Tissue-penetrating homing peptides, peptide conjugates and compositions comprising the same

Abstract

The present invention relates to peptides that home and penetrate to a target tissue of interest, as well as to peptide conjugates and compositions containing said homing peptides.

Description

[0001] TISSUE-PENETRATING HOMING PEPTIDES, PEPTIDE CONJUGATES AND COMPOSITIONS COMPRISING THE SAME

[0002] FIELD OF THE INVENTION

[0003] The present invention relates generally to the field of molecular medicine. More specifically, the invention relates to tissue-penetrating homing peptides. The invention also relates to peptide conjugates and compositions containing said tissue penetrating homing peptides.

[0004] BACKGROUND OF THE INVENTION

[0005] A general limitation in systemic drug delivery is that only a small fraction of drug reaches its desired location and systemic side effects are encountered in other organs. Thus, a critical goal of modern drug development is to generate drugs to be target organ-selective, with minimal adverse effects in the other parts of the body. This goal could be achieved by developing drugs that recognize a specific epitope expressed in the affected organ. Alternatively, drugs can be converted to be target-specific by conjugation with an affinity ligand such as a vascular homing peptide that recognizes tissue- or target specific molecular features in the blood vessels in the given organ. These peptides utilize the large heterogeneity in blood vessels of different organs and diseases.

[0006] WO 2008 / 136869 discloses the CRK peptide as a specific homing element for targeted delivery of decorin into skin wounds. However, the CRK peptide does not enable tissue penetration.

[0007] On the other hand, the best tissue or disease-selective homing peptides are also cell and tissue penetrating peptides capable of delivering the payload deep into organ parenchyma. This highlights the importance of not just selecting for the target selective homingpeptides but also identifying peptides capable of cell and tissue penetration for efficient drug delivery.

[0008] SUMMARY

[0009] Provided is an isolated peptide which peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12 and conservative sequence variants and peptidomimetics thereof.

[0010] Also provided is a conjugate which comprises the peptide set forth above conjugated to a cargo moiety.

[0011] Furthermore, provided is a pharmaceutical composition comprising the conjugate set forth above and a pharmaceutically acceptable carrier.

[0012] Also provided is the conjugate or the pharmaceutical composition set forth above for use in treating an angiogenesis-related condition.

[0013] Embodiments and details of the above-mentioned aspects are disclosed in the following figures, detailed description, examples, and dependent claims.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying drawings, which are included to provide a further understanding of the invention and constitute a part of this specification, illustrate embodiments of the invention and together with the description help to explain the principles of the invention.

[0016] Figure 1 is a schematic illustration of the present approach for screening and identifying homing and tissue-penetrating peptides. The approach combines in vivo phage display with microdialysis-based parenchymal recovery (additional selection step) and high-throughput sequencing.

[0017] Figure 2 demonstrates that Microdialysis-cap-tured peptides penetrate the cell membrane. The threenovel peptides (CDD, CPK, CYH) identified from in vivo phage display screen performed in combination with microdialysis accumulate in the CHO-K cells as does CAR peptide (amino acid sequence CARSKNKDC; SEQ ID NO: 13), previously shown to internalize these cells. CHO-K cells were grown on chamber slides. FAM-labeled CAR, CDD, CPK, or CYH peptide (20 pM) or medium (vehicle) was added to the cells and the cells were incubated for 3 hours. After that, the chamber slide was rinsed, fixed, and stained with DAPI. The cells were imaged with Zeiss LSM780 inverted confocal microscope. Scale bar 20 pm. Images representative from three individual experiments.

[0018] Figure 3 demonstrates that microdialysis-cap-tured peptides penetrate endothelial cells. The three novel peptides (CDD, CPK, CYH) identified from in vivo phage display screen performed in combination with microdialysis accumulate in the human umbilical vein endothelial cells (HUVECs) as does the cell and tissue penetrating peptide CAR peptide previously shown to internalize into these cells. FAM-labeled CAR, CDD, CPK, or CYH peptide (rows 2-5, respectively, 20 pM) or medium (vehicle; row 1 ) was added to HUVECs and the cells were incubated for 3 hours. After that, the chamber slide was rinsed, fixed, and stained with anti-VE cadherin antibody and DAPI. Lane 1 shows the FAM-labelled peptides binding and internalizing the HUVECS, lane 2 shows the anti-VE cadherin antibody staining, whereas lane 3 shows both staining results together for DAPI for cell visualization. The cells were imaged with Zeiss LSM780 inverted confocal microscope. Scale bar 20 pm. Images representative from three individual experiments.

[0019] Figure 4 demonstrates that microdialysis-cap-tured peptides home to skin wounds and localize in granulation tissue. The homing peptides enriched in the mirodialysis screens (CDD, CPK and CYH) accumulated in wound regions containing blood vessels and insurrounding extravascular granulation tissue. Six- mm diameter Full-thickness wounds 6 mm in diameter were cut in the back skin of 8-week old male mice. After 7 days, the peptides or PBS were inj ected systemically (tail vein). The peptides were allowed to circulate for 150 minutes before the vasculature was thoroughly perfused. The wound tissue was fixed and microscopic sections were stained with anti-FAM and hematoxylin. Magnifications of areas marked in panel a are represented in panel b with adj acent sections stained with anti-CD31 to visualize the blood vessels. Scale bar is 500 pm for panel a and 125 pm for panel b. Images representative of three separate experiments.

[0020] Figure 5 demonstrates that microdialysis-cap-tured peptides home to skin wounds and penetrate bloodretina barrier in retinopathy. Systemically administered homing peptides CDD, CPK, and CYH accumulate in skin wounds and penetrate blood-retina barrier in retinopathy. Full-thickness wounds, 6 mm in diameter, in the back skin of 8-week old male mice were used. After 7 days, each peptide, or PBS, was inj ected into the tail vein. For retinopathy-experiments, oxygen-induced retinopathy (OIR) was induced in using by placing mouse pups at day P7in a hyperoxia chamber for five days. On P17, when the angiogenesis peaks in this OIR model, the peptides were inj ected into the tail vein. The peptides circulated for 150 min (wounds) and 120 min (OIR) before the vasculature was thoroughly perfused. The skin wounds and eyeballs were fixed, and histological samples stained with anti-FAM and hematoxylin. Peptide homing was quantified with automated image analysis system. (A) The microdialysis-identified peptides home to skin wounds. (B and C) The peptides do not home to normal retina (B), but significant homing is detected in the retina in the OIR model (0). D) All three microdialysis-identified peptides also accumulate substantially in the vitreous in the OIR model. * p < 0.05, ** p<0.01,***<0.001. Each datapoint represents an individual wound, retina or vitreous.

