Combination comprising a CD63-binding molecule and a drug and / or a tracer and uses thereof

Abstract

The present invention pertains to combination comprising a CD63-binding molecule and a drug and / or a tracer for use in a method of therapy and / or in a method of in-vivo diagnosis, wherein the drug and / or the tracer is delivered to a myofibroblast.

Description

[0001] Prof. Jurgen Schrader

[0002] Moorenstrasse 5. 40225 Dusseldorf

[0003] “Combination comprising a CD63-binding molecule and a drug and / or a tracer and uses thereof”

[0004] FIELD OF THE INVENTION

[0005] The present invention pertains to a combination for use in a method of therapy and / or in-vivo diagnosis, wherein myofibroblasts are targeted. In particular, the combination for use is a combination comprising a CD63-binding molecule and a drug and / or a tracer. BACKGROUND OF THE INVENTION

[0006] After myocardial infarction (Ml), the heart tissue undergoes a complex process of healing and remodeling and fibroblasts play a crucial role in this process. They are responsible for the synthesis and deposition of collagen forming the extracellular matrix that provides structural support, but over time also can lead to impaired cardiac function and finally heart failure. Fibroblasts also facilitate the healing process by promoting the formation of new blood vessels. In addition, they influence the inflammatory response that occurs after an Ml, which may result in a prolonged inflammation that contributes to pathological remodeling. Overall, fibroblasts are integral to the repair and remodeling of the heart following Ml and understanding their role and mechanisms of action can potentially lead to the development of novel diagnostic and therapeutic strategies.

[0007] After Ml, resident cardiac fibroblasts differentiate first into activated fibroblasts and then into myofibroblasts. Hence, it is possible to predict the functional consequences of Ml by detecting myofibroblasts. Myofibroblasts may also be prevalent in atrial fibrillation, a cardiac disease, wherein inflammation may contribute to the differentiation of fibroblasts into myofibroblasts.

[0008] Interestingly, myofibroblasts also play a role in tumors where they may promote angiogenesis and stimulate the expansion of tumors and thus the proliferation of cancer cells and metastasis. Myofibroblasts also differentiate from resident fibroblasts and become activated for example by cytokines secreted into the tumor microenvironment and / or by inflammation.

[0009] Hence, there is a medical need in the art to deliver a drug and / or a tracer to myofibroblasts in different diseases and pathologies. Specific targeting of myofibroblasts can be used in a method of therapy and / or used for specific imaging, thereby in a method of in-vivo diagnosis.

[0010] SUMMARY OF THE INVENTION

[0011] The present inventors have identified CD63 as a novel marker for myofibroblasts. Hence, in combination with a CD63-binding molecule, a drug and / or a tracer can be delivered to myofibroblasts, for example after myocardial infarction, in other cardiac diseases or fibrotic diseases or after activation within a tumor or tumor microenvironment.

[0012] In a first aspect, a combination comprising a CD63-binding molecule and a drug and / or a tracer for use in a method of therapy and / or in a method of in-vivo diagnosis is provided, wherein the drug and / or the tracer is delivered to a myofibroblast.

[0013] In one embodiment, the myofibroblast is a cardiac or tumor myofibroblast. For example, the myofibroblast is activated by inflammation and / or myocardial infarction.

[0014] In a further embodiment, the drug and / or the tracer is comprised in and / or coupled to a carrier and / or the drug and / or the tracer is coupled to the CD63-binding molecule.

[0015] In a further embodiment, the CD63-binding molecule is coupled to a carrier.

[0016] The carrier is for example a cell, a protein, a lipid, a glycolipid, a liposome, a nanoemulsion, for example a nanoemulsion comprising perfluorocarbons, a bead, a nanoparticle, a virus-like- particle (VLP) and a molecule, such as a sugar molecule, and any combination thereof, preferably a nanoemulsion comprising perfluorocarbons. The drug is preferably an active pharmaceutical ingredient (API), in particular a small molecule, a biological or a cytostatic, preferably the biological is selected form the group consisting of a protein, a peptide or a nucleic acid, in particular selected from the group consisting of nucleic acids encoding a desired protein such as mRNA, cDNA, a plasmid or vector, inhibitory nucleic acids such as siRNA or miRNA and nucleic acids having catalytic activity such as a ribozyme.

[0017] In one embodiment, the drug is an anti-fibrotic and / or an anti-inflammatory drug.

[0018] In one embodiment, the tracer is non-radioactive, such as19Fluorine, Gadolinium, Iron Oxide, Iron Platinum or Mangan, or radioactive, such as68Gallium,11Carbon,18Fluorine,13Nitrogen or82Rubidium.

[0019] The CD63-binding molecule is preferably a protein, a peptide, a carbohydrate, an antibody, an aptamer, a small molecule ligand and / or any combination thereof, in particular the CD63- binding molecule comprises or consists of the peptide EP1 as defined by SEQ ID NO: 1 , EP2 as defined by SEQ ID NO: 2, EP3 as defined by SEQ ID NO: 3, EP7 as defined by SEQ ID NO: 4 or EP9 as defined by SEQ ID NO: 5, preferably EP9.

[0020] In a preferred embodiment, the method of therapy and / or the method of in-vivo diagnosis comprises treating, preventing and / or in-vivo diagnosing a fibrotic disease, a cardiac disease and / or a tumor disease, such as a fibrotic tumor, more preferably myocardial infarction and / or cardiac fibrosis.

[0021] In another preferred embodiment, the method of in-vivo diagnosis comprises in-vivo imaging, preferably by positron emission tomography (PET), a fluorescence-based method, ultrasound, magnetic resonance imaging (MRI) or nuclear magnetic resonance (NMR) spectroscopy, more preferably by MRI.

[0022] The combination is preferably used as a drug delivery system.

[0023] In another preferred embodiment, the combination is used in a method of concomitant in-vivo imaging and delivery of the drug. In a second aspect of the invention, a contrast agent comprising a CD63-binding molecule and a tracer and optionally a carrier is provided.

[0024] In a third aspect of the invention, a use of the contrast agent for imaging a myofibroblast is provided, in particular wherein the contrast agent is used in MRI.

[0025] In a fourth aspect of the invention, a pharmaceutical composition comprising a CD63-binding molecule and a drug and optionally a carrier is provided.

[0026] Other objects, features, advantages and aspects of the present invention will become apparent to those skilled in the art from the following description and appended claims. It should be understood, however, that the following description, appended claims, and specific examples, which indicate preferred embodiments of the application, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following.

[0027] DEFINITIONS

[0028] As used herein, the following expressions are generally intended to preferably have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise.

[0029] The expression "comprise", as used herein, besides its literal meaning also includes and specifically refers to the expressions "consist essentially of" and "consist of". Thus, the expression "comprise" refers to embodiments wherein the subject-matter which "comprises" specifically listed elements does not comprise further elements as well as embodiments wherein the subject-matter which "comprises" specifically listed elements may and / or indeed does encompass further elements. Likewise, the expression "have" is to be understood as the expression "comprise", also including and specifically referring to the expressions "consist essentially of" and "consist of". The term "consist essentially of", where possible, in particular refers to embodiments wherein the subject-matter comprises 20% or less, in particular 15% or less, 10% or less or especially 5% or less further elements in addition to the specifically listed elements of which the subject-matter consists essentially of. The protein “CD63” belongs to the superfamily of tetraspanins which are cell surface- associated membrane proteins usually characterized by four membrane domains. A main function of CD63 is preferably to enable the internalization of partner proteins. Mechanistically, the C-terminus of CD63 contains a tyrosine-based internalization motif that confers a fast rate of endocytosis, as well as a prominent localization in late endosomes. “CD63” in particular refers to the human CD63 protein, especially the mature human CD63 protein, including all its variants, in particular splice variants as described below. CD63 as used herein especially refers to human CD63 protein according to the UniProt entry P08962. CD63 in particular comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence which is at least 90%, especially at least 95% identical to SEQ ID NO: 6 over the entire length. At least three isoforms produced by alternative splicing are known (i.e. splice variants). The first one, also identified by UniProt entry P08962-1 , is identical to the amino acid sequence as set forth in SEQ ID NO: 6. The second one, also identified by UniProt entry P08962-2, misses amino acids 23-45 with respect to SEQ ID NO: 6 and thus is defined by SEQ ID NO: 7. The third one, also identified by UniProt entry P08962-3, misses amino acids 1-82 with respect to SEQ ID NO: 6 and thus is defined by SEQ ID NO: 8.

[0030] As used herein, a "CD63-binding molecule” preferably refers to a molecule that specifically binds to CD63, for example, a protein, a peptide, a carbohydrate, an antibody, an aptamer, a small molecule ligand and / or any combination thereof. Most preferably, the “CD63-binding molecule” is a peptide, an antibody or an aptamer. A CD63-binding molecule may have one or more binding sites to CD63. If there is more than one binding site, the binding sites may be identical to one another or they may be different.

[0031] "Specific binding" preferably means that a CD63-binding molecule such as an antibody (herein also generally referred to as an agent) binds stronger to CD63 (an epitope for which it is specific) compared to the binding to another target. An agent binds stronger to a first target compared to a second target if it binds to the first target with a dissociation constant (Kd) which is lower than the dissociation constant for the second target. Preferably the dissociation constant for the target to which the agent binds specifically is more than 10-fold, 30-fold, 100- fold or more than 500-fold lower than the dissociation constant for the target to which the agent does not bind specifically. Furthermore, the term "specific binding" in particular indicates a binding affinity between the binding partners with an affinity constant Kaof at least 105M’1, preferably at least 106M’1, more preferably at least 107M’1, for example at least 108M’1. An antibody specific for a certain antigen in particular refers to an antibody which is capable of binding to said antigen with an affinity having a Kaof at least 105M’1, preferably at least 106M’1, more preferably at least 107M’1.

