree of fibrosis in the cardiac tissue using a semi-quantitative score. Single data points represent individual mice n = 10 for non-immunized mice; n = 25 for immunized groups. *P < 0.05; **P < 0.01; ***P < 0.001; n.s. not significant; mean ± s.e.m.

Figure 3 shows that blocking midkine with an antibody targeting the N-domain of midkine (i.e., MK-Ab2) preserves cardiac function in an EAM mouse model of myocarditis (a) Echocardiographic images on day 63 after immunization. Representative M-Mode short axis view images depicting diastolic and systolic diameters of vehicle-treated animals (left, EAM + vehicle), as well as mice treated with an anti-N-MK antibody (MK-Ab2) (right, EAM + anti-N-MK). Parameters describing systolic function using (b) fractional shortening and (c) left ventricular ejection fraction (LVEF). n = 25 per group.

Figure 4 shows that administration of an Isotype IgG control antibody does not affect myocarditis (a) Degree of fibrosis in the cardiac tissue as analyzed histologically using semi- quantitative score on day 63 after induction of myocarditis. Mice were treated with IgGl isotype antibody from day 0 until day 21 of EAM myocarditis (immunized + isotype Ctrl or left untreated for control (immunized + vehicle) (b, c) Echocardiographic analysis of left ventricular systolic function on day 63. As indicators for systolic function (b) fractional shortening and (c) left ventricular ejection fraction were determined. N = 25. n.s. not significant. Mean +/- s.e.m DETAILED DESCRIPTION

Key to the Sequence Listing

SEQ ID NO: 1 - Human midkine protein sequence.

SEQ ID NO:2 - MK-Abl variable heavy chain protein sequence (version 1). SEQ ID NO:3 - MK-Abl variable light chain protein sequence.

SEQ ID NO:4 - MK-Abl variable heavy chain CDR1 protein sequence.

SEQ ID NO:5 - MK-Abl variable heavy chain CDR2 protein sequence.

SEQ ID NO:6 - MK-Abl variable heavy chain CDR3 protein sequence.

SEQ ID NO:7 - MK-Abl variable light chain CDR1 protein sequence.

SEQ ID NO:8 - MK-Abl variable light chain CDR2 protein sequence.

SEQ ID NO:9 - MK-Abl variable light chain CDR3 protein sequence.

SEQ ID NO: 10 - MK-Ab2 variable heavy chain protein sequence.

SEQ ID NO: 11 - MK-Ab2 variable light chain protein sequence.

SEQ ID NO: 12 - MK-Ab2 variable heavy chain CDR1 protein sequence.

SEQ ID NO: 13 - MK-Ab2 variable heavy chain CDR2 protein sequence.

SEQ ID NO: 14 - MK-Ab2 variable heavy chain CDR3 protein sequence.

SEQ ID NO: 15 - MK-Ab2 variable light chain CDR1 protein sequence.

SEQ ID NO: 16 - MK-Ab2 variable light chain CDR2 protein sequence.

SEQ ID NO: 17 - MK-Ab2 variable light chain CDR3 protein sequence.

SEQ ID NO: 18 - IP-13 variable heavy chain protein sequence.

SEQ ID NO: 19 - IP-13 variable light chain protein sequence.

SEQ ID NO:20 - MK-Ab3 variable heavy chain CDR1 protein sequence.

SEQ ID NO:21 - MK-Ab3 variable heavy chain CDR2 protein sequence.

SEQ ID NO:22 - MK-Ab3 variable heavy chain CDR3 protein sequence.

SEQ ID NO:23 - MK-Ab3 variable light chain CDR1 protein sequence.

SEQ ID NO:24 - MK-Ab3 variable light chain CDR2 protein sequence.

SEQ ID NO:25 - MK-Ab3 variable light chain CDR3 protein sequence.

SEQ ID NO:26 - MK-Ab4 variable heavy chain CDR1 protein sequence.

SEQ ID NO:27 - MK-Ab4 variable heavy chain CDR2 protein sequence.

SEQ ID NO:28 - MK-Ab4 variable heavy chain CDR3 protein sequence.

SEQ ID NO:29 - MK-Ab4 variable light chain CDR1 protein sequence.

SEQ ID NO:30 - MK-Ab4 variable light chain CDR2 protein sequence.

SEQ ID NO:31 - MK-Ab4 variable light chain CDR3 protein sequence.

SEQ ID NO:32 - Amino acid sequence for synthetic cardiac peptide aMyHC. SEQ ID NO:33 - MK-Abl variable heavy chain protein sequence (version 2). General

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality ( i.e . one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter.

Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the present disclosure.

Any example of the present disclosure herein shall be taken to apply mutatis mutandis to any other example of the disclosure unless specifically stated otherwise.

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley- Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

The description and definitions of variable regions and parts thereof, immunoglobulins, antibodies and fragments thereof herein may be further clarified by the discussion in Kabat Sequences of Proteins of Immunological Interest , National Institutes of Health, Bethesda, Md., 1987 and 1991, Bork et al., (1994) J. Mol. Biol. 242:309-320, Chothia and Lesk (1987) J. Mol. Biol. 196:901 -917, Chothia et al. (1989) Nature 342:877-883, and/or Al-Lazikani et al, (1997) J. Mol. Biol. 273:927-948.

The term“and/or”, e.g.,“X and/or Y” shall be understood to mean either“X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

Throughout this specification the word“comprise”, or variations such as“comprises” or“comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Selected Definitions

The skilled person will be aware that an“antibody” is generally considered to be a protein that comprises a variable region made up of a plurality of immunoglobulin chains, e.g., a polypeptide comprising a VL and a polypeptide comprising a VH. An antibody also generally comprises constant domains, some of which can be arranged into a constant region or constant fragment or fragment crystallisable (Fc). A VH and a VL interact to form an Fv comprising an antigen binding region that is capable of specifically binding to one or a few closely related antigens. Generally, a light chain from mammals is either a k light chain or a l light chain and a heavy chain from mammals is a, d, e, g, or m. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGi, IgGi, IgGs, IgG4, IgAi and IgA ₂ ) or subclass. The term“antibody” also encompasses humanized antibodies, de-immunized antibodies, non-depleting antibodies, non-activating antibodies, primatized antibodies, human antibodies and chimeric antibodies. As used herein, the term“antibody” is also intended to include formats other than full-length, intact or whole antibody molecules, such as Fab, F(ab')2, and Fv which are capable of binding the epitopic determinant. These formats may be referred to as antibody“fragments”. These antibody formats retain some ability to selectively bind to human midkine, examples of which include, but are not limited to, the following:

(1) Fab, the fragment which contains a monovalent binding fragment of an antibody molecule and which can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;

(2) Fab', the fragment of an antibody molecule which can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;

(3) (Fab') ₂ , the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab)2 is a dimer of two Fab' fragments held together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains;

(5) Single chain antibody ("SC A"), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule; such single chain antibodies may be in the form of multimers such as diabodies, triabodies, and tetrabodies etc which may or may not be polyspecific (see, for example, WO1994/007921 and WO 1998/044001); and

(6) Single domain antibody, typically a variable heavy domain devoid of a light chain.

Accordingly, an antibody in accordance with the present disclosure includes separate heavy chains, light chains, Fab, Fab', F(ab')2, Fc, a variable light domain devoid of any heavy chain, a variable heavy domain devoid of a light chain and Fv. Such fragments can be produced by recombinant DNA techniques, or by enzymatic or chemical separation of intact immunoglobulins.

The terms "full-length antibody," "intact antibody" or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antigen binding fragment of an antibody. Specifically, whole antibodies include those with heavy and light chains including an Fc region. The constant domains may be wild-type sequence constant domains ( e.g human wild-type sequence constant domains) or amino acid sequence variants thereof. In some cases, the intact antibody may have one or more effector functions.

The antibody disclosed herein may be a humanized antibody. The term "humanized antibody", as used herein, refers to an antibody derived from a non-human antibody, typically murine, that retains or substantially retains the antigen-binding properties of the parent antibody but which is less immunogenic in humans.

The antibody disclosed herein may be a non-depleting antibody. The term“non depleting antibody”, as used herein, refers to an antibody that binds to its target but does not recruit the immune system’s effector functions which effect target cell lysis. The immune system’s effector functions are dependent on interactions of the Fc-domain with Clq, the first component of the complement cascade, and/or receptors (FcR). Complement-dependent cytotoxicity (CDC) is initiated by multiple Fc-domains interacting with Clq, which can ultimately result in lysis of target cells through the formation of the membrane attack complex (MAC). Additionally, cells of the immune system, such as granulocytes, macrophages, and NK cells, may interact via FcRs with mAbs bound to target cells. Lysis of target cells is triggered via antibody-dependent cell mediated cytotoxicity (ADCC) or phagocytosis. Non depleting antibodies include antibody fragments without an Fc domain, including for example, monovalent ( e.g ., Fab, scFv, nanobodies and dAbs), bivalent ( e.g ., F(ab’)2 and diabodies) and multivalent (e.g., triabodies and pentabodies) formats. In addition, non depleting antibodies include antibodies that have been modified to remove effector functions without impacting pharmokinetics, for example, amino acid residues in the Fc-domain that play a dominant role in interaction with Clq and FcRs could be modified, or the N-linked glycosylation site in the CH2 domain could be removed. As a skilled person is aware, the chances of engineering a non-depleting antibody are linked to the constant region used to produce the antibody. An IgG3 constant region is more likely to produce a depleting antibody than an IgGl constant region which in turn is more likely to produce a depleting antibody than an IgG2 constant region, whereas an IgG4 constant region will generally mean that the antibody is non-depleting. A skilled person would also understand that modifications to a constant region could convert a depleting antibody into a non-depleting antibody and vice versa.