[0021] Figure 6 demonstrates that microdialysis-cap-tured peptides localize in the regions of granulation tissue rich in angiogenic neovessels. The CDD, CPK, and CYH peptides accumulate in regions containing angiogenic neovessels and in the surrounding granulation tissue. The experiments and the histological analysis were carried out as described in the examples. Scale bar 200 pm. The images are representative of three separate experiments.

[0022] Figure 7 demonstrates that microdialysis-cap-tured peptides do not home to normal organs. The CDD, CPK, and CYH) peptides do not accumulate in normal, healthy organs except in the kidneys, presumably because peptides are excreted through the kidneys. The experiments and the histological analysis were carried out as described in the legend of Figure 4. Scale bar 125 pm.

[0023] Figure 8 demonstrates that microdialysis-cap-tured peptides home to skin wounds in diabetic mice. The CDD, CPK, and CYH peptides home and penetrate to the granulation tissue in compromised wound healing in diabetic (BKS (D) -Leprdb / JOrlRj ) mice. Full-thickness wounds 6 mm in diameter were cut in the back skin of 14-week old male diabetic mice. After 7 days, 1, 5 mg / kg of each peptide or PBS were inj ected systemically. The peptide circulated for 150 minutes before the vasculature was perfused. The wounds were fixed with 4% PFA in PBS, and microscopic sections were stained with anti-FAM and hematoxylin, a, images of entire wounds, b, magnifications of areas marked in A with anti-CD31 staining of the adj acent sections. Scale bars 500 pm for a and 125 pm for b.

[0024] Figure 9 demonstrates that microdialysis-cap-tured peptides home and penetrate to retina in retinopathy. The CDD, CPK, and CYH peptides accumulate in the retina and vitreous in oxygen-induced retinopathy(OIR). OIR model was carried out as described previously. At P 17, the peptides were inj ected into the tail vein and circulated for 120 minutes before the vasculature was thoroughly perfused. The eyeballs were fixed and histological sections stained with anti-FAM antibody and hematoxylin. A) None of the tested peptides homed to normal retina after the systemic administration at P17. B) The three microdialysis-identified homing peptides homed and penetrated into retina in OIR at the peak of angiogenesis at P17. The peptides also accumulate in the vitreous in the OIR model. C) High magnification images of peptide accumulation in OIR retina. Scale bar 100 pm for A and B and 20 pm for C. Images representative of N = 6 and N = 8 for normal and OIR retinas.

[0025] DEFINITIONS

[0026] It is to be understood that, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It is further to be noted that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0027] As used herein and in the appended claims, the singular forms "a", "an" and "the" mean one or more. Thus, a singular noun, unless otherwise specified, carries also the meaning of the corresponding plural noun, and vice versa. As such, the terms "a", "an", "one or more" and "at least one" can be used interchangeably.

[0028] As used herein, the term "microdialysis" refers to an in vivo technique than can be used to monitor biochemical phenomena in living tissues. This is based on sampling endogenous material from intracellular and extracellular fluids in animal tissues.Microdialysis works by slowly pumping a physiological solution (the "perfusate") through a microdialysis probe (i. e. a catheter) inserted into a living tissue. Diffusible molecules in the tissue diffuse by a concentration gradient across a dialysis membrane into the solution (typically saline) as it is pumped through the probe. The solution can then be collected as the "dialysate" and analysed for determining the identities and concentrations of molecules that were in the living tissue. The pumping of the perfusate and the dialysate may be carried out using one or more, typically two, pumps. For example, a syringe pump may be employed to push, and a peristaltic pump may be used to pull the perfusion fluid and the dialysate through the probe.

[0029] As used herein, the term "peptide-expressing phage display library" refers to a collection of random or defined peptide sequences displayed on bacteriophages as a genetic fusion to a bacteriophage coat protein. The term is thus interchangeable with the expression "a library of bacteriophages each displaying a different peptide on their surface" or "phage display library" for short. Also the terms "bacteriophage" and "phage" are interchangeable.

[0030] As used herein, the term "cyclic peptide" refers to a peptide chain possessing a cyclic ring structure. Herein, the ring structure is formed by linking one end of the peptide to the other with a disulfide bond through terminal cysteine residues at both ends of the peptide.

[0031] As used herein, the term "homing" refers to an ability of a substance to selectively home to, i. e., target, specific cells or tissue in vivo in preference to other cells or tissues. Accordingly, the term "homing peptide" refers broadly to any peptide that has such a homing ability. However, the homing peptides disclosed herein are more specifically cyclic homing peptides, although sometimes referred to only as "homing peptides"or "peptides" for brevity. A "homing" peptide can also be a linear peptide

[0032] As used herein, the term "extravasation" refers broadly to an ability of a substance to escape into the extravascular space, either by leakage from a vessel or by direct infiltration.

[0033] As used herein, the term "tissue penetration" refers broadly to an ability of a substance to reach and enter a given tissue by penetrating the cells in the tissue, i. e. being a cell penetrating peptide.

[0034] As used herein, the term "granulation tissue" refers broadly to vascularized connective tissue that forms a wound or an injured tissue during the healing process and contain numerous neovessels.

[0035] The term "conservative sequence variant", as used herein, refers to amino acid sequence modifications, which do not significantly alter the biological properties of the peptide in question. Conservative peptide variants include variants arising from one or more amino acid substitutions with similar amino acids well known in the art (e. g., amino acids of similar size or with similar charge properties).

[0036] As used herein, the term "peptidomimetic" refers to a peptide-like molecule designed to mimic a given peptide without altering its activity, such as homing specificity. Non-limiting examples of pep-tidomimetics include chemically modified peptides, D-peptide peptidomimetics, peptide-like molecules comprising non-naturally occurring amino acids, peptoids and p-peptides. Also molecules that resemble peptides, but which are not connected via a natural peptide linkage are included in the term. Means and methods for producing peptidomimetics are readily available in the art.

[0037] As used herein, the term "conjugate" refers generally to a molecular entity where one molecule or agent is physically or chemically conjugated to, i. e.,coupled or adhered or attached to or incorporated into, another molecule or agent. In an embodiment, the present conjugates are cyclic peptide conjugates comprising a cyclic homing peptide physically or chemically conjugated to a cargo moiety. In other embodiments, the present conjugates are linear peptide conjugates comprising a linear homing peptide physically or chemically conjugated to a cargo moiety.

[0038] As used herein, the term "functional group" refer to distinct, definable portions or units of a molecule, and to units that perform some function or activity and are reactive with other molecules or portions of molecules.

[0039] As used herein, the term "angiogenesis" refers to a formation of new blood vessels or capillaries. It is a normal component of various growth and healing processes, but sometimes angiogenesis can play a role in diseases such as cancer. The expression "tissue that exhibits angiogenesis", and any linguistic variations thereof, refers to a tissue in which development of new blood vessels and capillaries is taking place. Non-limiting examples of such tissues include granulation tissues, ulcers, wounds with angiogenic vessels such as surgical wounds and diabetic wounds, retina affected by a retinopathy, inflammatory conditions, tumors and cancer tissues.