[0032] A “myofibroblast” typically differentiates from a resident fibroblast. Differentiation is typically induced by an event such as a myocardial infarction (Ml) or inflammation, the latter for example in a tumor or in atrial fibrillation. For example, on about day 4-7 after Ml, some or most of the resident fibroblasts have differentiated into myofibroblasts. Myofibroblasts can for example be distinguished from resident fibroblasts by one or more of the following markers: alpha-smooth muscle actin, platelet derived growth factor receptor alpha (PDGFR-a), fibroblast activation protein alpha (FAP). Some morphological features (myofibroblasts are for example described as cytoplasmic stress fiber forming contractile web-like fusiform cells) are also preferably characteristic for myofibroblasts. Optional further markers are prolyl-4-hydroxylase, periostin, fibroblast-specific protein 1 (FSP1), extra domain-A (ED-A) fibronectin, and / or vimentin. In a preferred embodiment, the “myofibroblasts” are CD63-positive myofibroblasts.

[0033] The population of “myofibroblasts” preferably does not comprise epicardium-derived stromal cells (EpiSC). In other words, a myofibroblast is not an EpiSC.

[0034] “Inflammation” in the sense of the present invention is a response to, e.g. pathogens or damaged cells. The response typically involves immune cells, blood vessels and molecular mediators, such as cytokines. Cytokines are usually pro- or anti-inflammatory, and the balance between these determines the outcome of an inflammatory response. An example for a pro- inflammatory cytokine is IL-6.

[0035] “Coupling” as used herein refers to covalent or non-covalent coupling. The coupling may be direct or indirect via for example a spacer or a linker. Coupling thereby refers to any type of binding, attachment, linkage, or interaction between two or more molecules (compounds), for example between a protein and a peptide or a peptide and a peptide or a particle and a peptide. A conjugate is for example a result of coupling. The coupling may be performed by a coupling process, for example a chemical coupling process. If coupling is performed with more than two molecules (compounds), the coupling may be in a chain conformation (one molecule attached to the next molecule), or several molecules (compounds) may be attached to one molecule (compound). A “small molecule” is preferably an organic compound of < 1000 Daltons.

[0036] A “peptide” is intended to mean two or more amino acids joined together by peptide bonds. A peptide according to the invention preferably has a maximum length of 50 amino acids. In a particular embodiment, a peptide has a maximum length of 30 amino acids, preferably a maximum length of 25 amino acids. A “protein” is intended to have a length of more than 50 amino acids. In one embodiment, a protein comprises 100 or more amino acids. A protein sequence may be defined by a GenBank entry. A protein sequence may also be defined by a UniProtKB / Swiss-Prot entry and / or by a GenPept entry. A protein may also be defined by any other database known to the skilled person.

[0037] A peptide and / or protein can be functionalized at one end or both ends and / or within the peptide and / or protein, e.g. by a His Tag. A His Tag preferably comprises two to ten histidine residues. The peptide and / or protein can also have other modifications such as one or more N- or C- terminal modifications or modifications at a side chain of an amino acid.

[0038] A “carbohydrate” in the sense of the present invention refers to a molecule consisting of carbon, hydrogen and oxygen atoms and is used synonymously with the term “saccharide”. Carbohydrates can occur as monosaccharides, disaccharides, oligosaccharides (usually three to ten monosaccharides) or polysaccharides (usually more than ten monosaccharides). In the sense of the present invention, carbohydrates include naturally-occurring carbohydrates as well as modified carbohydrates.

[0039] The term "vector" is used here in its most general meaning and comprises any intermediary vehicle for a nucleic acid which enables said nucleic acid, for example, to be introduced into prokaryotic and / or eukaryotic cells and, where appropriate, to be integrated into a genome. Vectors of this kind are preferably replicated and / or expressed in the cells. Vectors comprise plasmids, phagemids, bacteriophages or viral genomes. The term "plasmid" as used herein generally relates to a construct of extrachromosomal genetic material, usually a circular DNA duplex, which can replicate independently of chromosomal DNA.

[0040] The term "antibody" in particular refers to a protein comprising at least two heavy chains and two light chains connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The heavy chainconstant region comprises three or - in the case of antibodies of the IgM- or IgE-type - four heavy chain-constant domains (CH1 , CH2, CH3 and CH4), wherein the first constant domain CH1 is adjacent to the variable region and may be connected to the second constant domain CH2 by a hinge region. The light chain-constant region only consists of one constant domain. The variable regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR), wherein each variable region comprises three CDRs and four FRs. The antibody can be, e.g., a humanized, human or chimeric antibody.

[0041] The term "antibody" as used herein includes the full-length antibody as well as fragments and derivatives of said antibody. A "fragment or derivative" of an antibody in particular is a protein or glycoprotein which is derived from said antibody and is capable of binding to the same antigen, in particular to the same epitope as the antibody. Thus, a fragment or derivative of an antibody herein generally refers to a functional fragment or derivative. In particularly preferred embodiments, the fragment or derivative of an antibody comprises a heavy chain variable region. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody or derivatives thereof. Examples of fragments of an antibody include (i) Fab fragments, monovalent fragments consisting of the variable region and the first constant domain of each the heavy and the light chain; (ii) F(ab)2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments consisting of the variable region and the first constant domain CH1 of the heavy chain; (iv) Fv fragments consisting of the heavy chain and light chain variable region of a single arm of an antibody; (v) scFv fragments, Fv fragments consisting of a single polypeptide chain; (vi) (Fv)2 fragments consisting of two Fv fragments covalently linked together; (vii) a heavy chain variable domain; and (viii) multibodies consisting of a heavy chain variable region and a light chain variable region covalently linked together in such a manner that association of the heavy chain and light chain variable regions can only occur intermolecular but not intramolecular. Derivatives of an antibody in particular include antibodies which bind to the same antigen as the parent antibody, but which have a different amino acid sequence than the parent antibody from which it is derived. These antibody fragments and derivatives are obtained using conventional techniques known to those with skill in the art. The term “aptamer” refers to artificial nucleotide sequences (e.g. DNA or RNA) that bind to a target, for example to a peptide or protein. Aptamers typically comprise or consist of 20 to 80 nucleotides but they can also be shorter or longer.

[0042] Fibrosis is a pathologic process, which typically includes scar formation and overproduction of extracellular matrix proteins by the connective tissue and occurs in response to tissue damage and / or inflammation. Fibrosis formation preferably includes interaction between many cell types and cytokines when the balance between profibrotic and antifibrotic mediators is disturbed. Fibrosis is a feature of the pathology of a wide range of diseases across multiple organ systems. “Fibrotic diseases” in the sense of the present invention typically share an uncontrolled and progressive accumulation of fibrotic tissue in affected organs essentially causing their dysfunction and ultimate failure. Examples of fibrotic diseases are systemic fibrotic diseases such as systemic sclerosis (SSc), graft vs. host disease, and nephrogenic systemic fibrosis, as well as organ-specific disorders including radiation-induced fibrosis and cardiac, pulmonary, liver, and kidney fibrosis.

[0043] “Cardiac disease”, herein synonymously termed “cardiovascular diseases” are diseases of the heart and blood vessels and typically include (amongst others) coronary artery diseases (e.g. angina, myocardial infarction (Ml)), heart failure, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, arrhythmia, congenital heart disease, valvular heart disease, carditis, aortic aneurysms, peripheral artery disease, thromboembolic disease, and venous thrombosis.

[0044] “Myocardial infarction” (Ml) typically occurs when blood flow in one of the coronary arteries of the heart decreases or stops, for example caused by the blockage of a coronary artery which may in turn be caused by a rupture of an atherosclerotic plaque. The critical decrease or cessation of blood flow causes an infarction, i.e. death of the affected heart muscle. Typically, during Ml, resident fibroblasts differentiate into myofibroblasts.

[0045] One cardiac disease may for example be atrial fibrillation (AF). AF, a prevalent arrhythmic condition, is intricately associated with atrial fibrosis, a major pathological contributor. Central to the development of atrial fibrosis is for example myocardial inflammation, and the presence of immune cells (inflammation) may trigger the conversion of normal fibroblasts into myofibroblasts. These myofibroblasts and the resulting fibrosis may be instrumental to trigger AF.

[0046] By "tumor" is meant a group of cells or tissue that is formed by dysregulated cellular proliferation. Tumors may show partial or complete lack of structural organization and functional coordination with the normal tissue, and usually form a distinct mass of tissue, which may be either benign or malignant. A “tumor disease” preferably refers to a lesion or swelling formed by an abnormal growth of cells, i.e. tumor cells.

[0047] Tumors according to the invention in particular comprise leukemias, seminomas, melanomas, teratomas, lymphomas, neuroblastomas, gliomas, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, cancer of the brain, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, intestine cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophagus cancer, colorectal cancer, pancreas cancer, ear, nose and throat (ENT) cancer, oral cancer, bladder cancer, breast cancer, prostate cancer, cancer of the uterus, ovarian cancer and lung cancer and the metastases thereof. The term cancer according to the invention also comprises cancer metastases. The term cancer further also refers to and / or includes cancer stem cells, especially the cancer stem cells of the specific types of cancer listed above.

[0048] In the context of the invention, the term “drug delivery system” refers to a composition (comprising the present combination for use comprising a CD63-binding molecule, a drug and optionally a tracer) to be preferably administered to a subject in the need thereof, in particular to a human or animal. A drug delivery system, advantageously, enables the delivery of the drug contained therein or attached thereto to a site of interest, e.g. a selected organ, tissue, cell type, or microcompartment of a cell, etc., preferably in a human or animal. The site of interest is preferably a site with higher or concentrated CD63 expression. Preferably, the delivery is selective for the target, i.e. more of the drug is delivered to or in the target than to other sites of the body or organ.

[0049] The term “delivery” is also used in the general context of the drug and / or tracer being “delivered” to or near a myofibroblast. The “delivery” preferably enables the drug and / or tracer to be transported to a site of interest, i.e. to or in a vicinity of a myofibroblast, preferably by binding of the CD63-binding molecule to CD63 on the myofibroblast. The term “delivery” also includes that the drug and / or tracer is delivered into the myofibroblast. For example, the drug and / or tracer may bind to CD63 and subsequently be taken up by the myofibroblast. Thus, the drug and / or tracer may thereby accumulate in the myofibroblast. Thereby, the drug and / or the tracer can exert their effect at or in the myofibroblast or in their vicinity.