The antibody disclosed herein may be a non-activating antibody. As used herein, a “non-activating antibody” refers to antibodies that bind cell surface receptors and negate or block the action of endogenous ligands.

The term“EU numbering system of Kabat” will be understood to mean the numbering of an immunoglobulin heavy chain is according to the EU index as taught in Kabat et al, 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda. The EU index is based on the residue numbering of the human IgGl EU antibody.

As used herein,“variable region" refers to the portions of the light and/or heavy chains of an antibody as defined herein that is capable of specifically binding to an antigen and, for example, includes amino acid sequences of CDRs; i.e., CDR1, CDR2, and CDR3, and framework regions (FRs). For example, the variable region comprises three or four FRs (e.g., FR1, FR2, FR3 and optionally FR4) together with three CDRs. VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain. The amino acid positions assigned to CDRs and FRs can be defined according to Kabat (1987 and 1991, supra ) or other numbering systems in the performance of methods according to the present disclosure, e.g., the hypervariable loop numbering system of Clothia and Lesk (1987 and/or 1989, supra and/or Al-Lazikani et al, 1997, supra).

As used herein, the term "complementarity determining regions” (syn. CDRs; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable domain that form loops between the FRs the sequence of which vary between antibodies. Some or all of the CDRs confer the ability to bind antigen on the antibody. Each variable domain typically has three CDR regions identified as CDR1, CDR2 and CDR3. Each complementarity determining region may comprise amino acid residues from a "complementarity determining region" as defined by Kabat et al, (1991) and/or those residues from a "hypervariable loop" Chothia and Lesk (1987), or any other known numbering technique or combination thereof, including the IMGT numbering system (Lefranc et al, (2003) Dev. Comp. Immunol., 27(1)55-77).

"Framework regions" (hereinafter FR) are those variable domain residues other than the CDR residues.

The term“constant region” or“fragment crystalizable” or“Fc” or“Fc region” or“Fc portion” (which can be used interchangeably herein) as used herein, refers to a portion of an antibody comprising at least one constant domain and which is generally (though not necessarily) glycosylated and which is capable of binding to one or more Fc receptors and/or components of the complement cascade. The heavy chain constant region can be selected from any of the five isotypes: a, d, e, g, or m. Furthermore, heavy chains of various subclasses (such as the IgG subclasses of heavy chains) are responsible for different effector functions and thus, by choosing the desired heavy chain constant region, proteins with desired effector function can be produced. Preferably, the constant regions of the antibodies of the disclosure are derived from human immunoglobulins. Exemplary heavy chain constant regions are gamma 1 (IgGl), gamma 2 (IgG2), gamma 3 (IgG3), gamma 4 (IgG4), or hybrids thereof. The light chain constant region can be of the kappa or lambda type, preferably of the kappa type.

A“constant domain” is a domain in an antibody the sequence of which is highly similar in antibodies/antibodies of the same type, e.g., IgG or IgM or IgE. A constant region of an antibody generally comprises a plurality of constant domains, e.g., the constant region of g, a and d heavy chains comprises two constant domains.

As will be appreciated by the person skilled in the art, the term“residue” as used herein refers to an amino acid residue. Thus, the word“residue” may be used interchangeably with the term“amino acid”.

The term "recombinant" in the context of an antibody refers to the antibody when produced by a cell, or in a cell-free expression system, in an altered amount or at an altered rate compared to its native state. In one embodiment, the cell is a cell that does not naturally produce the antibody or immunoglobulin chain. However, the cell may be a cell which comprises a non-endogenous gene that causes an altered, preferably increased, amount of the polypeptide to be produced. A recombinant antibody of the disclosure includes polypeptides which have not been separated from other components of the transgenic (recombinant) cell, or cell-free expression system, in which it is produced, and an antibody produced in such cells or cell-free systems which are subsequently purified away from at least some other components.

The antibody disclosed herein may specifically bind to midkine protein (such as human midkine protein). As used herein, the term“specifically binds” shall be taken to mean a protein reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with midkine or a specified epitope thereof than it does with alternative antigens or epitopes. As such, "specific binding" does not necessarily require exclusive binding or non-detectable binding of another antigen. The term specifically binds” is used interchangeably with“selectively binds” herein.

By“overlapping” in the context of two epitopes shall be taken to mean that two epitopes share a sufficient number of amino acid residues to permit an antibody that binds to one epitope to competitively inhibit the binding of an antibody that binds to the other epitope. For example, the two epitopes share at least 1 or 2 or 3 or 4 or 5 or 6 or more amino acids.

Reference herein to“monoclonal antibody MK-Abl” or to“MK-Abl” is a reference to the monoclonal antibody which has a variable heavy chain sequence as shown in SEQ ID NO:2 or 33 and a variable light chain sequence as shown in SEQ ID NO:3. In one example, the variable heavy chain sequence is set forthi n SEQ ID NO: 2. In one example, the variable heavy chain sequence is set forth in SEQ ID NO: 33. The two versions of the MK-Abl variable heavy chain sequence set forth in SEQ ID NOs: 2 and 33 respectively comprise identical CDRs.

Reference herein to“monoclonal antibody MK-Ab2” or to“MK-Ab2” is a reference to the monoclonal antibody which has a variable heavy chain sequence as shown in SEQ ID NO: 10 and a variable light chain sequence as shown in SEQ ID NO: 11.

Reference herein to“monoclonal antibody MK-Ab3” or to“MK-Ab3” is a reference to the monoclonal antibody which has a variable heavy chain sequence as shown in SEQ ID NO: 18 and a variable light chain sequence as shown in SEQ ID NO: 19.

Reference herein to“monoclonal antibody MK-Ab4”,“MK-Ab4” or“murine MK- Ab4” is a reference to the monoclonal antibody which has a variable heavy chain sequence comprising CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 26, 27 and 28, respectively, and a variable light chain sequence comprising CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 29, 30 and 31, respectively.

As used herein, the terms "treating", "treat" or "treatment" and variations thereof, refer to clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. An individual is successfully "treated", for example, if one or more symptoms associated with a disease/condition ( e.g ., myocarditis or cardiomyopathy) and/or injury (e.g., cardiac injury associated with myocarditis or cardiomyopathy) are mitigated or eliminated or the clinical outcome or prognosis of the disease/condition or injury is improved. For example, since myocarditis often results in impaired cardiac pumping function or arrhythmias, treatment may result in a reduction in cardiac inflammation and a return or approach to normal cardiac function or rhythm. In another example, treatment may prevent cardiac hypertrophy and/or prevent cardiac fibrosis and/or improve heart function in the subject.

As used herein, the terms "preventing", "prevent” or "prevention" or variations thereof, refers to the provision of prophylaxis with respect to occurrence or recurrence of a disease or condition in an individual. An individual may be predisposed to or at risk of developing the disease/condition or disease/condition relapse but has not yet been diagnosed with the disease/condition or the relapse. The term prevention does not require absolute prevention but includes inhibiting the progression of the disease or condition to some extent or delaying the onset on the disease or condition or a symptom/injury associated with the disease/condition. For example, prevention of myocarditis and/or cardiomyopathy may include reducing cardiac inflammation and/or preventing cardiac hypertrophy and/or preventing cardiac fibrosis and/or preventing injury to the myocardium and/or improving heart function in the subject.

An "effective amount" refers to at least an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. An effective amount can be provided in one or more administrations. In some examples of the present disclosure, the term "effective amount" is meant an amount necessary to effect treatment of a disease or condition as hereinbefore described. The effective amount may vary according to the disease or condition to be treated and also according to the weight, age, racial background, sex, health and/or physical condition and other factors relevant to the mammal being treated. Typically, the effective amount will fall within a relatively broad range (e.g. a "dosage" range) that can be determined through routine trial and experimentation by a medical practitioner. The effective amount can be administered in a single dose or in a dose repeated once or several times over a treatment period.

A "therapeutically effective amount" is at least the minimum concentration required to effect a measurable improvement of a particular disease or condition (e.g., myocarditis or cardiomyopathy). A therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the protein to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the protein are outweighed by the therapeutically beneficial effects.

The term "effective concentration 50%" (abbreviated as "ECso") represents the concentration of an antibody of the disclosure that is required for 50% of a given effect of the molecule the antibody targets (e.g. inhibiting/displacing binding of human midkine to a target thereof). It will be understood by one in the art that a lower EC so value corresponds to a more potent antibody. The“mammal” treated according to the present disclosure may be a human, primate, livestock ( e.g . sheep, horses, cattle, pigs, donkeys), companion animal ( e.g . pets such as dogs and cats), laboratory test animal (e.g. mice, rabbits, rats, guinea pigs), performance animal (e.g. racehorses, camels, greyhounds) or captive wild animal. In one example, the mammal is a human.

Proteins which bind selectively to the MK N-domain

As described herein, isolated or recombinant proteins comprising an antigen binding domain of an antibody which binds selectively to the N-domain of MK are contemplated for use in the method of the present disclosure. Antibodies which bind selectively to the N- domain of MK are known in the art, including, but not limited to, those anti-MK antibodies as described in W02008/059616, WO2012/122590 and W02014/070642. Further anti-MK antibodies are described in Sun X. Z, et ah, (1997) J. Neuropathol. Exp. Neurol. 56(12): 1339- 48 and Muramatsu H., et ah, (2004) J. Biochem., 119: 1171-77). However, other isolated or recombinant proteins suitable for use in methods of the disclosure, including antibodies which bind selectively to the N-domain of MK and proteins comprising antigen binding domains thereof, may be produced by methods known in the art.