[0040] As used herein, the term "angiogenesis-related medical condition" refers to any disease, disorder, or pathological or other condition in which angiogenesis is involved. Non-limiting examples of such conditions include cancers, retinopathies, tissue injuries and traumas, surgical wounds, diabetic wounds, inflammation, and brain diseases or pathological conditions such as brain tumors, brain metastases, brain diseases, or traumatic brain conditions.

[0041] As used herein, the term "pharmaceutical composition" refers broadly to a preparation of one or moreof active ingredients and pharmaceutically acceptable components such as carriers, adjuvants and / or excipients. In the context of the present invention, said "active ingredient" refers particularly to a conjugate of the invention comprising a therapeutic agent, i. e., a cargo moiety accountable for a biological or therapeutical effect. The purpose of a pharmaceutical composition is to facilitate administration of the conjugate to a subj ect or organism.

[0042] As used herein, the terms "pharmaceutically acceptable" and "physiologically acceptable" are interchangeable and refer to a material that is suitable for administration to a subj ect or organism without undue adverse side effects such as toxicity, significant irritation and / or allergic responses. In other words, the benefit / risk ratio must be reasonable.

[0043] As used herein, the term "treatment" or "treating" refers to the administration of the conjugate or the pharmaceutical composition comprising the same to a subj ect in need thereof for a purpose which may include ameliorating, lessening, inhibiting, or curing the disease or condition.

[0044] As used herein, the term "efficient amount" refers to an amount by which harmful effects of the disease or condition are, at a minimum, ameliorated.

[0045] As used herein, the term "subj ect" refers broadly to a mammalian subj ect, including both human and non-human subj ects unless specified otherwise. Accordingly, the term "human subj ect" refers solely to human beings, whereas the term "non-human subj ect" refers to a mammalian animal excluding human subj ects. Non-limiting examples of non-human mammalian animals include laboratory animals, especially rodents such as mice and rats.

[0046] DETAILED DESCRIPTIONThe invention is based on utilization of a novel approach to identify peptides capable of vascular homing and tissue penetration to reach therapeutically relevant cells in the tissue parenchyma. The approach combines in vivo phage display with microdialysis-based parenchymal recovery and high-throughput sequencing as schematically illustrated in Figure 1.

[0047] As explained in more detail in the experimental part, it was first demonstrated in skin wounds that the approach can selectively distinguish wound homing peptides capable of extravasation and tissue penetration from those that do not display such capabilities.

[0048] After the applicability of the novel approach was verified with known wound homing peptides with known extravasation and tissue penetration capabilities along with additional peptides as negative controls, the approach was applied for screening of a naive peptide library to identify novel peptides that home and penetrate to extravascular tissues that exhibit angiogenesis.

[0049] The novel approach can be described as an in vivo method for screening and / or identifying a peptide that not only homes to a target tissue of a subject but also penetrates cells or tissues for efficient accumulation, the method comprising:

[0050] (a) implanting a microdialysis probe into the target tissue of a non-human subject or providing a nonhuman subject having a microdialysis probe already implanted into a target tissue;

[0051] (b) administering a library of bacteriophages each displaying a peptide on their surface to said non-human subject systemically;

[0052] (c) collecting those bacteriophages that have homed and penetrated to the target tissue of said non-human subject along with a dialysate; and

[0053] (d) analysing the dialysate to identify the peptide displayed by the collected bacteriophage.The peptide identified in step (d) has the ability to home and penetrate to the target tissue in question. Notably, said target tissue is preferably a human target tissue although the method is based on identification of bacteriophage-expressed peptides that home to a corresponding target tissue in a non-human subject. In other words, the approach may be applied for screening and / or identifying a peptide that homes to a target tissue of a human subj ect by subjecting a non-human subject to the present microdialysis-based method, namely to steps (a) - (c) set forth above.

[0054] Applicability of the new approach for identifying peptides with vascular homing and tissue-penetrating activity is demonstrated in the Examples hereinbelow. As a result of the disclosed experiments, 12 peptides having amino acid sequences depicted in SEQ ID NOs: 1-12 were found in the wound microdialysates in multiple copies at two different time points, and thus recognised as vascular homing peptides with a tissuepenetrating activity.

[0055] Accordingly, some embodiments of the present invention concern cell-penetrating homing peptides comprising an amino acid sequence selected from SEQ ID NOs: 1-12, preferably from SEQ ID NOs: 1-5, more preferably from SEQ ID NOs: 1-3. Some other embodiments concern cell-penetrating homing peptides consisting of an amino acid sequence selected from SEQ ID NOs: 1-12, preferably from SEQ ID NOs: 1-5, more preferably from SEQ ID NOs: 1-3. It is also envisaged that in some embodiments, the cell-penetrating homing peptide may comprise or consists of a conservative sequence variant and or a pep-tidomimetic of said amino acid sequences. However, neither the conservative sequence variation nor the pep-tidomimetic variation should change the homing and tissue penetration properties of the present homing peptides.In an embodiment, the homing peptide comprising or consisting of an amino acid sequence depicted in any one of SEQ ID NOs: 1-12 have the ability to home and penetrate into a human tissue exhibiting angiogenesis, such as a granulation tissue, preferably a granulation tissue of a skin wound or injured tissue in a human body, or retina affected by a retinopathy.

[0056] As the present homing peptides are capable of penetrating blood-retina barrier (BRB) in pathological conditions such as retinopathies, especially diabetic retinopathies, it is envisaged that the homing peptides are also capable of penetrating a structurally corresponding barrier, namely a blood-brain barrier (BBB), in brain diseases or pathological conditions such as brain tumors, brain metastases, brain diseases, or traumatic brain conditions.

[0057] The present homing peptides may be produced by any means, methods or techniques available in the art, for example, by an automated peptide synthesizer, or produced by genetic engineering technologies. In some preferred embodiments, the present homing peptides are synthetic homing peptides.

[0058] Owing to their homing ability, the present peptides may be used as vehicles for targeted delivery of an agent of interest. Accordingly, said agent of interest may be delivered to its target tissue as a cargo moiety in a cyclic peptide conjugate disclosed herein, wherein said cargo moiety is conjugated to a cyclic homing peptide also disclosed herein. However, linear peptide conjugates are also envisaged as already explained.