[0050] The term "pharmaceutical composition" particularly refers to a composition suitable for administering to a human or animal, i.e. a composition containing components which are pharmaceutically acceptable. Preferably, a pharmaceutical composition comprises an active compound or a salt or prodrug thereof together with a diluent or pharmaceutical excipient such as buffer, preservative and tonicity modifier.

[0051] The term “treatment” as used herein refers a course of action (such as administering a combination or pharmaceutical composition of the present invention) initiated after the onset of a clinical manifestation of a disease state or condition so as to eliminate or reduce such clinical manifestation of the disease state or condition. Such treating need not be absolute or entirely successful to be useful.

[0052] The term “prevention” as used herein refers to a course of action (such as administering a combination or pharmaceutical composition of the present invention) initiated prior to the onset of a clinical manifestation of a disease state or condition so as to prevent or reduce such clinical manifestation of the disease state or condition. Such preventing and suppressing need not be absolute or entirely successful to be useful.

[0053] The term “in-vivo diagnosis” as used herein refers to a course of action (such as administering a combination or pharmaceutical composition or contrast agent of the present invention) initiated prior to or after the onset of a clinical manifestation of a disease state or condition to evaluate whether such disease state or condition is present. Diagnosis also includes predicting the consequences of a disease state or condition.

[0054] “In-vitro diagnosis” does not comprise administering a combination or pharmaceutical composition or contrast agent of the present invention but instead preferably comprises providing body fluid, tissue and / or cells previously isolated from a subject, preferably a human subject, and then performing diagnostic tests on the body fluid, tissue and / or cells. The term “concomitant” in the sense of the present invention means “essentially at the same time”. In the sense of the present invention, if the combination comprises a CD63-binding molecule and both a drug and a tracer, the combination can be used for “concomitant” “in-vivo diagnosis” and “therapy” which means that both methods are performed after the administration of the combination. In that case it is preferably not necessary to separately administer another tracer or drug to perform the “in-vivo diagnosis” and “therapy”, respectively.

[0055] “Body fluids” of a subject typically include blood, urine, lymph, cerebrospinal fluid, peritoneal fluid, pleural fluid or synovial fluid or any other body fluid known in the art.

[0056] “Cells” in the sense of the present invention include cells isolated from a subject which are further processed or not further processed. Further processing includes sorting for and / or isolating a specific cell type, and / or purification of the sample including the cells. Cells may undergo cell culturing after isolation from the subject. “Tissue” typically refers to an assembly of similar cells and their extracellular matrix from the same embryonic origin that preferably together carry out a specific function.

[0057] In the sense of the present invention, a subject is preferably an animal or a human being, more preferably a human being.

[0058] DETAILED DESCRIPTION OF THE INVENTION

[0059] In the present invention, it has been found that infarct-activated cardiac myofibroblasts can specifically be visualized with 19F-MRI using a peptide-decorated nanoemulsion loaded with perfluorocarbons. This specific targeting of the infarct area was surprising. CD63 has been identified as a novel myofibroblast receptor which is in particular highly expressed on myofibroblasts after myocardial infarction (Ml). A 7-amino acid peptide termed EP9 (SEQ ID NO: 5) has a particularly strong affinity to CD63. Further, the peptide EP9 binds specifically to and is internalized by myofibroblasts after Ml. Furthermore, luciferase mRNA was efficiently taken up and expressed by myofibroblasts, in particular when the liposomes (containing the luciferase mRNA) were coupled to EP9. 1. Combination comprising a CD63-binding molecule and a drug and / or a tracer

[0060] A combination comprising a CD63-binding molecule and a drug and / or a tracer for use in a method of therapy and / or in a method of in-vivo diagnosis is provided, wherein the drug and / or the tracer is delivered to a myofibroblast.

[0061] Hence, the combination may comprise or consist of a CD63-binding molecule and a drug. Or the combination may comprise or consist of a CD63-binding molecule and a tracer. Or the combination may comprise or consist of a CD63-binding molecule, a drug and a tracer.

[0062] The combination is for example a conjugate between the CD63-binding molecule and the drug and / or the tracer, i.e. the drug and / or the tracer is for example coupled to the CD63-binding molecule. Coupling includes covalent or non-covalent coupling. In one embodiment, covalent coupling is preferred. The coupling may be direct or indirect via for example a spacer or a linker. Coupling may for example include a spacer between the CD63-binding molecule and the drug and / or the tracer. The spacer preferably allows more flexibility in binding of the CD63- binding molecule to CD63 because the drug and / or tracer is spaced apart.

[0063] In case the combination comprises or consists of a CD63-binding molecule, a drug and a tracer, the drug and the tracer may both be coupled to the CD63-binding molecule; or the CD63-binding molecule may be coupled to the drug and the drug is coupled to the tracer; or the CD63-binding molecule may be coupled to the tracer and the tracer is coupled to the drug. A combination of these embodiments is also possible.

[0064] In one embodiment, the drug and / or the tracer is comprised in and / or coupled to a carrier. Hence, in one embodiment the drug is comprised in a carrier and / or the drug is coupled to a carrier. In another embodiment, the tracer is comprised in a carrier and / or the tracer is coupled to a carrier. In another embodiment, the drug and the tracer are both comprised in a carrier (the same carrier or different carriers) and / or the drug and the tracer are both coupled to a carrier (the same carrier or different carriers). In another embodiment, the CD63-binding molecule is coupled to a carrier. A combination of these embodiments is also possible as long as the CD63-binding molecule is capable of binding to CD63. For example, in one preferred embodiment, a tracer and / or a drug is comprised in a carrier, preferably a nanoemulsion comprising perfluorocarbons, and the CD63-binding molecule is coupled to a carrier, preferably the same carrier, preferably a nanoemulsion comprising perfluorocarbons. In another embodiment, the tracer and the CD63-binding molecule are both coupled to a carrier, either directly or indirectly. Binding of the CD63-binding molecule to CD63 can be detected by standard methods.

[0065] A carrier in the sense of the present invention is preferably adapted to or is capable of carrying or transporting the CD63-binding molecule, the drug and / or the tracer. In order to carry or transport the CD63-binding molecule, the drug and / or the tracer, the carrier is preferably directly or indirectly coupled to the CD63-binding molecule, the drug and / or the tracer. The drug and / or the tracer may also be comprised in the carrier, for example partly or fully incorporated in the inside of the carrier.

[0066] In one embodiment, the carrier is a cell, a protein, a lipid, a glycolipid, a liposome, a nanoemulsion, for example a nanoemulsion comprising perfluorocarbons, a bead, a nanoparticle, a virus-like-particle (VLP) and a molecule, such as a sugar molecule, and any combination thereof, preferably a nanoemulsion comprising perfluorocarbons.

[0067] A nanoemulsion comprising perfluorocarbons, herein also termed perfluorocarbon nanoemulsions (PFC-NE), can for example be prepared by microfluidization technology. For example, phospholipid E80S (phosphatidylcholine content: approx. 70%, from soybean), perfluoro- 15-crown-5 ether, and DSPE-PEG (2000) maleimide (1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000]) are pre-emulsified in phosphate-buffered saline (PBS) using a homogenizer. Afterwards, the pre-emulsion can be processed in a high-pressure homogenizer on a Low Volume Microfluidizer. Nanoemulsions preferably contain about 20% w / w of perfluoro-15-crown-5 ether or perfluoro octylbromide (PFOB).

[0068] Preferably, the carrier is biodegradable in-vivo or is at least applicable to living subjects and broken down in-vivo or is eliminated from the subject to which the carrier is applied.

[0069] The combination of the present invention may be comprised in a composition, for example a pharmaceutical composition. Hence, the present invention also pertains to a pharmaceutical composition comprising a CD63-binding molecule and a drug and optionally a carrier. The present invention also pertains to a pharmaceutical composition comprising a CD63-binding molecule and a tracer and optionally a carrier. Preferably, the pharmaceutical composition comprises a CD63-binding molecule and a drug and optionally a carrier.

[0070] The CD63-binding molecule is preferably a protein, a peptide, a carbohydrate, an antibody, an aptamer, a small molecule ligand and / or any combination thereof.

[0071] In one embodiment, the CD63-binding molecule comprises or consists of (preferably consists of) the peptide EP1 as defined by SEQ ID NO: 1 , EP2 as defined by SEQ ID NO: 2, EP3 as defined by SEQ ID NO: 3, EP7 as defined by SEQ ID NO: 4 or EP9 as defined by SEQ ID NO: 5, preferably EP9. In another embodiment, any combination of EP1 , EP2, EP3, EP7 and / or EP9 is also possible.

[0072] The peptide sequences are also depicted in Table 1.

[0073] Table 1 : Preferred peptide sequences

[0074] In one embodiment, the CD63-binding molecule is not EP1 as defined by SEQ ID NO: 1. In one embodiment, the CD63-binding molecule is not EP2 as defined by SEQ ID NO: 2. In one embodiment, the CD63-binding molecule is not EP3 as defined by SEQ ID NO: 3. In one embodiment, the CD63-binding molecule is not EP7 as defined by SEQ ID NO: 4. In one embodiment, the CD63-binding molecule is not EP9 as defined by SEQ ID NO: 5.

[0075] In another preferred embodiment, the CD63-binding molecule is an antibody. The antibody in particular binds to CD63 and thereby enables the delivery of the drug and / or tracer of the combination to the myofibroblast. 2. Medical Use

[0076] In one embodiment, the myofibroblast is a cardiac or tumor myofibroblast, optionally the myofibroblast is activated by inflammation and / or myocardial infarction.

[0077] Hence, in one preferred embodiment a drug is delivered in particular to a cardiac myofibroblast for use in a therapy of a cardiac disease. In another preferred embodiment, a tracer is delivered in particular to a cardiac myofibroblast for use in a method of in-vivo diagnosis of a cardiac disease. In a particularly preferred embodiment, the cardiac disease is cardiac fibrosis.

[0078] In another preferred embodiment, a drug is delivered in particular to a tumor myofibroblast for use in a therapy of a tumor disease. In another preferred embodiment, a tracer is delivered in particular to a tumor myofibroblast for use in a method of in-vivo diagnosis of a tumor disease.