Methods for producing isolated or recombinant proteins suitable for use in a method of the disclosure, including antibodies which bind selectively to the N-domain of MK and binding fragments thereof, are described herein. Furthermore, functional assays for determining MK binding activity of a protein and its suitability for use in a method of the disclosure are also described herein.

An isolated or recombinant protein contemplated for use in a method of the disclosure is protein comprising an antigen binding domain of an antibody which binds specifically to an epitope located within the N-domain of MK e.g., as defined by amino acid residues 1-61 of the sequence set forth in SEQ ID NO: l, and thereby inhibits or reduces a function of MK. For example, an isolated or recombinant protein contemplated for use in a method of the disclosure recognizes at least a portion of a high electrostatic potential cluster located at amino acid residues 1-61 of the sequence set forth in SEQ ID NO: 1.

Suitable proteins for use in a method of the disclosure may bind specifically to a conformational epitope located within the N-domain of MK formed by the amino acid sequence set forth in SEQ ID NO: l, wherein the epitope includes at least two residues selected from the group consisting of 18W, 20W, 34F, 35R, 36E, 38T, 43T, 45R, 47R and 49R. In one example, the conformational epitope is defined by residues 18W, 20W, 35R and 49R. In one example, the conformational epitope is defined by residues 18W, 20W, 36E, 38T, 43T and 45R. In one example, the conformational epitope is defined by residues 18W, 20W, 34F, 36E, 45R and 47R. According to one particular example, an isolated or recombinant protein contemplated for use in a method of the disclosure comprises:

(i) a VH comprising the sequence set forth in SEQ ID NO: 2 or 33 or a sequence exhibiting 95% or greater identity thereto;

(ii) a VL comprising the sequence set forth in SEQ ID NO: 3 or a sequence exhibiting 95% or greater identity thereto;

(iii) a VH comprising the sequence set forth in SEQ ID NO: 2 or 33 or a sequence exhibiting 95% or greater identity thereto and a VL comprising the sequence set forth in SEQ ID NO: 3 or a sequence exhibiting 95% or greater identity thereto;

(iv) a VH comprising CDR1, CDR2 and CDR3 comprising the sequences set forth in SEQ

ID NO: 4, 5 and 6 respectively, and a VL comprising CDR1, CDR2 and CDR3 comprising the sequences set forth in SEQ ID NO: 7, 8 and 9 respectively; or

(v) three CDRs comprised within the sequence set forth in SEQ ID NO: 2 or 33 and three CDRs comprised within the sequence set forth in SEQ ID NO: 3.

In one particular example, the protein for use in the method of the disclosure in accordance with this example comprises: a VH comprising CDR1, CDR2 and CDR3 comprising the sequences set forth in SEQ ID NO: 4, 5 and 6 respectively; and a VL comprising CDR1, CDR2 and CDR3 comprising the sequences set forth in SEQ ID NO: 7, 8 and 9 respectively. For example, the protein for use in the method of the disclosure in accordance with this example may be the antibody designated MK-Abl having a VH comprising the sequence set forth in SEQ ID NO: 2 and a VL comprising the sequence set forth in SEQ ID NO: 3. For example, the protein for use in the method of the disclosure in accordance with this example may be the antibody designated MK-Abl having a VH comprising the sequence set forth in SEQ ID NO: 33 and a VL comprising the sequence set forth in SEQ ID NO: 3.

According to another particular example, an isolated or recombinant protein contemplated for use in a method of the disclosure comprises:

(i) a VH comprising the sequence set forth in SEQ ID NO: 10 or a sequence exhibiting 95% or greater identity thereto;

(ii) a VL comprising the sequence set forth in SEQ ID NO: 11 or a sequence exhibiting 95% or greater identity thereto;

(iii) a VH comprising the sequence set forth in SEQ ID NO: 10 or a sequence exhibiting 95% or greater identity thereto and a VL comprising the sequence set forth in SEQ ID NO: 11 or a sequence exhibiting 95% or greater identity thereto;

(iv) a VH comprising CDR1, CDR2 and CDR3 comprising the sequences set forth in SEQ ID NO: 12, 13 and 14 respectively, and a VL comprising CDR1, CDR2 and CDR3 comprising the sequences set forth in SEQ ID NO: 15, 16 and 17 respectively; or (v) three CDRs comprised within the sequence set forth in SEQ ID NO: 10 and three CDRs comprised within the sequence set forth in SEQ ID NO: 11.

In one particular example, the protein for use in the method of the disclosure in accordance with this example comprises: a VH comprising CDR1, CDR2 and CDR3 comprising the sequences set forth in SEQ ID NO: 12, 13 and 14 respectively; and a VL comprising CDR1, CDR2 and CDR3 comprising the sequences set forth in SEQ ID NO: 15, 16 and 17 respectively. For example, the protein for use in the method of the disclosure in accordance with this example may be the antibody designated MK-Ab2 having a VH comprising the sequence set forth in SEQ ID NO: 10 and a VL comprising the sequence set forth in SEQ ID NO: 11.

According to another particular example, an isolated or recombinant protein contemplated for use in a method of the disclosure comprises:

(i) a VH comprising the sequence set forth in SEQ ID NO: 18 or a sequence exhibiting 95% or greater identity thereto;

(ii) a VL comprising the sequence set forth in SEQ ID NO: 19 or a sequence exhibiting 95% or greater identity thereto;

(iii) a VH comprising the sequence set forth in SEQ ID NO: 18 or a sequence exhibiting 95% or greater identity thereto and a VL comprising the sequence set forth in SEQ ID NO: 19 or a sequence exhibiting 95% or greater identity thereto;

(iv) a VH comprising CDR1, CDR2 and CDR3 comprising the sequences set forth in SEQ ID NO: 20, 21 and 22 respectively, and a VL comprising CDR1, CDR2 and CDR3 comprising the sequences set forth in SEQ ID NO: 23, 24 and 25 respectively; or

(v) three CDRs comprised within the sequence set forth in SEQ ID NO: 18 and three CDRs comprised within the sequence set forth in SEQ ID NO: 19.

In one particular example, the protein for use in the method of the disclosure in accordance with this example comprises: a VH comprising CDR1, CDR2 and CDR3 comprising the sequences set forth in SEQ ID NO: 20, 21 and 22 respectively; and a VL comprising CDR1, CDR2 and CDR3 comprising the sequences set forth in SEQ ID NO: 23, 24 and 25 respectively. For example, the protein for use in the method of the disclosure in accordance with this example may be the antibody designated MK-Ab3 having a VH comprising the sequence set forth in SEQ ID NO: 18 and a VL comprising the sequence set forth in SEQ ID NO: 19. Another particulartly preferred protein for use in the method of the disclosure in accordance with this example is the antibody designated MK-Ab3 having a VH comprising the sequence set forth in SEQ ID NO: 18 and a VL comprising the sequence set forth in SEQ ID NO: 20.

The % identity of an immunoglobulin chain of an antibody disclosed herein is determined by GAP (Needleman and Wunsch, (1970) J. Mol. Biol., 48(3):443-453) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. The query sequence is at least 50 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 50 amino acids. Even more preferably, the query sequence is at least 100 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. Most preferably, the two sequences are aligned over their entire length.

With regard to a defined isolated or recombinant protein of the disclosure e.g., such as an immunoglobulin chain of an antibody, it will be appreciated that % identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that the isolated or recombinant protein comprises an amino acid sequence which is at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO.

In another embodiment, one residue is added to the nominated SEQ ID NO, one residue is deleted from the nominated SEQ ID NO, one residue is added and one residue is deleted compared to the nominated SEQ ID NO, two residues are added to the nominated SEQ ID NO, two residues are deleted from the nominated SEQ ID NO, one residue is changed from the nominated SEQ ID NO, two residues are changed from the nominated SEQ ID NO, one residue is changed and one residue is deleted from the nominated SEQ ID NO, or one residue is changed and one residue is added to the nominated SEQ ID NO, or any combination thereof.

In a preferred embodiment, there are no gaps in the alignment. More specifically, the algorithm does not need to create a gap in a contiguous stretch of amino acids to obtain an optimal (highest % identity) alignment.

Amino acid sequence mutants of the isolated or recombinant protein contemplated for use in the method of the present disclosure can be prepared by introducing appropriate nucleotide changes into a nucleic acid of the present disclosure, or by in vitro synthesis of the desired polypeptide. Such mutants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence. A combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final polypeptide product possesses the desired characteristics.

Mutant (altered) polypeptides can be prepared using any technique known in the art. For example, a polynucleotide of the disclosure can be subjected to in vitro mutagenesis. Such in vitro mutagenesis techniques include sub-cloning the polynucleotide into a suitable vector, transforming the vector into a "mutator" strain such as the E. coli XL-1 red (Stratagene) and propagating the transformed bacteria for a suitable number of generations. Products derived from mutated/altered DNA can readily be screened using techniques described herein to determine if they have receptor-binding and/or -inhibitory activity.

In designing amino acid sequence mutants, the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified. The sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site.

Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues.