[0059] The target tissue is not particularly limited and may be generally referred to as any tissue that exhibits angiogenesis or as any tissue affected by a medical condition that exhibits angiogenesis. Non-limiting examples of such tissues include granulation tissue in injuries and traumas, ulcers, wounds withangiogenic vessels such as surgical and diabetic wounds and wounds caused by trauma, tumors and cancer tissues. In an embodiment, the target tissue is a granulation tissue, such as the granulation tissue of a skin wound, such as a diabetic wound or a surgical wound. In a further embodiment, the target tissue is a tumor or a cancer tissue. In a still further embodiment, the target tissue is retina affected by a retinopathy. In some further embodiments, the target tissue is brain tissue affected by a medical condition such as a brain tumor, brain metastases, a brain disease, or a traumatic brain condition. In some still further embodiments, the target tissue exhibits inflammation, i. e. is a tissue affected by a medical condition exhibiting angiogenesis, namely inflammation.

[0060] The cargo moiety can be of any type, including any natural or non-natural physical, chemical or biological material, for example a peptide, an oligopeptide, a polypeptide, a protein (e. g., an antibody, an enzyme or a fluorescent protein), a proteoglycan (e. g., decorin), a lipid, or a nucleic acid (e. g., RNA or DNA, double stranded or single stranded) in the form of an oligonucleotide or a polynucleotide, such as small interfering RNA (siRNA) or any other RNA interfering molecule (RNAi molecule). As further non-limiting examples, the cargo moiety can be a chemical compound (e. g., a detectable agent or a radioactive isotope), a small molecule (e. g., a drug, a prodrug, a chemotherapeutic agent, or a cytotoxic agent), a liposome, a vector, a cell, a ribozyme, a virus (e. g., a retrovirus, an adenovirus, an adeno-associated virus), a phage, a particle (e. g., a gold particle, a virus-like particle (VLP), or a nanoparticle such as a drug-containing nanoparticle).

[0061] The cargo moiety may be conjugated to the homing peptide by any suitable protocol, such that the homing peptide retains its homing activity and specificity even when conjugated to said cargo moiety.Typically, the cargo moiety and the homing peptide are conjugated covalently, either directly or indirectly via a linker, but non-covalent linkages are also envisaged. If the cargo moiety possesses amino or carboxyl functional groups, standard peptide coupling reactions can be used. Also functional groups (e. g., thiol, hydroxyl, amino, carboxyl, etc. ) present in many small molecules are suitable for direct conjugation to the homing peptide, e. g. via a linker. Various forms of "click" chemistry may also be used for the conjugation. Selection of the type of chemistry to be used for the conjugation is largely determined by the type of the cargo moiety in question, e. g., depending on functional groups contained therein, which selection is within the knowledge of a skilled person in the pertinent art.

[0062] Any suitable linker may be used for conjugating the cargo moiety of interest to the homing peptide disclosed herein. Examples of suitable linkers include, but are not limited to, peptide linkers, lipid linkers, polyethylene glycol linkers, and aliphatic chain linkers. In some embodiments, the linker may contain two functional groups (e. g., two different functional groups), one at each end, wherein one functional group is for reacting with the cargo moiety, and the other is for reacting with the homing peptide.

[0063] As the present homing peptide detects angiogenic blood vessels and capillaries, it may be employed for visualizing formation and / or presence of new blood vessels, for example, in diabetic and other wounds and in diseases with neovasculature formation such as in the retinopathies and cancers. In other words, the homing peptide may be employed for detecting any tissue that exhibits angiogenesis. In such embodiments, the cargo moiety is a detectable agent, i. e. a molecule capable of releasing a signal that can be detected, either directly or indirectly, preferably by a non-invasive and / or in vivo visualization technique. Non-limitingexamples of detectable agents as suitable cargo moieties include optical agents such as fluorescent agents including a variety of inorganic and / or organic small molecules (e. g. fluorescein amide, FAM) and a variety of fluorescent proteins (e. g. green fluorescent protein, GFP) and derivatives thereof, phosphorescent agents, luminescent agents such as chemiluminescent agents, and chromogenic agents; radiolabels such as radionuclides that emit gamma rays, positrons, beta or alpha particles, or X-rays; non-radioactive isotopes such as Gadolinium (Gd); ionic and non-ionic contrasting agents such as iodine-based contrasting agents; electromagnetic agents such as magnetic, ferromagnetic, paramagnetic, and / or superparamagnetic agents; upconverting nanoparticles (UCNP), resonance particles, quantum dots, and gold particles. Further suitable detectable agents are available in the art. Those skilled in the art can readily select an appropriate imaging technique depending on the type and species of the detectable agent employed in the conjugate. Such techniques include, but are not limited to, radiological techniques, isotope techniques such as to positron emission tomography, ultrasound imaging and magnetic resonance imaging (MRI ).

[0064] For therapeutic purposes, the cargo moiety is a therapeutic agent such as an anti-inflammatory agent, an anti-angiogenic agent, a regenerative agent, a pro-angiogenic agent, a cytotoxic agent, a pro-apoptotic agent, an anti-apoptotic agent, an antimicrobial agent (e. g. an anti-bacterial agent, an anti-viral agent, an anti-fungal agent, or an anti-protozoan agent), an anti-fibrotic agent, an anti-transmitter, a pro-transmitter (such as histamine), a cytokine, or a cytokine inhibitor (e. g. an antagonist, a soluble receptor, a cytokinebinding molecule, or a cytokine that blocks other cytokines) to mention some non-limiting examples of potential therapeutic moieties to be included in a peptide conjugate.In some embodiments, the therapeutic agent is conjugated to the homing peptide via a covalent bond, either directly or via a linker. In some embodiments, the linker may be a cleavable linker thus controlling the release of the therapeutic agent. In some other embodiments, the therapeutic agent may be provided in a nanoparticle, such as a controlled release or sustained release nanoparticle, that is conjugated to the homing peptide either directly or via a suitable linker.

[0065] In a further aspect, the invention relates to a composition comprising the conjugate described above. In some embodiments, the composition comprises the conjugate and a pharmaceutically or physiologically acceptable carrier to enable administration in vivo. Such a composition may be, for example, a diagnostic composition, a composition for visualizing or imaging of the target, or a pharmaceutical composition. In all cases the composition must be physiologically acceptable.

[0066] A composition disclosed herein, regardless of whether it is a diagnostic composition, a composition for visualizing or imaging of the target or a pharmaceutical composition, may be formulated as desired, for example as a semisolid or solid preparation, solution, dispersion, or suspension, using means and methods readily available in the art, for example by means of conventional mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping, lyophilizing or similar processes.

[0067] Pharmaceutically acceptable carriers, i. e., carrier substances or diluents with which the active ingredient is combined to facilitate administration and that is physiologically acceptable to the recipient, are readily available in the art and. The selected carrier should not abrogate the biological activity and properties of the active ingredient but minimize any degradation of thereof as wells as minimize adverse side effects in the recipient.Excipients are preferably inert substances added to a pharmaceutical composition to further facilitate administration of an active ingredient. Typical examples of different types of excipients, without limitation, include stabilizers, preservatives, pH modifiers, fillers, thickeners, viscosity modifiers, lubricants, solubilizers, surfactants, sweeteners, taste masking agents, and the like.