[0079] In another preferred embodiment, a drug is delivered in particular to a myofibroblast for use in a therapy of a fibrotic disease. In another preferred embodiment, a tracer is delivered in particular to a myofibroblast for use in a method of in-vivo diagnosis of a fibrotic disease. In a particularly preferred embodiment, the fibrotic disease is cardiac fibrosis.

[0080] Hence, the combination for use of the present invention is provided, wherein the method of therapy and / or the method of in-vivo diagnosis comprises treating, preventing and / or in-vivo diagnosing a fibrotic disease, a cardiac disease and / or a tumor disease, such as a fibrotic tumor, more preferably myocardial infarction and / or cardiac fibrosis.

[0081] The combination may also be used in a method of concomitant in-vivo imaging and delivery of the drug. In one embodiment, the combination comprises both a drug and a tracer; and a CD63-binding molecule. In another embodiment, one combination is administered comprising a CD63-binding molecule and a tracer, preferably a non-radioactive tracer, and another combination is concomitantly administered comprising a CD63-binding molecule and a drug. Thereby, “in-vivo diagnosis” and “therapy” can be performed at essentially the same time after administration of the combination.

[0082] By binding of the CD63-binding molecule to CD63, a drug and / or tracer can be specifically delivered to a myofibroblast. In a preferred embodiment, the “myofibroblasts” are CD63-positive myofibroblasts.

[0083] In one embodiment, the pharmaceutical composition of the present invention is used in a method of therapy and / or in a method of in-vivo diagnosis, wherein the drug and / or the tracer is delivered to a myofibroblast.

[0084] Preferably, the therapy or in-vivo diagnosis is initiated or performed at least two days, more preferably at least three days, more preferably at least four days, even more preferably at least five days after the onset or initiation of a myocardial infarction.

[0085] The delivery of the drug and / or tracer with the inventive combination is particularly fast, i.e. an effect can be achieved less than 30 minutes, preferably less than 10 minutes, preferably less than 5 minutes, more preferably less than 1 minute after administration of the combination, in particular if the effect relies on binding of the CD63-binding molecule to CD63 such as in imaging. In case the effect relies on subsequent uptake and potentially intracellular accumulation of the drug and / or tracer, such as for a therapeutic use of a drug, an effect can be achieved within hours or days. Hence, in case of a therapeutic use, an effect is preferably achieved less than one week, preferably less than four days, more preferably less than two days, more preferably less than 24 hours after administration of the combination.

[0086] Another advantage of the combination of the present invention is the specific therapy or specific in-vivo diagnosis of an Ml.

[0087] 2a. Therapeutic use

[0088] The combination comprising a CD63-binding molecule and a drug and / or tracer may be used in a method of therapy, wherein the drug and / or the tracer is delivered to a myofibroblast. Preferably, a combination comprising a CD63-binding molecule and a drug may be used in a method of therapy, wherein the drug is delivered to a myofibroblast.

[0089] In a particular embodiment, the CD63-binding molecule and the drug are preferably coupled to and / or comprised in a carrier, such as a liposome or specifically a nanoemulsion comprising perfluorocarbons. For example, the drug is comprised in the liposome or the phospholipids forming the nanoemulsion comprising perfluorocarbons, and the CD63-binding molecule is attached to the liposomes or the perfluorocarbons. Concomitant delivery together with a combination comprising a CD63-binding molecule and a non-radioactive tracer as disclosed herein is also possible.

[0090] In another particular embodiment, the CD63-binding peptide is preferably attached or coupled to the drug.

[0091] A drug for example includes an active pharmaceutical ingredient (API), in particular a small molecule, a biological or a cytostatic, preferably wherein the biological is selected form the group consisting of a protein, a peptide or a nucleic acid, in particular selected from the group consisting of nucleic acids encoding a desired protein such as mRNA, cDNA, a plasmid or vector, inhibitory nucleic acids such as siRNA or miRNA and nucleic acids having catalytic activity such as a ribozyme, optionally wherein the drug is an anti-fibrotic and / or an antiinflammatory drug. mRNA is preferred.

[0092] In one embodiment, the drug is mRNA and the mRNA is modified, i.e. one or more nucleotides have been deleted, changed, added and / or chemically modified. Any combination thereof is possible.

[0093] Examples for common drugs with anti-fibrotic activity include classical therapeutic targets such as TGF-B signaling pathway, adrenergic receptor system and renin-angiotensin-aldosterone pathway. More recent approaches include targeting epigenetic mechanisms in particular histone acetylase and histone deacetylase. Potential drugs are also Fibromodulin (FMOD), Slit homolog 2 protein (SLIT2), Secreted frizzled-related protein 1 (SFRP1), WNT1-inducible- signaling pathway protein 2 (Wisp 2); and / or DnaJ homolog subfamily B member 11 (DNAJB11). All proteins are selectively secreted by myofibroblasts. For example, the drugs can be selectively overexpressed in myofibroblasts by administering a combination of the present invention, wherein the drug is mRNA encoding one of the proteins above.

[0094] The drug may be delivered to or into the myofibroblast. For example, in case the drug is an mRNA, delivery into the myofibroblast enables protein expression based on the delivery of the mRNA. In a particularly preferred embodiment, the drug is an mRNA coding for an anti-fibrotic and / or anti-inflammatory drug. Hence, the combination according to the present invention may be used as a drug delivery system so that a drug is delivered to a site of interest. In particular, the binding of the CD63- binding molecule to CD63 surprisingly allows the specific targeting of the myofibroblasts.

[0095] 2b. In-vivo diagnostic use

[0096] The combination comprising a CD63-binding molecule and a drug and / or tracer may be used in a method of in-vivo diagnosis, wherein the drug and / or the tracer is delivered to a myofibroblast. Preferably, a combination comprising a CD63-binding molecule and a tracer may be used in a method of in-vivo diagnosis, wherein the tracer is delivered to a myofibroblast.

[0097] Diagnosis also includes predicting the consequences of a disease state or condition. For example, if myofibroblasts are detected by an imaging technique using the combination of the present invention, this detection may allow the conclusion, potentially together with other standard methods, that an Ml is present and may also allow the prediction of heart failure, depending on the extent of the Ml.

[0098] Tracers are preferably compounds that are used as markers to show the location of a substance or to follow the pathway of a substance, i.e. for example to show the location of CD63-binding molecule, thereby following the pathway of the CD63-binding molecule preferably to CD63. Therefore, tracers can be used in in-vivo imaging methods. Tracers may thereby also be termed imaging tracers.

[0099] In one embodiment, the tracer is non-radioactive, such as19Fluorine, Gadolinium, Iron Oxide, Iron Platinum or Mangan, or radioactive, such as68Gallium,11Carbon,18Fluorine,13Nitrogen or82Rubidium.

[0100] Non-radioactive tracers can for example be used in imaging methods such as fluorescence methods or magnetic resonance imaging (MRI) or nuclear magnetic resonance (NMR) spectroscopy. Contrast media, such as MRI or NMR contrast media, may comprise the nonradioactive tracers.

[0101] Radioactive tracers can for example be used in positron emission tomography (PET). Hence, in one embodiment the method of in-vivo diagnosis comprises in-vivo imaging, preferably by positron emission tomography (PET), a fluorescence-based method, ultrasound or magnetic resonance imaging (MRI) or nuclear magnetic resonance (NMR) spectroscopy, more preferably by MRI.

[0102] In one particular embodiment, the combination comprising a CD63-binding molecule and a non-radioactive tracer is used in a method of in-vivo diagnosis, wherein the tracer is delivered to a myofibroblast and wherein the method of in-vivo diagnosis comprises fluorescence methods or magnetic resonance imaging (MRI) or nuclear magnetic resonance (NMR) spectroscopy. Non-radioactive tracers are or comprise for example19Fluorine, Gadolinium, Iron Oxide, Iron Platinum or Mangan. Contrast media, such as MRI or NMR contrast media, may comprise the non-radioactive tracers. Myofibroblasts are preferably cardiac or tumor myofibroblasts, more preferably cardiac myofibroblasts. In this particular embodiment, the CD63-binding molecule and the non-radioactive tracer are preferably coupled to and / or comprised in a carrier, such as a liposome or specifically a nanoemulsion comprising perfluorocarbons. For example, the non-radioactive tracer is comprised in the liposome or the phospholipids forming the nanoemulsion comprising perfluorocarbons, and the CD63-binding molecule is attached to the liposomes or the perfluorocarbons. Concomitant delivery together with a combination comprising a CD63-binding molecule and a drug as disclosed herein is also possible.

[0103] In another particular embodiment, the combination comprising a CD63-binding molecule and a radioactive tracer is used in a method of in-vivo diagnosis, wherein the tracer is delivered to a myofibroblast and wherein the method of in-vivo diagnosis comprises PET. Radioactive tracers are or comprise for example68Gallium,11Carbon,18Fluorine,13Nitrogen or82Rubidium. Myofibroblasts are preferably cardiac or tumor myofibroblasts, more preferably cardiac myofibroblasts. In this particular embodiment, the CD63-binding peptide is preferably attached or coupled to the radioactive tracer.

[0104] In one embodiment, the in-vivo imaging comprises the following steps:

[0105] - administering to a subject, preferably a human subject, a combination comprising a CD63- binding molecule and a tracer,

[0106] - allowing the CD63-binding molecule to bind to CD63,

[0107] - detecting signals emitted by said tracer and - generating an image representative of the location of the tracer and / or amount of said signals.

[0108] The method of in-vivo diagnosis may also comprise the step of evaluating the generated image.

[0109] The subject is preferably known or suspected to have a fibrotic disease, a cardiac disease, a tumor, such as a fibrotic tumor, more preferably myocardial infarction and / or cardiac fibrosis.

[0110] In one embodiment, myocardial infarction is diagnosed by imaging myofibroblasts. In particular, Ml is diagnosed at least two days, more preferably at least three days, more preferably at least four days, even more preferably at least five days after the onset or initiation of a myocardial infarction. In other words, it can be diagnosed that an Ml has occurred by imaging myofibroblasts.