Substitution mutants have at least one amino acid residue in the antibody and/or immunoglobulin chain molecule removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include sites identified as important for antigen binding. These sites, especially those falling within a sequence of at least three other identically conserved sites of human antibodies and/or immunoglobulin chains, are preferably substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 1 under the heading of "exemplary substitutions".

Table 1. Exemplary substitutions

Furthermore, if desired, unnatural amino acids or chemical amino acid analogues can be introduced as a substitution or addition into the antibody and/or immunoglobulin chain of the present disclosure. Such amino acids include, but are not limited to, the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, 6-amino hexanoic acid, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as a-methyl amino acids, Ca-methyl amino acids, Na- methyl amino acids, and amino acid analogues in general.

The isolated or recombinant protein contemplated for use in a method of the disclosure may comprise a heavy chain variable domain (VH) and a light chain variable domain (VL) of an anti-MK antibody described herein. In one example, the VH and the VL are provided in a single polypeptide chain. Alternatively, the VH and the VL may be provided in separate polypeptide chains.

According to an example in which the VH and the VL are provided in a single polypeptide chain, the isolated or recombinant protein of the disclosure may be provided in the form of:

(i) a single chain Fv fragment (scFv);

(ii) a dimeric scFv (di-scFv); or

(iii) at least one of (i) and/or (ii) linked to a Fc or a heavy chain constant domain (CH) 2 and/or CH3.

According to a different example in which the VH and the VL are provided in as separate polypeptide chains, the isolated or recombinant protein of the disclosure may be provided in the form of:

(i) a diabody;

(ii) a triabody;

(iii) a tetrabody;

(iv) a Fab;

(v) a F(ab’)2;

(vi) a Fv; or

(iv) one of (i) to (iii) linked to a Fc or a heavy chain constant domain (CH) 2 and/or

CH3.

According to an example in which the isolated or recombinant protein of the disclosure is an antibody, the antibody may be a chimeric, de-immunized, humanized or human antibody.

In one example, the isolated or recombinant protein may also comprise a human or non-human primate heavy chain immunoglobulin constant region selected from a group consisting of IgGl, IgG2, IgG3, IgG4, IgM, IgE and IgA. For example, the isolated or recombinant protein may comprise a human or non-human primate heavy chain immunoglobulin constant region from an IgGl. For example, the isolated or recombinant protein may comprise a human or non-human primate heavy chain immunoglobulin constant region from an IgG4. For example, the isolated or recombinant protein may comprise a human or non-human primate heavy chain immunoglobulin constant region from an IgG2.

In a preferred embodiment, an isolated or recombinant protein described herein is an immunoglobulin light chain variable region joined directly to an immunoglobulin light chain constant region described herein. Similarly, in a further preferred embodiment an immunoglobulin heavy chain variable region described herein is joined directly to an immunoglobulin heavy chain constant region described herein. A skilled person will understand that the variable and constant regions of an immunoglobulin heavy or light chain can be joined as described by using standard recombinant DNA technology to create a polynucleotide (encoding the joined variable and constant domains) that can be expressed in a suitable host (to produce the said immunoglobulin chain(s)) or by using peptide chemistry to synthesise the joined variable and constant domains.

In accordance with an example in which the isolated or recombinant protein is a humanised anti-MK antibody or binding fragment thereof, the humanised antibody or binding fragment will retain a significant proportion of the binding properties of the parent or precursor antibody or fragment. Suitable humanised antibodies will retain the ability to specifically bind MK protein e.g., human MK and/or mouse MK, recognized by the parent or precursor antibody used to produce such antibodies. Preferably a humanised antibody for use in a method of the disclosure exhibits substantially the same or improved binding affinity and avidity as the parent or precursor antibody. Ideally, the affinity (KD) of the antibody for midkine will be greater than the parent antibody affinity for midkine.

Binding affinity can be determined by association (Ka) and dissociation (Kd) rate. Equilibrium affinity constant, K, is the ratio of Ka/Kd. Association (Ka) and dissociation (Kd) rates can be measured using surface plasmon resonance (SPR) (Rich and Myszka, (2000) Curr. Opin. Biotechnol. 11 :54; Englebienne P, (1998) Analyst. 123(7): 1599-1603). Instrumentation and methods for real time detection and monitoring of binding rates are known and are commercially available (BiaCore 2000, Biacore AB, Upsala, Sweden; and Malmqvist M (1999), Biochem. Soc. Trans. 27:335-340). Methods for assaying binding affinity are well known in the art and include half-maximal binding assays, competition assays, and Scatchard analysis.

As the skilled person will appreciate,“avidity” relates to the overall strength of interaction between two molecules, such as an antibody and antigen. Avidity depends on both the affinity and the valency of interactions. Furthermore,“affinity” relates to the strength of the binding between a single binding site of a molecule (e.g., an antibody) and a ligand (e.g., an antigen). The affinity of a molecule X for a ligand Y is represented by the dissociation constant (Kd), which is the concentration of Y that is required to occupy the combining sites of half the X molecules present in a solution. A smaller Kd indicates a stronger or higher affinity interaction, and a lower concentration of ligand is needed to occupy the sites.

An anti-MK antibody suitable for use in a method of the disclosure may also be a heteroconjugate antibody. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (US 4,676,980), and for treatment of HIV infection (W01991/000360; WO1992/200373; EP 586505). It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.

It may be desirable to modify an antibody of the disclosure with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating a disorder described herein, such as cardiomyopathy or myocarditis. For example, cysteine residue(s) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) (Caron et al, (1992) J. Exp. Med., 176(4): 1191-1195; Shopes B. (1992) J. Immunol., 148(9):2918-2922). Homodimeric antibodies with enhanced activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al. (1993). Alternatively, an antibody can be engineered that has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities (Stevenson et al, (1989) JAMA, 261 :884- 888).

Isolated or recombinant proteins for use in the method of the disclosure may be produced by the intervention of man e.g., as described herein. In a preferred embodiment, the isolated or recombinant protein of the disclosure is "substantially purified" or "purified". By "substantially purified" or "purified" we mean an isolated or recombinant protein e.g., an antibody or binding fragment thereof, that has been separated from one or more lipids, nucleic acids, other polypeptides, or other contaminating molecules with which it is associated in its native state. It is preferred that the substantially purified polypeptide is at least 60% free, more preferably at least 75% free, and more preferably at least 90% free from other components with which it is naturally associated. In another embodiment, "substantially purified" or "purified" means that the molecule that is the predominant species in the composition wherein it is found with respect to the class of molecules to which it belongs ii.e., it makes up at least about 50% of the type of molecule in the composition and typically will make up at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more of the species of molecule, e. g., peptide, in the composition).

Production of proteins which bind MK

Isolated or recombinant proteins useful in a method of the present disclosure can be produced using methods available in the art, examples of which are described herein.

Production of antibodies

In one example, an isolated or recombinant protein useful in a method of the present disclosure is an antibody that binds the N-domain of MK. Methods for generating antibodies are known in the art and/or described in Harlow and Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988). Generally, in such methods a MK protein or immunogenic fragment or epitope thereof or a cell expressing and displaying same (i.e., an immunogen), optionally formulated with any suitable or desired carrier, adjuvant, or pharmaceutically acceptable excipient, is administered to a non-human animal, for example, a mouse, chicken, rat, rabbit, guinea pig, dog, horse, cow, goat or pig. The immunogen may be administered intranasally, intramuscularly, subcutaneously, intravenously, intradermally, intraperitoneally, or by other known route.

The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. One or more further immunizations may be given, if required to achieve a desired antibody titer. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal is bled and the serum isolated and stored, and/or the animal is used to generate monoclonal antibodies (Mabs).

Monoclonal antibodies are one exemplary form of MK-binding protein contemplated for use in a method of the present disclosure. The term“monoclonal antibody" or“mAb” refers to a homogeneous antibody population capable of binding to the same antigen(s), for example, to the same epitope within the antigen. This term is not intended to be limited as regards to the source of the antibody or the manner in which it is made.

For the production of mAbs any one of a number of known techniques may be used, such as, for example, the procedure exemplified in US Patent No. 4,196,265 or Harlow and Lane (1988), supra.

For example, a suitable animal is immunized with an immunogen under conditions sufficient to stimulate antibody producing cells. Rodents such as rabbits, mice and rats are exemplary animals. Mice genetically-engineered to express human immunoglobulin proteins and, for example, do not express murine immunoglobulin proteins, can also be used to generate an antibody which is suitable for use in a method of the present disclosure ( e.g as described in W02002/066630).

Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsies of spleens, tonsils or lymph nodes, or from a peripheral blood sample. The B cells from the immunized animal are then fused with cells of an immortal myeloma cell, generally derived from the same species as the animal that was immunized with the immunogen.

Hybrids are amplified by culture in a selective medium comprising an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary agents are aminopterin, methotrexate and azaserine. The amplified hybridomas are subjected to a functional selection for antibody specificity and/or titer, such as, for example, by flow cytometry and/or immunohistochemistry and/or immunoassay ( e.g . radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, dot immunoassay, and the like).

Alternatively, ABL-MYC technology (NeoClone, Madison WI 53713, USA) is used to produce cell lines secreting MAbs (e.g., as described in Largaespada et al, (1996) J. Immunol. Methods. 197:85-95).

Antibodies can also be produced or isolated by screening a display library, e.g., a phage display library, e.g., as described in US Patent No. 6,300,064 and/or US Patent No. 5,885,793.