[0068] Useful stabilizing excipients include, but are not limited to, surfactants such as polysorbate 20, polysorbate 80 and poloxamer 407; polymers such as polyethylene glycols and povidones; carbohydrates such as sucrose, mannitol, glucose and lactose; sugar alcohols such as sorbitol, glycerol, propylene glycol and ethylene glycol; proteins such as albumin; amino acids such as glycine and glutamic acid; fatty acids such as ethanolamine; antioxidants such as ascorbic acid; chelating agents such as EDTA salts; and metal ions such as Ca, Ni, Mg and Mn. Among useful preservative agents, without limitation, are benzyl alcohol, chlorbutanol, benzalkonium chloride and possibly parabens. Among useful buffering excipients are, without limitation, sodium and potassium phosphates, citrate, acetate and carbonate or glycine buffers depending on the targeted pH- range. The use of sodium chloride as a tonicity adjuster is also useful. Non-limiting examples of further excipient materials include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. As readily understood by those skilled in the art, a given excipient may serve more than one function.

[0069] Pharmaceutical compositions disclosed herein can be administered in a number of ways depending on different variables, such as on the area to be treated or imaged. Preferably, the route of administration is parenteral. In some embodiments, systemic administration by intravenous, intraperitoneal, subcutaneous,intradermal or intramuscular injection or by sublingual administration is preferred.

[0070] Parenteral administration of the composition, if used, is generally applied by injection, for example intravenously, intraperitoneally, subcutaneously, intradermally or intramuscularly. Preparations for parenteral administration are typically sterile aqueous or non-aqueous solutions, suspensions or emulsions, but the preparation may also be provided in a concentrated form or in a form of a powder to be reconstituted on demand. Slow release or sustained release formulation are also contemplated. Means and methods for formulating compositions for parenteral administration are readily available in the art, and those skilled in the art can easily select appropriate physiologically suitable carriers, adjuvants and / or excipients depending on the desired specifics of the composition.

[0071] Non-limiting examples of aqueous carriers for parenteral compositions include sterile water, water-alcohol solutions, saline, and buffered solutions at physiological pH. Parenteral vehicles include sodium chloride solution, Ringer ' s dextrose solution, dextrose plus sodium chloride solution, Ringer ' s solution containing lactose, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer ' s dextrose solution, and the like.

[0072] Non-limiting examples of non-aqueous carriers for parenteral include solvents such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, fish oils, and injectable organic esters such as ethyl oleate.

[0073] If the parenteral composition is provided as a concentrated solution or dispersion, or as a powder, aqueous or non-aqueous carriers mentioned above may be used for reconstitution. A solution for the reconstitution may be provided in the same package as theconcentrate or powder. If lyophilization is used for preparing the powder, it may be beneficial to use cryoprotectants including, without limitation, polymers (e. g., povidones, polyethylene glycol, dextran), sugars (e. g., sucrose, glucose, lactose), amino acids (e. g., glycine, arginine, glutamic acid) and albumin.

[0074] Some of the compositions can be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.

[0075] Amounts and regimens for administration of a conjugate or a pharmaceutical composition disclosed herein can be determined readily by those with ordinary skill in the clinical art of treating angiogenesis-related conditions. Generally, dosing will vary depending on considerations such as: age, gender and general health of the subject to be treated; kind of concurrent treatment, if any; frequency of treatment and nature of the effect desired; severity and type of disease or condition in question; causative agent of the disease and other variables to be adjusted by the individual physician. A desired dose can be administered in one or more applications to obtain the desired results. For example, the pharmaceutical composition may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of e. g., two, three or four times daily. The pharmaceutical composition maybe provided, for example, in unit dosage forms or in extended-release formulations.

[0076] Accordingly, provided herein is a method of treating an angiogenesis-related condition in a subj ect in need thereof by administering an efficient amount of a conjugate, or a pharmaceutical composition of the invention to said subj ect. This aspect of the invention may also be expressed as use of the present conjugate, or the pharmaceutical composition for manufacturing a medicament for treating an angiogenesis-related condition. Accordingly, one aspect of the invention provides the present conjugate or the present pharmaceutical composition for use in treating an angiogenesis-related condition.

[0077] Also disclosed is a method of delivering a desired cargo moiety to a site of angiogenesis in a recipient subj ect. The method comprises or consists of administering a herein disclosed conjugate or pharmaceutical composition to the subj ect. Owing to the targeting specificity of the homing peptide contained in the conjugate, the conjugate will selectively home to a tissue that exhibits angiogenesis, in which location the cargo moiety can serve its function. If the cargo moiety is a detectable agent, the target site can be imaged or visualized. Likewise, if the cargo moiety is a therapeutic agent, it will exert its therapeutic effect at the target site. This aspect of the disclosure may also be expressed as use of a conjugate or a pharmaceutical composition disclosed herein for targeted delivery of a desired cargo moiety to a site of angiogenesis in the recipient' s body. Such a site can be, without limitation, a tumor, an ulcer or a wound, including surgical wounds, wounds cause by a trauma and diabetic wounds.

[0078] EXAMPLES

[0079] It is obvious to a person skilled in the art that with the advancement of technology, the basic ideaof the invention may be implemented in various ways. The invention and its embodiments are thus not limited to the examples described below, instead they may vary within the scope of the claims.

[0080] EXAMPLE 1. Identification and characterization of novel tissue-penetrating homing peptides

[0081] Materials and Methods

[0082] Reagents

[0083] Phosphate-buff ered saline (PBS), tween, paraformaldehyde (PFA) and bovine serum albumin (BSA) were purchased from Thermo Fisher Scientific (Waltham, MA). Cell culture reagents including Dulbecco' s Modified Eagle Medium (DMEM) were purchased from Gibco ( Invitrogen, Waltham, MA) and cell culture plastics from Sarstedt (Numbrecht, Germany) unless otherwise stated.

[0084] Phages and peptide synthesis

[0085] The phage library used in the in vivo screen with the microdialysis probe was in the 10-3b T7 backbone ( 5-15 peptide copies per phage), grown in BLT5403 E. coli strain, CsCl-purified, dialyzed in PBS, and filter-sterilized. The library was constructed via trinucleotide synthesis to prevent bias in the amount of different amino acids (Kayushin et al, 1996). The diversity of the library was 1 x 109and the titer 5 x 1010per ml. Novagen T7 Select System (Merck Millipore) was used according to the instructions provided by the manufacturer.