[0111] The in-vivo imaging may also be performed by using a contrast agent. Hence, in one embodiment a contrast agent is provided comprising a CD63-binding molecule and a tracer, preferably a non-radioactive tracer, and optionally a carrier. Use of such contrast agent for imaging a myofibroblast is also provided, in particular wherein the contrast agent is used in MRI.

[0112] A contrast agent is preferably used in a method of imaging such as ultrasound or MRI or NMR.

[0113] 3. In-vitro diagnostic use

[0114] In one embodiment, a method of in-vitro diagnosis of a fibrotic disease, a cardiac disease and / or a tumor disease is provided comprising the following steps: providing body fluid, tissue and / or cells previously isolated from a subject, preferably a human subject, adding a CD63-binding molecule, and detecting the binding of the CD63-binding molecule to CD63.

[0115] Preferably, by detecting the binding of the CD63-binding molecule to CD63, a myofibroblast can be detected. The person skilled in the art knows various methods how to detect the binding, for example by binding a fluorescently marked antibody directed against the CD63- binding molecule and detecting the fluorescence. This method also allows an in situ detection. Providing the cells may also comprise culturing the cells before providing them.

[0116] The subject is preferably known or suspected to have a fibrotic disease, a cardiac disease, a tumor, such as a fibrotic tumor, more preferably myocardial infarction and / or cardiac fibrosis.

[0117] FIGURES

[0118] Figure 1 : Coupling of peptide EP9 with a perfluorocarbon-containing nanoemulsion (PFC-NE). A: General reaction scheme of preparing EP9-decorated PFC-NE. Pre-manufactured nanoemulsions, composed of phospholipids that encapsulate perfluoro-15-crown-5 ether (19F), were PEGylated (PEG2000) on the surface and in addition DSPE-PEG (2000) maleimide (mal) was attached. EP9 contains a cysteine amino acid leaving on its end a reactive thiol (SH) group. B: In an aqueous buffer, the maleimide and the thiol react and form a stable thioether bond, in this way firmly coupling the peptide to the nanoemulsions.

[0119] Figure 2: Targeting of EP9-PFC-NE to the infarct heart as visualized in-vivo and ex-vivo by1H / 19F MRI.

[0120] A: Anatomically corresponding representative in-vivo1H and19F images from the mouse thorax recorded on day 6 after Ml showing accumulation of19F signal within the infarcted region (upper row). The EP9-targeted nanoemulsion was injected on day 5 after Ml. Images in the lower row are from the control experiments with MUT-PFC-NE. Images are representative from 6 in-vivo experiments. B: Evaluation of in-vivo imaging data showed that the19F signal within the infarcted heart was EP9-specific (n=3, unpaired t test, *p<0.05). C: Time course of the19F signal intensity in circulating blood after intravenous injection of EP9-PFC-NE showed a decrease over time with a calculated half-life of 20 hours. SNR, signal-to-noise ratio. D: In-vitro1H and19F images of explanted hearts taken 12 vs 24 hours after injection of EP9-PFC-NE showed an increase of signal intensity over time.

[0121] Figure 3: Cellular EP9-PFC-NE uptake into cells of the infarcted heart evaluated by transmission electron microscopy (TEM).

[0122] A: Representative images of the experiments show fibroblast-like cells (myofibroblasts) heavily loaded by PFC-NE within the infarcted area of the heart, 5 days following injection of EP9- NEs. Fibroblasts of the non-infarcted heart did not show uptake of EP9-PFC-NE. B: Cells of the epicardial layer contained PFC-NEs under condition described in (A); no PFC-NE were found in cardiomyocytes. Blue dashed line: border between cardiomyocytes and epicardial cell layer. C: Representative monocyte containing no visible PFC-NEs. D: Size distribution of injected PFC-NEs measured by dynamic light scattering (DLS) as compared to size distribution assessed by TEM was similar. (n=6).

[0123] Figure 4: In-vitro peptide binding to various mouse and human cells.

[0124] A: Freshly isolated fibroblasts bound EP9 but highest binding was observed in epicardial stromal cells (EpiSCs) from infarcted mouse hearts. (n=3) B: Among the human cells, primary fibroblasts (myofibroblasts) isolated from transplanted hearts showed highest binding as compared to dermal fibroblasts and monocytes. (n= 3) MFI, mean fluorescence intensity.

[0125] Figure 5: Receptor identification of EP9 by photoaffinity labeling.

[0126] A: Structure of the synthetized trifunctional probe exibiting the following functional groups: diazirine moiety for receptor capture upon UV light irradiation, maleimide for peptide coupling through reaction with thiol, and biotin for affinity purification with streptavidin. B: Volcano plot of the transmembrane proteins identified several proteins and CD63 was the most prominent receptor. C: The protein abundance measured by the Intensity Based Absolute Quantitation (iBAQ) of the CD63 protein was high in the EP9 probe samples, but negligable in the MUT probe samples as control.

[0127] Figure 6: CD63 expression in freshly isolated cardiac cells evaluated by flow cytometry.

[0128] A: Fibroblasts, immune cells and endothelial cells were isolated from non-infarcted (control) hearts and after myocardial infarction (Ml) (50 min ischemia / reperfusion) 5 days after Ml by a fast enzymatic procedure and analyzed by flow cytometry (n= 4, t test, ***P < 0.001). In infarct- activated fibroblasts (i.e. myofibroblasts) CD63 was significantly upregulated. Cells isolated from the myocardial tissue were considered to be cardiac fibroblasts when PDGFR-O+ / CD31- / CD45- , endothelial cells when CD31+, and immune cells when CD45+. Gating strategy is shown in Figure 7. B: Representative histograms of the experiments shown in A) comparing CD63 expression between the cell populations of infarct-activated myofibroblasts, endothelial and immune cells from infarcted hearts.

[0129] Figure 7: Gating strategy in flow cytometric analysis of cells isolated from control and infarcted hearts. A: Fibroblasts (CD31-, CD45-), endothelial cells (CD31+) and immune cells (CD45+) were gated with the appropriate FSC and SSC settings. B: The PDGFR-O+ (CD140a+) population of fibroblasts (myofibroblasts) was identified. CD63 was quantified for PDGFR-O+ fibroblasts (CD140a+, CD31-, CD45-), endothelial cells and immune cells populations as shown in Fig. 6A.

[0130] Figure 8: Gene expression analysis of CD63 and FAP in post-MI hearts

[0131] A: Expression of CD63 at the gene level was analyzed in the infarcted heart (50 min ischemia / reperfusion; 5 days post Ml) using mouse scRNAseq data previously published (Hesse, J. et al. (2021); Single-cell transcriptomics defines heterogeneity of epicardial cells and fibroblasts within the infarcted murine heart', eLife 10: e65921). B: Expression profile of fibroblast activation protein (FAP) using the same scRNAseq data as under A. In all data shown, the fibroblast subpopulation CF_1-9 refers to myofibroblasts of the infarcted heart.

[0132] Figure 9: Protein expression of CD63 and FAP in healthy and failing human heart

[0133] A: Unsupervised Uniform Manifold Approximation and Projection (UMAP) embedding plot with weighted nearest neighbor clustering (RNA and protein) of CITE-seq data clustering from 22 patients and 143,804 cells. B: Violin plots generated from the integrated dataset displaying the CD63 and FAP marker genes in the cardiac fibroblast cell population in 6 healthy donors (Donor), 4 acute Ml patients (AMI, <3 months post-MI), 6 ischemic cardiomyopathy (ICM, >3 months post-MI) patients, and 6 non-ischemic cardiomyopathy (NICM, idiopathic dilated cardiomyopathy) patients.

[0134] Figure 10: Expression of luciferase in control cardiac fibroblasts (cCFs) and infarct activated fibroblasts (miCF)

[0135] Luminescence of fibroblasts incubated with EP9- or mutated EP9-coupled liposomes containing luciferase mRNA after exposition to a luciferase substrate.

[0136] EXAMPLES

[0137] Example 1 : In-vivo labeling of cardiac cells post Ml with an EP9-targeted nanoemulsion The potential of EP9 (SEQ ID NO: 5) for in-vivo imaging of the post-MI mouse heart was assessed. To this end, EP9 was coupled with a19F-containing nanoemulsion (EP9-PFC-NE) designed for the in-vivo labeling of cardiac cells (Figure 1). Five days post Ml (50 minutes of ischemia / reperfusion), 150 pl of the targeted nanoemulsion was administered into the tail vein. A mutated EP9 (MUT-PFC-NE; SEQ ID NO: 10; cf. Table 1) served as control. Twenty-four hours following nanoemulsion injection, in-vivo visualization using1H / 19F-MRI was performed. As shown in Fig. 2A and B, a strong19F signal was found to be closely associated with the infarcted area after EP9-PFC-NE injection. In contrast, the19F signal in the MUT-PFC-NE control group was significantly reduced (Fig. 2A, B) and lacked specific localization in the heart.

[0138] To investigate the temporal changes of the19F signal following injection of EP9-PFC-NE, both the accumulation of19F in explanted hearts and the changes in the19F signal in the blood after injection over time were assessed. Representative images (of six in-vivo experiments) merged1H / 19F data sets, shown in Figure 2D, demonstrate that the19F signal was transmurally distributed within the infarcted area and increased over time (12 and 24 hours after EP9-PFC- NE injection). As illustrated in Fig. 2C, the19F signal measured in blood after intravenous injection only slowly declined with a calculated half-life of about 20 hours. The extended availability of the EP9-PFC-NE in the blood stream can explain the continuous increase of the19F signal in the infarcted heart over time (Fig. 2D). Hence, the infarcted tissue has been identified as primary site of labeling.

[0139] Example 2: Identification of cardiac cells which preferentially take up EP9-PFC-NE

[0140] It was next aimed to identify the cardiac cell type(s) which preferentially has taken up EP9- PFC-NE. To this end, transmission electron microscopy (TEM) was performed on the paraformaldehyde-fixed tissue samples that were prepared to visualize ex-vivo the1H / 19F signal (see above). Tissue samples from various locations (non-infarcted, infarcted, border zone, epicardium) of the left ventricle were analyzed 6 days after Ml and 1 day after EP9-PFC- NE injection.