Chimeric Antibodies

In one example, an isolated or recombinant protein useful in a method of the present disclosure is a chimeric antibody which binds the N-domain of MK. The term“chimeric antibody” refers to antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species (e.g., murine, such as mouse) or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species (e.g., primate, such as human) or belonging to another antibody class or subclass. Typically chimeric antibodies utilize rodent or rabbit variable regions and human constant regions, in order to produce an antibody with predominantly human domains. Methods for producing chimeric antibodies are described in, e.g., US Patent No. 4,816,567; and US Patent No. 5,807,715.

The present disclosure also contemplates the use of a chimeric immunoglobulin, e.g., in which a variable region from one species is fused to a region of a protein from another species. For example, the disclosure contemplates the use of an immunoglobulin comprising a variable region from a T cell receptor of one species fused to a T cell receptor constant domain from a separate species.

Humanized and Human Antibodies

In one example, an isolated or recombinant protein useful in a method of the present disclosure may be humanized or human antibody or protein that binds the N-domain of MK.

The term "humanized antibody” shall be understood to refer to a subclass of chimeric antibodies having an antigen binding site or variable region derived from an antibody from a non-human species and the remaining antibody structure of the molecule based upon the structure and/or sequence of a human antibody. The antigen-binding site comprises the complementarity determining regions (CDRs) from the non-human antibody grafted onto appropriate FRs in the variable domains of a human antibody and the remaining regions from a human antibody. Antigen binding sites may be wild type or modified by one or more amino acid substitutions. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues.

Methods for humanizing non-human antibodies or parts thereof ( e.g ., variable regions) are known in the art. Humanization can be performed following the method of US Patent No. 5,225,539, or US Patent No. 5,585,089. Other methods for humanizing an antibody are not excluded.

The term "human antibody" as used herein in connection with antibodies refers to antibodies having variable regions (e.g. VH, VL) and, optionally constant regions derived from or corresponding to sequences found in humans, e.g. in the human germline or somatic cells. The "human" antibodies can include amino acid residues not encoded by human sequences, e.g. mutations introduced by random or site directed mutations in vitro (in particular mutations which involve conservative substitutions or mutations in a small number of residues of the antibody, e.g., in 1, 2, 3, 4 or 5 of the residues of the antibody, e.g., in 1, 2, 3, 4 or 5 of the residues making up one or more of the CDRs of the antibody). These“human antibodies” do not actually need to be produced by a human, rather, they can be produced using recombinant means and/or isolated from a transgenic animal (e.g., mouse) comprising nucleic acid encoding human antibody constant and/or variable regions (e.g., as described above). Human antibodies can be produced using various techniques known in the art, including phage display libraries (e.g., as described in US Patent No. 5,885,793).

Human antibodies which recognize a selected epitope can also be generated using a technique referred to as "guided selection." In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope (e.g., as described in US Patent No. 5,565,332).

De -immunized Antibodies

In another example, an isolated or recombinant protein useful in a method of the present disclosure may be a de-immunized antibody or protein which binds the N-domain of MK. De-immunized antibodies and proteins have one or more epitopes, e.g., B cell epitopes or T cell epitopes removed (i.e., mutated) to thereby reduce the likelihood that a mammal will raise an immune response against the antibody or protein. Methods for producing de- immunized antibodies and proteins are known in the art and described, for example, in W02000/034317, W02004/108158 and W02004/064724.

Methods for introducing suitable mutations and expressing and assaying the resulting protein will be apparent to the skilled artisan based on the description herein. Heavy Chain Antibodies

In another example, an isolated or recombinant protein useful in a method of the present disclosure may be a heavy chain of an antibody which binds the N-domain of MK. Heavy chain antibodies differ structurally from many other forms of antibodies, in so far as they comprise a heavy chain, but do not comprise a light chain. Accordingly, these immunoglobulins are also referred to as “heavy chain only antibodies”. Heavy chain immunoglobulins are found in, for example, camelids and cartilaginous fish (also called IgNAR).

The variable regions present in naturally occurring heavy chain antibodies are generally referred to as "VHH domains" in camelid antibodies and V-NAR in IgNAR, in order to distinguish them from the heavy chain variable regions that are present in conventional 4- chain antibodies (which are referred to as "VH domains") and from the light chain variable regions that are present in conventional 4-chain antibodies (which are referred to as "VL domains").

A general description of heavy chain antibodies from camelids and the variable regions thereof and methods for their production and/or isolation and/or use is found inter alia in the following references WO 1994/004678, and WO 1997/049805.

A general description of heavy chain immunoglobulins from cartilaginous fish and the variable regions thereof and methods for their production and/or isolation and/or use is found inter alia in W02005/118629.

Antibody Fragments

Single-Domain Antibodies

In some examples, an isolated or recombinant protein useful in a method of the present disclosure is or comprises a single-domain antibody (which is used interchangeably with the term“domain antibody” or“dAb”) which binds the N-domain of MK. A single-domain antibody is a single polypeptide chain comprising all or a portion of the heavy chain variable domain of an antibody. In certain examples, a single-domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., US Patent No. 6,248,516).

Diabodies, Triabodies, Tetrabodies

In some examples, an isolated or recombinant protein which binds the N-domain of MK useful in a method of the present disclosure is or comprises a diabody, triabody, tetrabody or higher order protein complex such as those described in WO 1998/044001 and/or WO 1994/007921. For example, a diabody is a protein comprising two associated polypeptide chains, each polypeptide chain comprising the structure VL-X-VH or VH-X-VL, wherein VL is an antibody light chain variable region, VH is an antibody heavy chain variable region, X is a linker comprising insufficient residues to permit the VH and VL in a single polypeptide chain to associate (or form an Fv) or is absent, and wherein the VH of one polypeptide chain binds to a VL of the other polypeptide chain to form an antigen binding site, i.e., to form a Fv molecule capable of specifically binding to one or more antigens. The VL and VH can be the same in each polypeptide chain or the VL and VH can be different in each polypeptide chain so as to form a bispecific diabody {i.e., comprising two Fvs having different specificity).

A diabody, triabody, tetrabody, etc capable of inducing effector activity can be produced using an antigen binding domain capable of binding to IL-3Ra and an antigen binding domain capable of binding to a cell surface molecule on an immune cell, e.g., a T cell (e.g., CD3).

Single Chain Fv (scFv) Fragments

In some examples, an isolated or recombinant protein useful in a method of the present disclosure is or comprises a scFvs fragment which binds the N-domain of MK. The skilled artisan will be aware that scFvs comprise VH and VL regions in a single polypeptide chain and a polypeptide linker between the VH and VL which enables the scFv to form the desired structure for antigen binding {i.e., for the VH and VL of the single polypeptide chain to associate with one another to form a Fv). For example, the linker comprises in excess of 12 amino acid residues with (Gly4Ser)3 being one of the more favoured linkers for a scFv.

The present disclosure also contemplates the use of a disulfide stabilized Fv (or diFv or dsFv), in which a single cysteine residue is introduced into a FR of VH and a FR of VL and the cysteine residues linked by a disulfide bond to yield a stable Fv.

Alternatively, or in addition, the present disclosure encompasses the use of a dimeric scFv, i.e., a protein comprising two scFv molecules linked by a non-covalent or covalent linkage, e.g., by a leucine zipper domain {e.g., derived from Fos or Jun). Alternatively, two scFvs are linked by a peptide linker of sufficient length to permit both scFvs to form and to bind to an antigen, e.g., as described in US20060263367.

The present disclosure also contemplates the use of a dimeric scFv capable of inducing effector activity. In one example, the dimeric protein is a combination of a dAb and a scFv. Examples of bispecific antibody fragments capable of inducing effector function are described, for example, in US Patent No. 7,235,641. Recombinant Expression of MK-binding proteins

In some examples, an isolated or recombinant protein useful in a method of the present disclosure is produced by recombinant techniques.

In the case of a recombinant protein, a nucleic acid encoding same can be cloned into expression vectors, which are then transfected into host cells, such as E. coli cells, yeast cells, insect cells, or mammalian cells, such as simian COS cells, Chinese Hamster Ovary (CHO) cells, human embryonic kidney (HEK) cells, or myeloma cells that do not otherwise produce immunoglobulin protein. Exemplary cells used for expressing an immunoglobulin are CHO cells, myeloma cells or HEK cells. Molecular cloning techniques to achieve these ends are known in the art and described, for example in Ausubel et al, (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present) or Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). A wide variety of cloning and in vitro amplification methods are suitable for the construction of recombinant nucleic acids. Methods of producing recombinant antibodies are also known in the art. See e.g., US Patent No. 4,816,567 or US Patent No. 5,530,101.

Following isolation, the nucleic acid is inserted operably-linked to a promoter in an expression construct or expression vector for further cloning (amplification of the DNA) or for expression in a cell-free system or in cells.

As used herein, the term“promoter” is to be taken in its broadest context and includes the transcriptional regulatory sequences of a genomic gene, including the TATA box or initiator element, which is required for accurate transcription initiation, with or without additional regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers and silencers) that alter expression of a nucleic acid, e.g., in response to a developmental and/or external stimulus, or in a tissue specific manner. In the present context, the term“promoter” is also used to describe a recombinant, synthetic or fusion nucleic acid, or derivative which confers, activates or enhances the expression of a nucleic acid to which it is operably-linked. Exemplary promoters can contain additional copies of one or more specific regulatory elements to further enhance expression and/or alter the spatial expression and/or temporal expression of said nucleic acid.

As used herein, the term“operably-linked to" means positioning a promoter relative to a nucleic acid such that expression of the nucleic acid is controlled by the promoter.