[0086] Peptides were synthetized with an automated peptide synthesizer by using standard solid phase fluorenylmethoxycarbonyl chemistry. During synthesis, the peptides were labeled with fluorescein amide (FAM) using an amino-hexanoic acid spacer as previously described.

[0087] Microdialysis equipmentFor the in vivo microdialysis, CMA 402 Syringe Pump with Hamilton syringes was used to push and Harvard P70 peristaltic pump (Harvard Apparatus, Holliston, MA) to pull the CMA Perfusion Fluid (CMA Microdialysis AB) and dialysate. Each of the CMA 402 two outputs were connected to the inlet spike of CMA 12 custom-made 3 MDa probe (CMA Microdialysis AB) via CMA FEP tubing (CMA Microdialysis AB) and the outlet spike of CMA 12 probe was further connected to Harvard P70 peristaltic pump and further to CMA Probe Shaft Clip (CMA Microdialysis AB) via CMA connector tubing (CMA Microdialysis AB). From the CMA Probe Shaft Clip the dialysate was pumped to CMA sample tubes (CMA Microdialysis AB). Harvard 0.19 mm diameter tubing (Harvard Apparatus) and Harvard 3 collar 1.52 mm tubing (Harvard Apparatus) were used with the Harvard P70 peristaltic pump. Additionally, CMA tubing adaptors (CMA Microdialysis AB) were used for connecting tubes to syringes and probes. The system was flushed with 70 % ethanol before any use.

[0088] Animals and wound models

[0089] Male Wistar rats were used for the in vivo validation experiment and the library screen. The rats were 27 wk old and weighed 490 g. Two full-thickness circular wounds were created on a shaved and disinfected skin with a 12-mm-diameter biopsy punch and scissors under sevoflurane anesthesia (Sevoflurane Baxter, Baxter SA) and carprofen analgesia (50 mg / kg; Norocarp, Norbrook Laboratories). Seven days after the wounding, the microdialysis experiment was performed. The animal use was accepted by the National Animal Ethics Committee of Finland. The experiments were conducted according to the ARRIVE guidelines.

[0090] Male BALB / cJRj and diabetic male BKS (D) -Leprdb / JOrlRj mice were used for the peptide homing experiment. The BALB / cJRj mice were 8 wk old and weighed 24-28 g. The BKS (D) -Leprdb / JOrlRj mice were 14 wk oldand weighed 45-58 g, and their blood sugar levels were over 33 mmol / liter. Four full-thickness circular wounds were created on a shaved and disinfected skin with a 6-mm-diameter biopsy punch and scissors under isoflurane anesthesia (Attane vet 1, 000 mg / g; Piramal Critical Care B. V. ) and buprenorphine analgesia ( 0.15 mg / kg; Veterg-esic vet 0.3 mg / ml; Ceva Sante Animal). Seven days after the wounding, the homing experiment was performed. The animal use was accepted by the National Animal Ethics Committee of Finland.

[0091] Oxygen-induced retinopathy (OIR)

[0092] WT C57BL / 6Rj mice were used for the OIR experiments (Vahatupa, et al, 2020). The OIR model was performed as described in detail previously (Vahatupa, et al, 2020; De Rossi et al, 2021 ). Briefly, to induce retinopathy in the retina, the pups and their nursing mothers were exposed to 75% oxygen in a custom-made chamber (ProOx P110 oxygen controller; BioSpherix Ltd. ) at postnatal day 7 (P7) for 5 d until P12 when they were returned to normal room air. The homing experiments were performed at P17 (late hypoxic phase and the peak of neovascularization) (Vahatupa et al, 2021 ). Control animals were housed under normal room air conditions, and retinas were harvested on the corresponding day (P17). All retina experiments were conducted under the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research guidelines. The animal use for the OIR model was accepted by the National Animal Ethics Committee of Finland.

[0093] In vivo microdialysis

[0094] The tubing system was cleaned as described above. A 12-wk-old male rat with two 7-d-old wounds was anesthetized with inhaled sevoflurane. A small skin incision was made ~16 mm from the edge of the wound, and a 16-mm CMA guide cannula (CMA Microdialysis AB) was inserted and superglued in place in the fashion that thecannula ended at the edge of the wound. The metal pin was replaced by a CMA 12 custom-made 3-MDa probe. The microdialysis membrane extends 12 mm out of the cannula into the wound with a 12 mm diameter. Similarly, another CMA guide cannula and a CMA 12 custom-made 3-MDa probe were placed in normal skin far from wounds. The pump system was run at 0.5 µl / min for 45 min. ( If bleeding took place, i. e., dialysate turned red, the experiment was stopped. ) Then, 200 µl of the custom-made library or 2 ml of the screening library was inj ected via the tail vein. The first 30 min of collected dialysate was discarded as dead volume. The dialysate was collected for 1 h ( in vivo validation) or at timed intervals described in Table SI (library screen). When the dialysis was ready, the rat was anesthetized with subcutaneous medetomidine-ketamine inj ection ( 0.4 mg / kg, Domitor vet 1 mg / ml; Orion Corporation, and 60 mg / kg, Ketalar 50 mg / ml; Pfizer Oy) and thoroughly perfused with sterile 1% BSA in DMEM as described previously (Jarvinen, 2012 ). The dialysate from skin wound and normal skin, a sample of the inj ected library as well as the wound with the probe (proper probe placement and no visible hematoma around the probe were checked), the wound without a probe, and tissue pieces from liver and normal skin without a probe were collected as samples. In the library screen, the following samples were collected in addition to the aforementioned samples: eyes, Achilles and patellar tendons, gastrocnemius and soleus muscles, heart, kidney, spleen, and tissue pieces from liver, bladder, colon, lung, and small intestine.

[0095] Determination of phage-displayed peptide sequences The tissue samples harvested from animals were snap-frozen and later broken down by a hand-held homogenizer and suspended in LB / 1% NP-40. The peptide-encod-ing region of the bacteriophage genome was amplified by PCR using Phusion Green Hot Start II High-Fidelity DNAPolymerase (F537L; Thermo Fisher Scientific) (reaction volume: 25 µl). Cycling conditions were as follows: denaturation at 98 °C for 30 s, followed by 25 amplification cycles ( 10 s at 98 °C, 21 s at 72 °C), and final elongation (72°C for 5 min). PCR products were purified using the AMPure XP Bead Based Next-Generation Sequencing Cleanup system (A63881; Beckmann Coulter) using 1.8 µl of beads per 1 µl of PCR products. Purified PCR products were quantified using Agilent Bioanalyzer 2100 Instrument using High-sensitivity DNA Kit (5067-4626; Agilent). Ion Torrent Emulsion PCR and enrichment steps were performed using Ion PGM HiQ View OT2 Kit (A29900; Life Technologies). HTS was performed using Ion Torrent Personal Genome Machine ( Ion PGM) using Ion PGM HiQ View Sequencing Kit (A30044; Life Technologies) and Ion 316v2 chips (448149; Life Technologies). The FASTQ sequence files were converted to text files and translated using in-house-developed Python scripts.