[0141] As shown in Fig. 3A, TEM images revealed that in particular the elongated cells in the interstitial space between cardiomyocytes, phenotypically resembling cardiac fibroblasts, contained numerous intracellular vesicles. These cells in the infarcted heart comprise myofibroblasts which are known to differentiate from resting fibroblasts after Ml. The average size of the vesicles was measured to be 118 ± 47 nm (n=10) (Fig. 3D), which closely resembled the distribution of EP9-PFC-NE determined by dynamic light scattering (DLS): 124 ± 49.2 nm (Fig. 3D). Vesicles of similar size were also found in the cells which constitute the epicardial layer (Fig. 3B). In macrophages and cardiomyocytes examined by TEM, nanoemulsion-derived vesicular structures were not found (Fig. 3A,B,C). Together these observations indicate that EP9-PFC-NE were preferentially taken up by cardiac myofibroblasts.

[0142] Example 3: Cellular specificity of peptide binding

[0143] To further explore the cellular specificity of EP9 and other peptides, binding of these fluorescently labeled peptides was assessed to a broader spectrum of cells derived from both mice and humans (evaluated by flow cytometry after 30 minutes incubation in-vitro). As summarized in Figure 4A, all five small peptides exhibited binding to cultured cardiac fibroblasts and EpiSC from mice. Notably, EP9 consistently showed superior binding to EpiSC compared to the other peptides. As illustrated in Figure 4B, EP9 displayed high binding in primary human cardiac myofibroblasts isolated from a heart sample of a patient with terminal heart failure undergoing heart transplant surgery. Similarly, binding to a human dermal fibroblast cell line (NHDF) was found, albeit to a lesser extent, while no discernible binding was observed in human monocytes (THP1).

[0144] Example 4: Identification of the cellular receptor for EP9

[0145] To identify the cellular receptor for EP9, most likely a transmembrane protein, a trifunctional probe was employed, including a diazirine photolinker, a maleimide reactive site for peptide binding, and biotin for purification (see Fig. 5A). The ligand receptor capture experiment (LRC) was conducted on NHDF cells under conditions optimal for effective peptide-cell binding which enabled the identification of captured proteins by mass spectrometry. Using the trifunctional probe in the LRC experiments, 35 proteins were identified in either the targeted peptide samples (probe coupled to EP9, EP9 probe), the untargeted control (probe coupled to mutated peptide, MUT probe) samples, or both. By comparing the proteins captured by the EP9 probe and the MUT probe, EP9-specific proteins were identified. The volcano plot shown in Fig. 5B lists the identified transmembrane proteins that were enriched in the EP9 and MUT samples. Notably, CD63 emerged as the most significantly enriched protein in the EP9 samples (Figure 5B,C) while the signal was notably absent in the majority of MUT samples (Figure 5C). Together the findings strongly support the conclusion that CD63 is the primary candidate for the receptor of EP9.

[0146] In separate experiments, the expression of CD63 on cardiac fibroblasts, endothelial cells and immune cells isolated from infarcted and healthy (control) mouse hearts was analysed by flow cytometry. As shown in Fig. 6A, CD63 was significantly increased in cardiac fibroblasts after myocardial infarction, i.e. in myofibroblasts, while there was no difference in endothelial cells and immune cells between the Ml and control. Histograms of the experiments are shown in Fig. 6B. The gating strategy from the FACS analysis is shown in Figure 7.

[0147] Example 5: Comparison of gene expression of FAP and CD63 using mouse data

[0148] As shown in Figure 8, CD63 was highly expressed in most cardiac fibroblast (CF) cell populations, pericytes and epicardial stromal cells (EpiSC) compared to the relatively low presence in T cells and B cells. FAP also showed strong expression in all other cardiac fibroblast subpopulations, with little expression in epicardial cells / EpiSC. In general, the expression of CD63 was about 50-fold higher as compared to FAP.

[0149] Example 6: Comparison of protein expression of FAP and CD63 using human data

[0150] Expression of CD63 and FAP (CITE-seq) was analyzed in healthy and failing human heart using data previously published (Amrute, J. M. et al. (2023); Targeting Immune-Fibroblast Crosstalk in Myocardial Infarction and Cardiac Fibrosis. Res. Sq. rs.3.rs-2402606). CD63 is highly and preferentially expressed in the cardiac fibroblast fraction and exceeds the protein expression of FAP (Figure 9). Remarkably, CD63 in cardiac fibroblasts was significantly increased in the acutely infarcted heart and other cardiac pathologies, while changes in FAP were generally less pronounced.

[0151] Example 7: Expression of luciferase in control cardiac fibroblasts (cCFs) and infarct activated fibroblasts (miCF)

[0152] Fibroblasts (control cardiac fibroblasts (cCFs) and infarct activated fibroblasts (miCF)) (i.e. myofibroblasts) were incubated with EP9- or mutated EP9-coupled liposomes containing luciferase mRNA, and luminescence was measured after exposition to a luciferase substrate. As can be seen in Figure 10, luciferase mRNA was efficiently transported to miCFs and expressed therein, when the liposomes were coupled to EP9.

[0153] Materials and Methods

[0154] Synthesis of targeted peptides and control peptides

[0155] Five small peptides (EP1 , EP2, EP3, EP7, EP9) composed of 7 amino acids (Table 1 ; SEQ ID NOs: 1-5) were synthesized commercially (Genaxxon Bioscience GmbH, Ulrn, Germany). The fluorescent dye carboxyfluorescein (6-FAM) was coupled to the e-amino group of the lysine of a linker with the sequence GGGKC (SEQ ID NO: 11), i.e. (GGGK(FAM)C), to enable flow cytometry and fluorescence imaging. The initial glycine of the linker was used for conjugation to the terminal amino acid of EP9, and the terminal cysteine of the linker was used for conjugation to the perfluorocarbon nanoemulsion. The EP9 sequence (SEQ ID NO: 5) was used as a basis for scrambled and mutated peptides. As depicted in Table 1 (SEQ ID NOs: 9- 10), the amino acid positions were randomly interchanged and lysine was changed for glycine to obtain the scrambled peptide. The mutated peptide was obtained by changing the proline residue for glycine and alanine.

[0156] Animal experiments

[0157] Animal protocols were approved by the Federal State Office for Nature, the Environment and Consumer Protection of North Rhine Westphalia (l_ANUV) (Reference number: AZ SI- 02.04.2019. A466). For this study, male mice C57BL / 6J (Janvier Labs, Le Genest-Saint-lsle, France) were used with body weight: 20-25 g and age: 8-12 weeks. Mice were housed at the central animal facility of the Heinrich Heine University Dusseldorf (ZETT, Dusseldorf, Germany) on a 12 h light / dark cycle and were fed with a standard chow diet and received tap water ad libitum. Ischemia / reperfusion was induced as described in Bonner, F. et al. 2012 (Resident Cardiac Immune Cells and Expression of the Ectonucleotidase Enzymes CD39 and CD73 after Ischemic Injury; PLoS ONE 7: e34730) In brief, mice were anesthetized (isoflurane 1.5% via respiration), and the left anterior descending coronary artery was ligated for 50 minutes followed by reperfusion. Left anterior descending coronary artery occlusion was ensured by ST-segment elevation in ECG recordings. Mice were kept under analgesic treatment for 3 days (buprenorphin 0.1 mg / kg, 3 times / day).

[0158] Preparation of the perfluorocarbon nanoemulsions (PFC-NE) Perfluorocarbon nanoemulsions (PFC-NE) were manufactured as described in Kramer, W. et al. 2019 (Rational manufacturing of functionalized, long-term stable perfluorocarbon- nanoemulsions for site-specific 19F magnetic resonance imaging-, Eur J Pharm Biopharm 142: 114-122) by microfluidization technology. In brief, phospholipid E80S (phosphatidylcholine content: approx. 70%, from soybean), perfluoro- 15-crown-5 ether, and DSPE-PEG (2000) maleimide (1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-

[0159] [maleimide(polyethyleneglycol)-2000]) were pre-emulsified in phosphate-buffered saline (PBS) (Roth, Karlsruhe, Germany) for 5 minutes using an homogenizer (Ultra-Turrax, IKA T18 basic; IKA Works, Wilmington, USA). Afterwards, the pre-emulsion was processed in a high- pressure homogenizer on a Low Volume Microfluidizer (Microfluidics, Westwood, USA) for 9 cycles at 16,000 psi (=1 ,000 bar). Nanoemulsions contained 20% w / w of perfluoro- 15-crown- 5 ether.

[0160] Preparation of targeted PFC-NE

[0161] To generate PFC-NE for the in-vivo targeting experiments, EP9 (KLMLPRP; SEQ ID NO: 5) was coupled with a linker (SEQ ID NO: 11) attached to FAM to result in the targeted nanoemulsion EP9-PFC-NE. The mutated peptide (SEQ ID NO: 10) was used as control (MUT- PFC-NE). Briefly, 25 pg of peptide was incubated with 100 pl of the PFC-NE previously prepared overnight. As depicted in Fig. 1 , peptides were coupled to pre-formed PFC-NE, which carry a maleimide group on their surface. This group is part of DSPE-PEG(2000) maleimide, a molecule containing on one side a lipid that attaches to the lipid layer and on the other side exposing the reactive group. The peptide composed of EP9 and the linker (thus forming a peptide with SEQ ID NO: 12) comprises a cysteine amino acid with a reactive thiol group. In aqueous buffer, the maleimide and the thiol groups react and form a stable thioether bond, firmly coupling the peptide to the nanoemulsions surface as shown in Fig. 1 B.

[0162] Size distribution analysis of PFC-NE

[0163] To determine the hydrodynamic diameter of the PFC-NE, 20 pl of the PFC-NE were diluted in 980 pl of milliQ water and analyzed by dynamic light scattering (DLS; Nanotrac Wave II, Microtrac MRB, Haan, Germany). This technique also revealed the polydispersity index (PDI) and the ^-potential. In-vivo imaging of infarcted hearts by cardiac MR!