Many vectors for expression in cells are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, a sequence encoding an immunoglobulin (e.g., derived from the information provided herein), an enhancer element, a promoter, and a transcription termination sequence. The skilled artisan will be aware of suitable sequences for expression of an immunoglobulin. Exemplary signal sequences include prokaryotic secretion signals ( e.g ., pelB, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II), yeast secretion signals (e.g., invertase leader, a factor leader, or acid phosphatase leader) or mammalian secretion signals (e.g., herpes simplex gD signal).

Exemplary promoters active in mammalian cells include cytomegalovirus immediate early promoter (CMV-IE), human elongation factor 1-a promoter (EF1), small nuclear RNA promoters (Ula and Ulb), a-myosin heavy chain promoter, Simian virus 40 promoter (SV40), Rous sarcoma virus promoter (RSV), Adenovirus major late promoter, b-actin promoter; hybrid regulatory element comprising a CMV enhancer/ b-actin promoter or an immunoglobulin promoter or active fragment thereof. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture; baby hamster kidney cells (BHK, ATCC CCL 10); or Chinese hamster ovary cells (CHO).

Typical promoters suitable for expression in yeast cells such as for example a yeast cell selected from the group comprising Pichia pastoris, Saccharomyces cerevisiae and S. pombe , include, but are not limited to, the ADH1 promoter, the GAL1 promoter, the GAL4 promoter, the CUP1 promoter, the PH05 promoter, the nmt promoter, the RPR I promoter, or the TEF1 promoter.

Means for introducing the isolated nucleic acid or expression construct comprising same into a cell for expression are known to those skilled in the art. The technique used for a given cell depends on the known successful techniques. Means for introducing recombinant DNA into cells include microinjection, transfection mediated by DEAE-dextran, transfection mediated by liposomes such as by using lipofectamine (Gibco, MD, USA) transduction mediated by a viral delivery system, and/or cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation and microparticle bombardment such as by using DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA) amongst others.

The host cells used to produce the immunoglobulin may be cultured in a variety of media, depending on the cell type used. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMl-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing mammalian cells. Media for culturing other cell types discussed herein are known in the art.

Isolation/purification of MK-binding proteins

MK-binding proteins used in accordance with the method of the present disclosure are preferably isolated, and, more preferably, provided in a substantially purified form. Methods for isolating and purifying antibodies and proteins are known in the art and/or described herein. Where an antibody or antibody fragment is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.

The antibodies and antibody fragments prepared from the cells can be purified using, for example, ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography ( e.g ., protein A affinity chromatography or protein G chromatography), or any combination of the foregoing. These methods are known in the art and described, for example in WO 1999/057134 or Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988).

The skilled artisan will also be aware that a protein of the disclosure e.g., such as an antibody or fragment thereof, can be modified to include a tag to facilitate purification or detection, e.g., a poly-histidine tag, e.g., a hexa-histidine tag, or an influenza virus hemagglutinin (HA) tag, or a Simian Virus 5 (V5) tag, or a FLAG tag, or a glutathione S- transferase (GST) tag. The resulting protein is then purified using methods known in the art, such as, affinity purification. For example, an immunoglobulin comprising a hexa-his tag is purified by contacting a sample comprising the immunoglobulin with nickel-nitrilotriacetic acid (Ni-NTA) that specifically binds a hexa-his tag immobilized on a solid or semi-solid support, washing the sample to remove unbound antibodies, and subsequently eluting the bound antibodies. Alternatively, or in addition a ligand or antibody that binds to a tag may be used in an affinity purification method.

Conjugates

Isolated or recombinant proteins used in accordance with the present disclosure may be provided as conjugates of proteins e.g., antibodies or binding fragments thereof, as described herein according to any embodiment. For example, an isolate or recombinant protein of the disclosure can be modified to contain additional non-proteinaceous moieties that are known in the art and readily available. Preferably, the moieties suitable for derivatization of the protein are physiologically acceptable polymer, preferably a water soluble polymer. Such polymers are useful for increasing stability and/or reducing clearance (e.g., by the kidney) and/or for reducing immunogenicity of a protein of the disclosure. Non limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), polyvinyl alcohol (PVA), or propropylene glycol (PPG). In one example, an isolated or recombinant protein as described herein according to any embodiment is conjugated or linked to another protein, including another protein of the disclosure or a protein comprising an antibody variable region, such as an antibody or a protein derived therefrom, e.g., as described herein. Other proteins are not excluded. Additional proteins will be apparent to the skilled artisan and include, for example, an immunomodulator or a half-life extending protein or a peptide or other protein that binds to serum albumin amongst others.

Exemplary serum albumin binding peptides or protein are described in US20060228364 or US20080260757.

In one example, an isolated or recombinant protein used in accordance with the present disclosure comprises one or more detectable markers to facilitate detection and/or isolation. For example, the compound comprises a fluorescent label such as, for example, fluorescein (FITC), 5,6-carboxymethyl fluorescein, Texas red, nitrobenz-2-oxa-l,3- diazol-4- yl (NBD), coumarin, dansyl chloride, rhodamine, 4'-6-diamidino-2- phenylinodole (DAPI), and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, fluorescein (5-carboxyfluorescein-N- hydroxysuccinimide ester), rhodamine (5,6- tetramethyl rhodamine). The absorption and emission maxima, respectively, for these fluors are: FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm), Cy3.5 (581 nm; 588 nm), Cy5 (652 nm: 672 nm), Cy5.5 (682 nm; 703 nm) and Cy7 (755 nm; 778 nm).

Alternatively, or in addition, the isolated or recombinant protein as described herein according to any embodiment is labelled with, for example, a fluorescent semiconductor nanocrystal (as described, for example, in US Patent No. 6,306,610).

Alternatively, or in addition, the isolated or recombinant protein is labelled with, for example, a magnetic or paramagnetic compound, such as, iron, steel, nickel, cobalt, rare earth materials, neodymium-iron-boron, ferrous-chromium-cobalt, nickel-ferrous, cobalt- platinum, or strontium ferrite.

Assaying MK-binding activity of a protein

In vitro functional assays

In order to be suitable for use in a method of the disclosure, the isolated or recombinant protein must bind to the N-domain of MK and thereby inhibit or reduce MK activity and/or block transmission of a biological signal of MK. Accordingly, one or more functional assays may be performed to determine binding specificity and affinity of a candidate protein e.g., an antibody or fragment thereof, to the N-domain of human MK to thereby determine suitability of that protein for use in a method of the disclosure.

Various in vitro assays are available to assess the suitability of a recombinant or isolated protein of the disclosure, such an antibody, to bind to the N-domain of MK protein and/or inhibit or reduce MK activity and/or treat or prevent myocarditis and/or cardiomyopathy.

For example, binding specificity and affinity of an antibody or binding protein to human MK may be assessed by ELISA. In this way, the dissociation constant (Kd) of a candidate antibody may be determined.

In another example, the "Kd" or "Kd value" for a candidate protein or antibody may be measured by a radiolabelled MK binding assay (RIA) to determine its suitability for use in a method of the disclosure. This assay equilibrates the test protein or antibody with a minimal concentration of radioactive MK protein in the presence of a titration series of unlabelled MK protein. Following washing to remove unbound MK protein, the amount of radioactivity is determined, which is indicative of the Kd of the test protein or antibody.

According to another example the“Kd” or“Kd value” is measured by using surface plasmon resonance assays, e.g., using BIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, NJ) with immobilized IL-3Ra.

In yet another example, a chemotaxis assay can be used to assess the ability of an isolated or recombinant protein of the disclosure to block binding of MK protein to a receptor thereof and/or inhibit function associated with binding of the MK to its receptor. These assays are based on the functional migration of cells in vitro or in vivo induced by a compound (chemoattractant). Chemotaxis can be assessed by any suitable means, for example, in an assay utilizing a 96-well chemotaxis plate, or using other art-recognized methods for assessing chemotaxis.

Generally, chemotaxis assays monitor the directional movement or migration of a suitable cell into or through a barrier (e.g., endothelium, a filter), toward increased levels of a compound, from a first surface of the barrier toward an opposite second surface. Membranes or filters provide convenient barriers, such that the directional movement or migration of a suitable cell into or through a filter, toward increased levels of a compound, from a first surface of the filter toward an opposite second surface of the filter, is monitored. In some assays, the membrane is coated with a substance to facilitate adhesion, such as ICAM-1, fibronectin or collagen. Such assays provide an in vitro approximation of cell "homing".

For example, one can detect or measure inhibition of the migration of cells in a suitable container (a containing means), from a first chamber into or through a microporous membrane into a second chamber which contains a chemoattractant e.g., midkine protein, and an antibody to be tested, and which is divided from the first chamber by the membrane. A suitable membrane, having a suitable pore size for monitoring specific migration in response to compound, including, for example, nitrocellulose, polycarbonate, is selected. For example, pore sizes of about 3-8 microns, and preferably about 5-8 microns can be used. Pore size can be uniform on a filter or within a range of suitable pore sizes. To assess migration and inhibition of migration, the distance of migration into the filter, the number of cells crossing the filter that remain adherent to the second surface of the filter, and/or the number of cells that accumulate in the second chamber can be determined using standard techniques ( e.g ., microscopy and flow cytometry). In one embodiment, the cells are labelled with a detectable label (e.g., radioisotope, fluorescent label, antigen or epitope label), and migration can be assessed in the presence and absence of a candidate antibody by determining the presence of the label adherent to the membrane and/or present in the second chamber using an appropriate method (e.g., by detecting radioactivity, fluorescence, immunoassay). The extent of migration induced or inhibited can be determined relative to a suitable control (e.g., compared to background migration determined in the absence of the antibody, compared to the extent of migration induced by a second compound (i.e., a standard), compared with migration of untransfected cells induced by the antibody).