[0096] Peptide internalization in vitro

[0097] CHO-K cells were obtained from the American Type Culture Collection (ATCC). Cells were maintained in a-MEM and Earle' s salt supplemented with 10% FBS, 100 µg / ml streptomycin sulfate, 100 U / ml of penicillin G, and 292 µg / ml L-glutamine (Thermo Fisher Scientific). HUVECs (PromoCell) were grown as a monolayer in Endothelial Cell Growth Medium 2 (Promo cell). When the cells were about to reach confluence, they were subcultured to eight-well Nunc Lab-Tek IT Chamber Slide (Thermo Fisher Scientific) in the growth medium. After 3-4 d of incubation, peptides CAR, CDD, CPK, and CYH were added. The peptides were diluted in PBS to 4 mM concentration, and 1 µl of this solution was used to add the peptides to the growth medium to a final incubation concentration of 10 µM in 400 pl growth medium. Plaingrowth medium was used as a negative control. The chamber slides were incubated for 3 h at 37 °C, 5% CO2.

[0098] After the incubation, the chamber slide was rinsed three times with cell medium and the cells were fixed with 2% PFA for 10 min. The HUVECs were then rinsed two times with 0.05% Tween in PBS, and 0.2% Tween in PBS was added for 5 min. The slides were rinsed twice and incubated with 10% normal goat serum (Thermo Fisher Scientific) for 15 min. The primary antibody rabbit anti-VE-cadherin (Abeam) was added. After 60-min incubation at RT and two washes, the secondary antibody Alexa Fluor 594 (anti-rabbit, Jackson) was added and incubated for 25 min at RT. 1% BSA and 0.05% Tween-20 were used for primary and secondary antibody dilution.

[0099] The chamber slide was rinsed three times and dried on room air for 15-30 min. The chambers were removed according to the manufacturer' s instructions. VEC-TASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories, Maravai LifeSciences) was added, and the slip was covered with Menzel 1-mm Microscope Coverslip (Thermo Fisher Scientific). Images were acquired with Zeiss LSM780 Laser Scanning Confocal Microscope (Carl Zeiss AG) with a 63x oil immersion obj ective using diode laser 405 nm for DAPI, argon laser 488 nm for FAM, and HeNe laser 594 nm for Alexa Fluor 594.

[0100] Peptide homing

[0101] BALB / cJRj and diabetic male BKS (D) -Leprdb / JOrlRj mice with 7-d-old skin wounds were used for the peptide homing experiment. FAM-labeled peptides were dissolved in the PBS just before the experiment. 5 mg / kg (BALB / cJRj) or 1.5 mg / kg (BKS(D)-Leprdb / JOrlRj) of each peptide (CAR, CDD, CPK, CYH, and a control peptide) in PBS or plain PBS (control ) was inj ected on the tail vein under sevoflurane anesthesia. 150 min later, the body was perfused as after in vivo microdialysis and the wounds, normal skin from the upper back, heart, leftlung, left kidney, spleen, and a piece of liver were gathered in 4% PFA in PBS.

[0102] Normal or OIR C57BL / 6Rj mice were injected intraperitoneally with peptide solution (5 mg / kg) at P17 (Vahatupa et al, 2021 ). Two hours after inj ection, the mice were perfused with DMEM containing 1% BSA while under deep anesthesia. The eyeballs were dissected and placed in 4% PFA (Vahatupa et al, 2021 ).

[0103] Quantitative analysis of peptide homing

[0104] After 24-h (or 4 h for eyeballs) fixation in 4% PFA, the tissues were rinsed in 70% EtOH and embedded in paraffin. To avoid autofluorescence, the peptides were detected by staining the sections with rabbit anti-FITC / -FAM (71-1900; Thermo Fisher Scientific, 1: 400 BALB / cJRj and C57BL / 6Rj or 1: 100 BKS (D) -Leprdb / JOrlRj ) in Dako antibody diluent (Agilent) and Biocare Medical Rabbit on Rodent (RMR622H; Biocare Medical) with XM-Factor (XMF963C; Biocare Medical) as described previously (Urakami et al, 2011; Toba et al, 2014; Pemmari, et al, 2020). DAB (K3468; Agilent) was applied, and the slides were counterstained with hematoxylin ( 1.09253; Merck) and mounted. For CD31 stainings, rat anti-CD31 (550274; BD Pharmingen) in Dako antibody diluent and Histofine Simple Stain Mouse MAX PO (Nichirei) were used. DAB (K3468; Agilent) was applied, and the slides were counterstained with hematoxylin and mounted.

[0105] The brightfield images were acquired with Nano-Zoomer S60 (Hamamatsu Photonics). Slides were viewed and analyzed using QuPath software version 0.2.0 or later (Centre for Cancer Research and Cell Biology, Queen' s University Belfast, Belfast, Northern Ireland, UK, Bank-head et al, 2017 ). Analyzed areas were marked from high-magnification images using QuPath. Analysis algorithms were used, and the area of positive staining was determined as described previously (Vahatupa et al, 2021; Salomaa et al, 2022 ).Statistical analysis

[0106] For comparisons of multiple groups, statistical analysis was performed by two-way analysis of variance (ANOVA) complemented by the Bonferroni post hoc test for pairwise comparisons between the test groups. The possible difference in the homing of the different peptides was assessed using the log-transformed variables. P-values of less than 0.05 were considered statistically significant for all tests. The significance level shown refers to a two-tailed test.

[0107] Results

[0108] Microdialysis-based in vivo phage biopanning reveals new wound homing and penetrating peptides