[0164] 19F-MRI was used to investigate whether cardiac cells can be noninvasively visualized after Ml. For this purpose, 150 pl of EP9-PFC-NE were injected intravenously 5 days after Ml (50 minutes ischemia / reperfusion). In-vivo visualization by1H / 19F-MRI was carried out 24 hours after EP9-PFC-NE injection on a Bruker 9.4 Tesla AVANCEIII WB spectrometer (Bruker BioSpec, Ettlingen, Germany). ParaVision 5.1 was used to drive the instrument, which ran at frequencies of 400.21 MHz for1H measurements and 376.54 MHz for19F measurements. A 25-mm quadrature19F resonator with one channel adjustable to both1H and19F was used to collect the data. Mice were housed at 37 °C and anesthetized with 1.5% isoflurane. After morphological1H pictures were acquired, the resonator was tuned to19F and anatomically compatible19F images were captured as reported in Fidgel, II. et al. 2021 (Multi-targeted 1H / 19F MR! unmasks specific danger patterns for emerging cardiovascular disorders’, Nat Commun 12: 5847).

[0165] Ex-vivo MR!

[0166] For ex-vivo1H / 19F-MRI, in-vivo tissue fixation was performed by cardiac perfusion with paraformaldehyde (4%). Briefly, mice are anesthetized with Thiopental. Thereafter, the thorax was opened to expose the heart, and the left ventricle was punctured with a needle connected to the perfusion solutions while a small incision was done in the right atrium for venous outflow. Hearts were first perfused with 10 ml of PBS with a speed of ~1 ml / 10 s, followed by 3 ml of paraformaldehyde 4%. The explanted mice hearts were kept in paraformaldehyde (4%) until further processed.

[0167] For the1H / 19F-MRI, the hearts were embedded in agarose within a 2 ml-vial, which was carefully fixed in the center of a 25-mm birdcage resonator tunable to1H or19F. After acquisition of morphological1H images, the resonator was tuned to19F, and anatomically matching19F images were recorded by using a RARE sequence (RARE factor 32, 25.6 x 25.6 mm2, matrix 64 x 64 x 13, resulting in an in-plane voxel size of 0.4 x 0.4 mm2after zerofilling; slice thickness, 1 mm; TR 7500 milliseconds; TE, 6.98 milliseconds; 2500 averages; acquisition time, 10 hours and 25 minutes).

[0168] Transmission electron microscopy

[0169] Transmission electron microscopy (TEM) was performed to further confirm the localization and cellular uptake of PFC-NE. Sections of the explanted hearts were cut in small pieces of approximately 1 mm3. Tissue samples were first fixed in 4% paraformaldehyde, 2% glutaraldehyde in 0,1 M cacodylate buffer, pH 7,4, at 4°C overnight and then washed 3 times with 0.1 M cacodylate buffer. After that the samples were treated with 1 % osmium tetroxide for 60 minutes. The tissue blocks were dehydrated in a graded series of acetone, starting with 30% and increasing 10% until reaching to 100%. Samples were stained for 1 hour in 1 % uranylacetate and 0.5% phosphotungstic acid in 70% acetone. The rest of the dehydration was completed and the samples were embedded in Spurr's medium for ultrathin sections (70 nm; Ultracut). After being stained with uranyl acetate and lead citrate, the slices were examined by electron microscopy (TEM Hitachi H7100, Tokyo, Japan, http: / / www.hitachi.com) and digital images were acquired.

[0170] For the assessment of individual heart sections, samples of about 1 mm2with a depth of 70 nm were analyzed. The sample was placed on a grid with quadratic mesh and was imaged with a TEM JEOL JEM 2100plus. To reduce bias, ten arbitrarily chosen sites of each sample were selected, taking care that they were broadly spread across the sample. Each of the selected sites were imaged in high quality (stack of 70 slides per site) and each stack was systematically analyzed for the occurrence of liposome-derived nanoparticles in anatomically defined cell types.

[0171] Cell isolation and cultivation of mouse Epi SC and cardiac fibroblasts

[0172] For cell isolation, hearts were harvested 5 days after Ml (50 minutes ischemia / reperfusion). The isolation and cultivation of activated cardiac fibroblasts and EpiSC were performed by a Langendorff-based technique as described (Owenier, C. et al. 2020; Novel technique for the simultaneous isolation of cardiac fibroblasts and epicardial stromal cells from the infarcted murine heart', Cardiovasc Res 116: 1047-1058). In brief, hearts were rinsed with 3 ml (2 ml / min) of PBS via the coronary arteries, followed by digestion with collagenase type 2 (Worthington Biochem, Freehold, NJ, USA)-containing solution (1 ,200 U / ml) at 37 °C in <10 minutes. Digestion was performed by simultaneous perfusion with and immersion in collagenase solution under gentle rocking to obtain fibroblasts from the myocardium and EpiSC from the surface of one heart.

[0173] The EpiSC-containing cell suspension collected from the heart surface was centrifuged twice (55 *g, 2 min; 300 xg, 7 min) and the cells were resuspended in EpiSC culture medium [DM EM (high glucose; Sigma Aldrich Chemie GmbH, Munich, Germany) with 30% fetal bovine serum (FBS; Biochrom GmbH, Berlin, Germany), 1% sodium pyruvate (100 mM; Invitrogen GmbH, Meerbusch, Germany), 1 % penicillin-streptomycin (Biochrom GmbH, Berlin, Germany)]. EpiSC were plated in cell culture flasks (T75; Greiner Bio-One GmbH, Frickenhausen, Germany).

[0174] For the isolation of cardiac fibroblasts, collagen-treated hearts (see above) were removed from the cannula and were further mechanically dissociated. The resulting cell suspension was collected, passed through a 100 pm cell strainer, and centrifuged (55 x g, 1 min). Afterwards, the supernatant was passed through a 40 pm and centrifuged (300 x g, 7 min). Subsequently, magnetic bead depletion with Mojosort nanobeads (BioLegend, San Diego, CA, USA) was performed for CD31+ cells (endothelial cells) and CD45+ cells (immune cells). Remaining cells (fibroblasts) were resuspended in fibroblast culture medium [DM EM (high glucose) with 20% FBS, 1% penicillin-streptomycin and plated on cell culture dishes. After one day of culture in an incubator at 37 °C and 5% CO2 in a humidified atmosphere, cells were washed with PBS and fresh medium was added. For prolonged cultivation, the medium was replaced every 2 days, and cells were split at a confluence of about 80%.

[0175] Analysis of binding of peptides to different cell lines

[0176] The following cells were used to study in-vitro binding of peptide-targeted PFC-NE: mice EpiSC and mice cardiac fibroblasts prepared as described above, Normal Human Dermal Fibroblasts (NHDF) Juvenile foreskin, and human monocytes (THP-1). In addition, primary human cardiac fibroblasts were used which were prepared by explant culture technique from a heart sample of a patient with terminal heart failure undergoing orthotopic heart transplant surgery. In brief, a tissue sample weighing about ~5 g was removed from the failing heart's apex and placed in cooled BIOPS buffer to be divided into tiny pieces for subsequent culture to allow cell outgrowth in a 6-Well plate with DMEM (low glucose), 20% FBS, and 1% penicillin-streptomycin. The medium was replaced every 2 days and biopsy pieces were discarded after 5 days. After 14 days, cells reached confluence of about 80% and were then passed to T75 cell culture flasks.

[0177] The binding assay was performed as follows. Cells were detached with PBS-edtA (5 mM) for 10 minutes at 37°C, and centrifuged for 5 minutes at 500g. Afterwards 5 ug of the tested peptide was added to 800 uL (0.5 x 106cells) of the studied cells and incubated for 30 minutes. Afterwards, 200 uL of cells were taken up in 2 mL MACS buffer (2 % FCS and 1 mM EDTA in PBS) and centrifuged for 5 minutes at 500 g and supernatant was removed. One additional washing step was performed with 200 uL of MACS buffer and centrifuged again for 5 minutes at 500 g, the supernatant was removed. The sample was resuspended in 200 pl of 4'6- diamidino-2-phenylindole dichlorhydrate (DAPI) (Merck KgaA, Darmstadt, Germany) (1 pg / ml) to allow discrimination of dead cells.

[0178] Flow cytometric analysis was using a BD FACS Canto II flow cytometer (BD Biosciences, Franklin Lakes, NJ). To control the specific targeting of the selected peptide sequence, scrambled and mutated peptides were used as negative controls.

[0179] Receptor Identification and synthesis of the diazirine photolinker

[0180] For the identification of a possible binding partner for EP9, a trifunctional molecule (diazirine photolinker) was synthetized, combining a maleimide moiety for ligand-coupling, a diazirine moiety for the receptor capture after the irradiation with UV light, and a biotin group for affinity purification using streptavidin (see Fig. 5A). The biotin-labeled diazirine OSu-ester was prepared according to a procedure by Muskens, FM et al. 2019 (Design, synthesis, and evaluation of a diazirine photoaffinity probe for ligand-based receptor capture targeting G protein-coupled receptors, Mol Pharmacol 95: 196-209) from 3-(4-(3-(trifluoromethyl)-3H- diazirin-3-yl)phenyl)propanoic acid and 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1 -amine. Prior to OSu ester formation the free acid was purified by flash column chromatography on silica gel (dichloromethane / MeOH (15%) / acetic acid (2%)). From this, the OSu-ester was obtained by HATLI coupling followed by acidic workup as reported (Muskens, FM et al. 2019) and used without further purification. Identity and purity of all compounds were assessed by spectroscopic analysis (NMR, ESI-MS).