In one embodiment, a population of cells to which MK protein binds or which is capable if migrating to MK protein is placed in a chamber of a cell culture device that is in liquid communication with another chamber comprising MK protein (chemoattractant). The two chambers are separated by a suitable membrane, e.g., a membrane that mimics the extracellular matrix found in a subject. The amount of cell migration from one chamber to the other through the membrane is assessed in the presence or absence of candidate proteins or antibodies. A protein or antibody that prevents or reduces the amount of MK-mediated cell migration compared to a control sample (containing no protein or antibody) is considered to have MK inhibitory activity.

In vivo functional assays

In another example, in vivo assays may be employed to assess the usefulness of isolated or recombinant protein which binds the N-domain of MK in a method of the disclosure. For example, a candidate protein may be administered to a non-human mammal suffering from myocarditis e.g., such as the experimental autoimmune myocarditis (EAM) mouse model described in Example 1 or a viral infection-induced myocarditis mouse model as described in Fairweather and Rose (2007) Methods 41(1): 118-122. In accordance with this example, screening may involve evaluating the effects of the candidate protein or antibody on cardiac muscle structure and/or function in the animal model of myocarditis e.g., as described in Example 1 or using other methods known in the art.

Compositions

Suitably, in compositions or methods for administration of the isolated or recombinant proteins or conjugates of the disclosure to a mammal, the isolated or recombinant protein or conjugate is combined with a pharmaceutically acceptable carrier, diluent and/or excipient, as is understood in the art. Accordingly, one example of the present disclosure provides a pharmaceutical composition comprising the isolated or recombinant protein or conjugate thereof combined with a pharmaceutically acceptable carrier, diluent and/or excipient. Alternatively, the isolated or recombinant proteins or conjugates of this disclosure can be lyophilised for storage and reconstituted in a suitable carrier prior to use according to art- known lyophilisation and reconstitution techniques.

In another example, the disclosure provides a kit comprising a pharmaceutically acceptable carrier, diluent and/or excipient suitable for combining or mixing with the isolated or recombinant protein or conjugate prior to administration to the mammal. For example, the isolated or recombinant proteins or conjugates of this disclosure can be provided in a lyophilized form for combining or mixing with a pharmaceutically acceptable carrier, diluent and/or excipient prior to administration to the mammal. In this example, the kit may further comprise instructions for use e.g., in accordance with a method of the disclosure.

In general terms, by "carrier, diluent or excipient" is meant to be a solid or liquid filler, binder, diluent, encapsulating substance, emulsifier, wetting agent, solvent, suspending agent, coating or lubricant that may be safely administered to any mammal, e.g., a human. Depending upon the particular route of administration, a variety of acceptable carriers, diluents or excipients, known in the art may be used, as for example described in Remington's Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991).

By way of example only, the carriers, diluents or excipients may be selected from a group including sugars (e.g. sucrose, maltose, trehalose, glucose), starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulphate, oils inclusive of vegetable oils, synthetic oils and synthetic mono- or di-glycerides, lower alcohols, polyols, alginic acid, phosphate buffered solutions, lubricants such as sodium or magnesium stearate, isotonic saline and pyrogen-free water. For example, the carrier, diluent or excipient is compatible with, or suitable for, parenteral administration. Parenteral administration includes any route of administration that is not through the alimentary canal. Non-limiting examples of parenteral administration include injection, infusion and the like. By way of example, administration by injection includes intravenous, intra-arterial, intramuscular and subcutaneous injection. Also contemplated is delivery by a depot or slow-release formulation which may be delivered intradermally, intramuscularly and subcutaneously, for example.

Treatment indications

As described herein, the present disclosure provides a method of treating and/or preventing myocarditis or cardiomyopathy in a subject, comprising administering to the subject an isolated or recombinant protein comprising an antigen binding domain of an antibody which binds specifically to an epitope located within the N-domain of MK protein and which inhibits or reduces a function of MK.

As described herein, the aetiology of myocarditis can be due to a wide variety of injuries to the myocardium, including injury caused by toxins and drugs, including recreational and therapeutic drugs ( e.g ., cocaine and interleukin 2 respectively) or infectious agents, most commonly including viral (e.g., coxsackievirus, adenovirus, HIV and hepatitis C virus), bacterial (e.g., diphtheria, meningococcus, psittacosis and streptococcus), rickettsial (e.g., typhus and Rocky Mountain spotted fever), fungal (e.g., aspergillosis and candidiasis), and parasitic (Chagas disease, toxoplasmosis), as well as giant cell myocarditis, and hypersensitivity reactions to drugs such as antibiotics, sulfonamides, anticonvulsants, and anti-inflammatories. Therapeutic drugs may include, for example, immunotherapeutic drug e.g. immune checkpoint inhibitors. In some examples, myocarditis induced by an immune checkpoint inhibitor is referred to as immune checkpoint inhibitor (ICI)-associated myocarditis.

It is contemplated that the method of the disclosure will be suitable for treatment or prevention of myocarditis of any origin, including but not limited to those described hereinabove. The myocarditis may be acute, subacute, or chronic, and there may be either focal or diffuse involvement of the myocardium, involving any or all cardiac chambers. Severe diffuse myocarditis can result in dilatation of all cardiac chambers and there may be mural thrombus formation in any chamber. With subacute and chronic myocarditis, interstitial fibrosis may replace fiber loss, and myofiber hypertrophy may be seen.

In one example, administering the isolated or recombinant protein to the subject prevents cardiac hypertrophy and/or prevents cardiac fibrosis and/or improves heart function in the subject.

Alternatively, or in addition, and in accordance with an example in which the subject is suffering from myocarditis, administering the isolated or recombinant protein to the subject prevents development of cardiomyopathy.

The cardiomyopathy may be selected from hypertrophic cardiomyopathy, dilated cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular dysplasia, metabolic cardiomyopathy and unclassified cardiomyopathy. In one example, the cardiomyopathy is hypertrophic cardiomyopathy. In one example, the cardiomyopathy is dilated cardiomyopathy. In one example, the cardiomyopathy is restrictive cardiomyopathy. In one example, the cardiomyopathy is arrhythmogenic right ventricular dysplasia. In one example, the cardiomyopathy is metabolic cardiomyopathy (e.g., diabetic cardiomyopathy, alcoholic cardiomyopathy, or drug-induced cardiomyopathy). In one example, the cardiomyopathy is unclassified cardiomyopathy. The method of the present disclosure may further comprise the step of identifying a subject in need of treatment e.g., a subject suffering from, or who is likely to suffer from, myocarditis or cardiomyopathy. Methods for diagnosing myocarditis and cardiomyopathy are known in the art and contemplated for use herein.

Dosages and Regimens

For the treatment or prevention of myocarditis and/or cardiomyopathy as described herein, the appropriate dosage of an active agent (e.g., a protein or conjugate of the disclosure), will depend on the underlying cause of the myocarditis and/or cardiomyopathy to be treated, the severity and course of the myocarditis and/or cardiomyopathy, whether the active agent (e.g., a protein or conjugate of the disclosure) is administered for preventive or therapeutic purposes, previous therapy received by the patient (such as those described below under the heading“combination therapy”), the patient's clinical history and response to the active agent, and the discretion of the attending physician. More recently, myocarditis has been associated with injury to the myocardium caused by immunotherapeutic drugs e.g. immune checkpoint inhibitors. Myocarditis induced by, or associated with, an immune checkpoint inhibitor has been referred to as “immune checkpoint inhibitor-associated myocarditis” or“ICI-associated myocarditis”.

Some patients may have chronic and irreversible damage to the heart muscle requiring lifelong treatment, while other patients may require treatment for just a few months and then recover completely.

Typically, a therapeutically effective amount of the MK-binding protein or conjugate will be administered. The phrase "a therapeutically effective amount" refers to an amount sufficient to promote, induce, and/or enhance treatment or other therapeutic effect in the subject being treated. The therapeutically effective amount should be large enough to produce the desired effect but should not be so large as to cause adverse side effects. The particular dosage regimen, i.e., dose, timing, and repetition, will depend on the particular individual and that individual's medical history as assessed by a physician. Typically, a clinician will administer an active agent (e.g., MK-binding protein or conjugate comprising same) until a dosage is reached that achieves the desired result.

Generally, the dosage will vary with the age, condition, sex and extent of the disease, disorder and/or injury in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any complication. For in vivo administration of the MK-binding proteins or conjugates described herein, normal dosage amounts may vary from about lOng/kg up to about lOOmg/kg of an individual's body weight or more per day. Exemplary dosages and ranges thereof are described herein. For repeated administrations over several days or longer, depending on the severity of the disease, disorder and/or injury to be treated, the treatment can be sustained until a desired suppression of symptoms or treatment is achieved.