[0181] Ligand-receptor capture experiments using photoaffinity labeling

[0182] The diazirine photolinker (1 mM) was coupled to EP9 (1 mM) by incubation for 1 hour at room temperature to obtain the EP9 probe for the ligand-receptor capture (LRC) experiments. In parallel, the mutated peptide was used as a control ligand to control for unspecific binding (MUT probe). LRC experiments were performed according to the procedure by Muskens, FM et al. 2019 with modifications as follows: The experiment was made on NHDF cells that were grown in 150 mm-cell culture dishes with DM EM medium and 10% FBS to < 80% confluence. Cells were washed three times with 5 ml PBS to remove medium. The probe (EP9 probe or MUT probe) was diluted in Hank's balanced salt solution (HBSS) (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) to a final concentration of 1 pM and 6 ml were added directly to the NHDF cell dishes. After incubation in the dark under constant agitation in a VWR Rocking Platform 444-0142 for 30 minutes at 37°C, the cell dishes were then placed on ice and exposed to light of 365 nm-100 W (Everbeam, Surrey, CA, USA) for 30 minutes. For subsequent cell lysis, cells were washed with PBS and 1 ml / dish of lysis buffer (1X Protease inhibitor, 1%SDS in 50 mM Tris HCI at pH=8) was added. The lysates obtained from the previous step were vortexed for 5 minutes and centrifuged at 16,000 xg at 4°C for 5 minutes. The supernatant was recovered and frozen at -20°C. All further steps were performed at 4°C and buffers were supplemented with complete EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics, Mannheim, Germany). The complete lysate of a dish was added to 250 pl of Dynabeads M-280 Streptavidin (Invitrogen GmbH, Meerbusch, Germany), and the total volume was filled up to 3 ml using PBS supplemented with 1 % (v / v) NP40. The beads were incubated for 18 hours in the dark at 4°C. Afterwards the beads were washed four times with radioimmunoprecipitation assay buffer (RIPA buffer, Sigma-Aldrich), four times with PBS supplemented with 880 mM NaCI, and four times with PBS.

[0183] After pull down, the samples were washed sequentially with 1 ml PBS with 5% acetonitrile (ACN) and 1 ml HPLC-grade water, each for 5 minutes with end-over-end rotation at 4°C. Subsequently, the precipitated proteins were reduced, alkylated, and digested with 5 mM Tris(2-carboxyethyl) phosphin-hydrochlorid (TCEP), 27.5 mM chloroacetamide (CAA), and 10 ng / pl Lys-C, respectively, in 8 M Urea and 50 mM ammonium bicarbonate (ABC) for 3 h at room temperature. Afterward, samples were diluted with ABC to a final urea concentration of 2 M and subjected to trypsin digest overnight with a final concentration of 10 ng / pl at room temperature and 1000 rpm on a thermo shaker to prevent settling of beads. Peptides were then desalted and stored on SDB-RPS (styrenedivinylbenzene resin) stage tips until measurement.

[0184] LC-MS / MS Analysis

[0185] Peptides were eluted with 2% ammonium hydroxide and 80% ACN and dried in a vacuum concentrator. For measurement, dried peptides were resuspended in 2% ACN and 5% formic acid. Samples were measured on a Thermo QExactive PLUS instrument, coupled to a Thermo easy nLC 1000 instrument using a 60 min reverse phase elution gradient. Raw files were analyzed with MQ v2.0.1.0 using Uniprot KB database including reviewed and unreviewed entries as well as protein isoforms. Further analysis was done in Perseus v1.6.14.0 and InstantClue vO.12.2. CD63 expression analysis by flow cytometry

[0186] Cardiac cells were isolated by Langendorff-based procedure from hearts of infarcted and healthy mice as disclosed in Owenier, C. et al. (2020) and as described in the previous section. All cardiac cells were immediately resuspended after isolation without the separation of cell fractions in MACS buffer with the antibodies CD190, CD31 , CD45, and CD63 (Miltenyi Biotec, Bergisch Gladbach, Germany) , diluted 1 : 100) and incubated for 20 minutes at 4°C. Cells were centrifuged for 5 minutes, the supernatant was discarded and to omit dead cells, samples were stained with 1 pg / ml DAPI (Merck KgaA, Darmstadt, Germany). Flow cytometric analysis was using a BD FACS Canto II flow cytometer (BD Biosciences, Franklin Lakes, NJ).

[0187] Single-cell / single-nucleus RNA sequencing data analysis

[0188] Single-cell RNA sequencing (scRNAseq) and single-nucleus RNA sequencing (snRNAseq) data were processed using the R Seurat package26 (v3.0). For comparison of gene expression among cardiac cell types, a snRNAseq data set of cardiac cells isolated from mouse hearts analyzed 5 days post Ml (50 min ischemia / reperfusion) was used. Names of CF populations were annotated according to expression of CF population markers on basis of Farbehi, N. et al. (2019) (Single-cell expression profiling reveals dynamic flux of cardiac stromal, vascular and immune cells in health and injury. eLife 8: e43882). To assess gene expression of CD63 and FAP in the infarcted heart (50 min ischemia / reperfusion; 5 days post Ml) mouse scRNAseq data previously published were reanalyzed.

[0189] Protein expression of FAP and CD63

[0190] Cellular Indexing of Transcriptomes and Epitomes by sequencing (CITE-seq) on freshly explanted human hearts and Multiomic (paired single nucleus RNA + assay for transposable accessible chromatin, ATAC) sequencing from non-diseased donors, acutely infarcted, chronic ischemic, and non-ischemic cardiomyopathy patients. A panel of 279 antibodies was used including FAP and CD63 to phenotype cell states that emerge in disease to comprehensively define human cardiac fibroblast diversity and to uncover cell surface protein targets for diagnostic and therapeutic applications. Details of the methods are described in Amrute, J. M. et al. (2023) (Targeting Immune-Fibroblast Crosstalk in Myocardial Infarction and Cardiac Fibrosis. Res. Sq. rs.3.rs-2402606). Luciferase expression

[0191] Fibroblasts, grown in 24-well plates (cell density: 1x104cells per well), were incubated with peptide-coupled liposomes (EP9 or mutated EP9) containing modified RNA (1 g / pL) coding for luciferase. The mRNA for luciferase was kindly supplied by Dr. Mirko Volkers, Heidelberg. Fibroblasts were exposed to ViviRen™ (60pM), a luciferase substrate, 5 minutes prior to luminescent reading for 1 minute in an I VIS® In-Vivo Imaging Systems. Luminescent pictures were taken 6 hours after treatment with targeted liposomes.

[0192] Statistical information Data are presented as means ± SD; n indicates the number of replicates (experiments, samples). Data were statistically analyzed using GraphPad Prism with multiple t tests (comparison of differences between infarcted and healthy in cell populations). For comparison of two samples, unpaired t test was used (comparison of EP9 and control peptide in experiments). Correction for multiple testing was performed using the Holm-Sidak method with alpha = O' 05. The threshold for statistical significance was set at p<0 05.

Claims

CLAIMS1. Combination comprising a CD63-binding molecule and a drug and / or a tracer for use in a method of therapy and / or in a method of in-vivo diagnosis, wherein the drug and / or the tracer is delivered to a myofibroblast.

2. The combination for use according to claim 1 , wherein the myofibroblast is a cardiac or tumor myofibroblast, optionally wherein the myofibroblast is activated by inflammation and / or myocardial infarction.

3. The combination for use according to claim 1 or 2, wherein the drug and / or the tracer is comprised in and / or coupled to a carrier; and / or wherein the drug and / or the tracer is coupled to the CD63-binding molecule; and / or wherein the CD63-binding molecule is coupled to a carrier.

4. The combination for use according to claim 3, wherein the carrier is a cell, a protein, a lipid, a glycolipid, a liposome, a nanoemulsion, for example a nanoemulsion comprising perfluorocarbons, a bead, a nanoparticle, a virus-like-particle (VLP) and a molecule, such as a sugar molecule, and any combination thereof, preferably a nanoemulsion comprising perfluorocarbons.

5. The combination for use according to any of the preceding claims, wherein the drug is an active pharmaceutical ingredient (API), in particular a small molecule, a biological or a cytostatic, preferably wherein the biological is selected form the group consisting of a protein, a peptide or a nucleic acid, in particular selected from the group consisting of nucleic acids encoding a desired protein such as mRNA, cDNA, a plasmid or vector, inhibitory nucleic acids such as siRNA or miRNA and nucleic acids having catalytic activity such as a ribozyme, optionally wherein the drug is an anti-fibrotic and / or an anti-inflammatory drug.

6. The combination for use according to any of the preceding claims, wherein the tracer is non-radioactive, such as19Fluorine, Gadolinium, Iron Oxide, Iron Platinum or Mangan, or radioactive, such as68Gallium,11Carbon,18Fluorine,13Nitrogen or 82Rubidium.

7. The combination for use according to any of the preceding claims, wherein the CD63- binding molecule is a protein, a peptide, a carbohydrate, an antibody, an aptamer, a small molecule ligand and / or any combination thereof, in particular wherein the CD63- binding molecule comprises or consists of the peptide EP1 as defined by SEQ ID NO: 1 , EP2 as defined by SEQ ID NO: 2, EP3 as defined by SEQ ID NO: 3, EP7 as defined by SEQ ID NO: 4 or EP9 as defined by SEQ ID NO: 5, preferably EP9.

8. The combination for use according to any of the preceding claims, wherein the method of therapy and / or the method of in-vivo diagnosis comprises treating, preventing and / or in-vivo diagnosing a fibrotic disease, a cardiac disease and / or a tumor disease, such as a fibrotic tumor, more preferably myocardial infarction and / or cardiac fibrosis.

9. The combination for use according to any of the preceding claims, wherein the method of in-vivo diagnosis comprises in-vivo imaging, preferably by positron emission tomography (PET), a fluorescence-based method, ultrasound, magnetic resonance imaging (MRI) or nuclear magnetic resonance (NMR) spectroscopy, more preferably by MRI.

10. The combination for use according to any of the preceding claims, wherein the combination is used as a drug delivery system.

11. The combination for use according to any of the preceding claims, wherein the combination is used in a method of concomitant in-vivo imaging and delivery of the drug.

12. A contrast agent comprising a CD63-binding molecule and a tracer and optionally a carrier.

13. Use of the contrast agent of claim 12 for imaging a myofibroblast, in particular wherein the contrast agent is used in MRI.

14. A pharmaceutical composition comprising a CD63-binding molecule and a drug and optionally a carrier.

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