In some examples, a MK-binding protein or conjugate as described herein is administered at an initial (or loading) dose of between about lmg/kg to about 30mg/kg, such as from about lmg/kg to about lOmg/kg, or about 2mg/kg or about 3mg/kg or 4mg/kg or 5mg/kg. The MK-binding protein or conjugate can then be administered at a maintenance dose of between about O.OOOlmg/kg to about lmg/kg, such as from about 0.0005mg/kg to about lmg/kg, for example, from about O.OOlmg/kg to about lmg/kg, such as about 0.005mg/kg to about lmg/kg, for example from about 0. lmg/kg to about lmg/kg, such as about 0.2mg/kg or 0.3mg/kg or 0.4mg/kg or 0.5mg/kg. The maintenance doses may be administered every 7-30 days, such as, every 10-15 days, for example, every 10 or 11 or 12 or 13 or 14 or 15 days.

Dosages for a particular MK-binding protein or conjugate may be determined empirically in mammals that have been given one or more administrations of the respective MK-binding protein or conjugate. To assess efficacy of a dosage of MK-binding protein or conjugate of the disclosure, a clinical symptom of a disease, condition or injury being treated e.g., myocarditis and/or cardiomyopathy, can be monitored following administration. For example, efficacy of a dosage of MK-binding protein or conjugate of the disclosure in treatment of myocarditis and/or cardiomyopathy may be assessed based on one or more of the following criteria: the level of reduction in cardiac inflammation achieved; the level of improvement in heart function achieved; a return or approach to normal cardiac function or rhythm; prevention of or reduction in cardiac hypertrophy; and/or prevention of or reduction in cardiac fibrosis in the subject.

Administration of a MK-binding protein or conjugate or composition according to the methods of the present disclosure can be continuous or intermittent, depending, for example, on the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of a MK-binding protein or conjugate or composition may be essentially continuous over a preselected period of time or may be in a series of spaced doses.

A variety of routes of administration are possible including, but not necessarily limited to, oral, dietary, topical, parenteral (e.g., intravenous, intraarterial, intramuscular, subcutaneous injection), inhalation (e.g., intrabronchial, intraocular, intranasal or oral inhalation, intranasal drops), depending on the underlying cause of the myocarditis and/or cardiomyopathy, the severity of the myocarditis and/or cardiomyopathy, and the particular patient. Other suitable methods of administration can also include rechargeable or biodegradable devices and slow release polymeric devices. Combination therapies

In one example of a method described herein, the isolated or recombinant protein or conjugate or composition as described herein is administered in combination with one or more other compounds or agents useful for treating or preventing myocarditis and/or cardiomyopathy. The specific combination will depend upon the underlying cause of the myocarditis and/or cardiomyopathy and upon the individual patient to be treated. However, the isolated or recombinant protein or conjugate or composition as described may be conveniently administered in combination with one or more existing standard of care treatments for myocarditis and/or cardiomyopathy. For example, in the case of viral myocarditis, the isolated or recombinant protein or conjugate or composition as described herein may be administered in combination with antibiotics, heart protective agents, and antioxidant (such as high dose vitamin C, vitamin E and coenzyme Q10). In accordance with an example in which more aggressive treatment is necessary, the isolated or recombinant protein or conjugate or composition as described herein may be administered in combination with drugs to reduce the heart’s workload ( e.g ., ACE inhibitors, Angiotensin III receptor blockers (ARBs) or beta blockers), drugs to eliminate excess fluid (e.g., diuretics) and/or intravenous medication to improve the heart pumping function. In yet another example, the isolated or recombinant protein or conjugate or composition as described herein may be administered to a subject who has received or will receive a pump in the aorta (intra-aortic balloon pump) or a temporary artificial heart (e.g., ventricular assist device (VAD)), and/or who is in need of urgent heart transplantation. Other suitable treatment for myocarditis and/or cardiomyopathy which are contemplated are described in e.g., Maisch and Pankuweit (2013) Heart Fail. Rev. 18:761-795 and Fung et al, (2016) Circ. Res. 118:496-514, the full contents of which are incorporated herein.

The present invention will now be described more specifically with reference to the following non-limiting Examples.

EXAMPLES

Example 1 - Effect of midkine on myocarditis

In this example, the inventors evaluated the effects of intraperitoneally administered anti-midkine antibody on cardiac muscle structure and function in mice undergoing heart failure due to myocarditis. Methods

Mice

For the Experimental Autoimmune Myocarditis (EAM) model of myocarditis, 6-8 week old male WT mice on BALB/c background were purchased from Charles River (Sulzfeld, Germany). Animals were fed a standard chow diet ad libitum with free access of water. All animal experiments were institutionally approved by the Regierung von Oberbayern, Germany.

For the induction of EAM (immunized), purified synthetic cardiac peptide aMyHC ( Ac-RSLKLM ATLF ST YAS ADR (SEQ ID NO: 32) emulsified in complete Freund’s adjuvant (CFA (Sigma Aldrich, Taufkirchen, Germany) was administered subcutaneously (200 pg of cardiac peptide per mouse) at day 0 and day 7, respectively.

Treatment

The EAM mice then received one of the following treatments twice a week until day

21 :

(i) intraperitoneal injection of an antibody which binds the N-terminal domain of MK at a dose of 10 mg/kg body weight (/. _<? ., MK-Ab2 treatment);

(ii) intraperitoneal injection of an antibody which binds the C-terminal domain of MK at a dose of 10 mg/kg body weight (/. _<? ., MK-Ab4treatment);

(iii) intraperitoneal injection of isotype control IgG at a dose of 10 mg/kg body weight (/.<?., IgG isotype control); or

(iv) equal volumes of CFA + PBS administered intraperitoneally (/.<?., vehicle control).

Mice were euthanized on days 21 and 63 and hearts were removed for evaluation.

Histology and heart / body weight ratio

For histopathological evaluation of cardiac tissue, mouse hearts were removed, rinsed with saline and fixed in paraformaldehyde 4% for at least 24 hours. For analysis of the heart/body weight ratio, the pericardium, connective tissue and vascular remains were excised carefully before hearts were weighed using a microbalance (CP64-0CE, Sartorius, Gottingen, Germany). The hearts were then dehydrated in a graded series of ethanol concentrations, after which the tissues were embedded in paraffin. Cardiac sections were stained with Masson’s trichrome staining (day 63) to assess cardiac fibrosis.

To quantify fibrosis by morphometric evaluation, approximately 30 fields per section were chosen randomly. After assessment, the level of fibrosis was divided into scores ranging from 0 to 5 using a semi -quantitative score (0: 0-1%; 1 : 1-2%; 2: 2- 3%; 3: 3-4% and 5: >4% fibrosis). All analyses were performed in a blinded manner. Echocardiographic imaging

Transthoracic echocardiography was conducted using a Vevo2100 imaging system (VisualSonics, Toronto, Canada) with a 40 MHz transducer at day 63. Anesthesia was performed with 2% isoflurane. To ensure a stable body temperature in the range of 35.5- 36.5°C, mice were transferred to a heated platform (40°C). Parasternal long-axis M-mode was used to standardize mid-ventricular transducer positioning for short-axis M-mode imaging as previously described (Grabmaier et al (2014). PloS one , 9:e94615). For evaluation of cardiac function, three consecutive cycles were measured and averaged.

Statistical analysis

Data shown in Figures 1-4 represent the mean +/- s.e.m as indicated. Statistical significance (P < 0.05) was determined with Sigma Plot vlO.O (Systat Software, Chicago, IL) using Student's t test, Mann-Whitney rank sum test or Kruskal-Wallis ANOVA test, respectively.

Results

Following induction of cardiac muscle injury in the acute phase of myocarditis, the MK-Ab2 treatment resulted in significantly reduced heart/body weight ratio compared to EAM mice which received the vehicle control, suggesting that blocking MK via the N- domain protects the heart from the deteriorating hypertrophic effect of myocarditis (Figure 1). On the other hand, IgG isotype control did not protect against the increased heart weight elicited by myocarditis when compared to EAM mice which received the vehicle control.

In order to determine whether MK affects transition to fibrosis and dysfunction of the heart, the inventors evaluated cardiac fibrosis and dysfunction after the respective treatments at day 63 of EAM. The inventors evaluated the amount of collagen in cardiac muscle sections as an indicator for cardiac fibrosis after Masson’s tri chrome staining (Figure 2a). As expected, detailed analysis using a semi-quantitative fibrosis score revealed a significant increase of fibrotic tissue in the myocardium of EAM mice (immunized) compared to non- immunized control animals (Figure 2b). However, unexpectedly, MK blockade with MK- Ab2 significantly reduced fibrosis compared to vehicle-treated immunized mice (Figure 2b). No effect was seen for either the IgG isotype control compared to vehicle-treated mice (Figure 2b). These findings suggest that inhibition of MK with MK-Ab2 targeting the N- domain protects animals from cardiac fibrosis, whereas this is not the case for antibodies that target the C-domain of midkine e.g., such as MK-Ab4.

Next, the efficacy of blocking MK on cardiac systolic dysfunction was evaluated. Transthoracic echocardiography at day 63 (Figure 3a) revealed that Fractional Shortening (FS%, Figure 3b) as well as Left Ventricular Ejection Fraction (LVEF%, Figure 3c) were significantly improved in MK-Ab2 treated mice, but not in vehicle-treated mice. There was no difference between mice who received vehicle control, IgG isotype control (Figures 3b and 3c). These results indicate that blocking MK with MK-Ab2 targeting the N-domain prevents the progression of cardiac dysfunction in this model of myocarditis, while MK-Ab4 was not beneficial.

Collectively, these results show that blocking MK with MK-Ab2 targeting the N- domain ameliorated the risk of cardiac hypertrophy, fibrosis and heart failure in an animal model of myocarditis.

It will be appreciated by persons skilled in the art that numerous variations and/or

modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.