[0109] Microdialysis-based approach was adopted for the in vivo biopanning to identify new wound-penetrating peptides. We used C7XtrinucC peptide T7 phage library (diversity 3.9 x 108) in which random amino acids are encoded by equally represented trinucleotides to prevent codon bias. To be useful for long microdialysis experiments, the library was cloned into the genome of the T7 bacteriophage engineered for longer circulation halflife and reduced liver uptake. For biopanning, 3.9 x 1010phages displaying C7XtrinucC library was inj ected in the tail vein of rats with day 7 skin wounds. The microdialysate was collected from wounds or from normal skin for 5 h (Table 1). The majority of the phage recovery at the microdialysis probe happened during the first 2 h following library dosing - a significant extension from ~10 min half-life for wild-type T7 bacteriophage. The wound dialysates yielded almost all phages as only one phage clone was recovered from normal skin dialysate. We observed that the majority peptide-phage clones appeared in the microdialysate during the first 2 h after phage dosing. To obtain representative tissue biodistribution of phage clones in relation todialysates, the skin wound-bearing rats with no inserted dialysis probes were dosed with the naive CXVtrinucC peptide library, followed by 30 min circulation, perfusion, collection of organs and analysis of peptide landscapes. The two dialysates collected during the first 2 h were subj ected to HTS. Whole tissue samples from the wound with and without the probe ( 1 + 5 wounds) at 30 min and 5 h, as well as control samples from 14 different tissues (2 x 14 ) and from normal skin ( 1 x with and 2 x without probe), and two serum samples at the time of sacrifice (30 min and 5 h after the library inj ection) were also subjected to HTS and downstream analysis. The peptide sequences obtained from 1 and 2 h wound dialysates were then compared to 36 phage pools collected from tissue samples. First of all, we could not detect any homing induced enrichment of certain peptide sequences when wound samples were compared to healthy control organs in line with our previous experience after one round of in vivo phage screening. However, we identified 13 peptide sequences in the wound microdialysates that showed multiple copies at both dialysates (Table 2). When these sequences enriched at both dialysates were compared with the sequences from controls, it was found that one sequence (CKKNEINNC; SEQ ID NO: 8 ) was found in one skin wound sample, but all 13 were not detected in control organs and demonstrated enrichment over their presentation in the library (Table 2).

[0110] Based on the presence of multiple clones at both microdialysis analysis time points, and on sequence similarities to proteins involved in relevant pathways, we selected 3 of the enriched peptides [CDD (CDDYQQISC; SEQ ID NO: 1), CPK (CPKKHHLDC; SEQ ID NO: 2 ) and CYH (CYHDTYPNC; SEQ ID NO: 3 ) ] for further analysis.

[0111] Table 1. The number of rescued phage clones (the C7XC library screen) from the skin wound and normal skin microdialysates at different time points.Time Total phage in wound Total phage in normal skin dialysate dialysate

[0112] 60 ' 810 0

[0113] 120 ' 1920 0

[0114] 200 ' 200 0

[0115] 260 ' 45 15

[0116] 290 ' 22 0

[0117] Table 2. Screening of a naive peptide library by in vivo phage display and microdialysis yields enriched peptide sequences in skin wound dialysate. A custom-made, long circulating C7XtrinuclC T7 bacteriophage library with diversity of 3.9 x 108peptides was injected on a rat tail vein. Microdialysate from a seven days old wound was gathered during the first two hours after the library injection and peptide-encoding inserts in phage DNA was analyzed with Ion Torrent high throughput sequencing and compared to inserts from control organs harvested from the same animal. The frequency of clones detected is shown. The peptides chosen for further analysis are bolded.

[0118] Peptide SEQ ID First hour Second hour Total

[0119] NO: (counts) (counts) counts CDDYQQISC 1 10 11 21 CPKKHHLDC 2 19 23 42 CYHEITYPNC 3 14 14 28 CNYLVEKNC 4 14 9 23 CLSQTYRIC 5 19 2 21 CKMLYEYHC 6 1 17 18 CELLYNWSC 7 14 3 17 CKKNEINNC 8 6 10 16 CLTVLSEQC 9 8 7 15 CYWEDKNLC 10 6 9 15 “CFCQLMYQC 11 8 5 13

[0120] CRMKFYSEYC 12 2 9 11Newly identified, peptides bind cultured angiogenic endothelial cells and home to skin wounds

[0121] Short homing peptides are generally thought to be not species-specific as they target functionally important binding pockets highly conserved between species on target molecules. We next characterized the cellular uptake and in vivo biodistribution of the synthetic CDD, CPK and CYH peptides labeled with 6-carboxyfluorescein (FAM) on cultured Chinese hamster ovary (CHO-K) and human endothelial cells (HUVECs) in vitro and in mouse wound model in vivo. All three peptides bound to and were taken up by CHO-K and HUVEC cells, as demonstrated by confocal imaging (Figures 2 and 3).

[0122] We next studied homing of systemically administered CDD, CPK and CYH peptides in mouse skin excision wounds at the peak of neovascularization, 7 days after wounding. The peptides were injected into tail vein and allowed to circulate for 2.5 h. The systemically administered peptides were detected in the granulation tissue of the wounds outside of the blood vessels (Figures 4 and 6). Quantification showed that all three homed to skin wounds in significant amounts, the CYH peptide accumulated in wounds 9-fold (P=0.01 ), the CDD 12-fold (P=0.02 ) and CYH 5-fold (P=0.03) more than a control (Figure 7 ). In contrast, the peptides were undetectable in normal skin, heart, liver, lung, kidney, and spleen (Figure 7).

[0123] Next, we wanted to study the utility of the newly identified peptides as vehicles for targeting diabetic skin wounds. Compromised angiogenesis in diabetic wounds leads to deficient granulation tissue formation and poses an important hindrance to systemic drug delivery. Balb / cJRj and diabetic male BKS (D) -Leprdb / JOrlRj mice were wounded and systemically dosed with fluoro-phore-labeled peptides 7 days after the skin wounding.For all tested peptides we observed efficient peptide accumulation in the granulation tissue where angiogenesis takes place (Figure 8).

[0124] Homing to retinopathy and. penetration of blood-retina barrier

[0125] We wanted to explore whether the new wound-penetrating peptides externalize and penetrate tissues that have an additional barrier around the blood vessels. The retinal blood vessels have an additional element, blood-retinal barrier (BRB, equivalent of bloodbrain barrier, BBB) that hinders the access of systemically administered drugs to retina. Thus, we explored the homing of the new peptides to normal retina as well as to the retinal angiogenesis in oxygen-induced retinopathy (OIR) model. None of the peptides homed to normal retina (Figures 5 and 9), but a strong homing signal was detected in OIR retinas around retinal blood vessels and the pathological preretinal blood vessels (tufts) with the new homing peptides, CDD, CPK, and CYH, but not with a control peptide (Figure 9). There was also a strong accumulation of three peptides in OIR vitreous, whereas almost no control peptide was detected in the vitreous (Figure 9). Next, we quantified the area of positive staining in the retina. There was 9-fold more of CYH (P<0.01 ) and 120-fold and 88-fold more of CDD and CPK than of a control peptide in OIR retinas (P<0.0001 for the CDD and CPK peptides) (Figure 5). The peptide accumulation in the vitreous of the OIR retina was 16.9 x (P=0.0005), 67.2 x (P<0.0001 ) and 80.3 x (P=0.0002 ) higher than that of control peptide for CYH, CDD and CPK peptides, respectively (Figure 5).

[0126] References

[0127] Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ ( 1990) Basic local alignment search tool. J Mol Biol 215: 403-410. doi: 10. 1016 / S0022-2836 ( 05 ) 80360-2Bankhead P, Loughrey MB, Fernandez JA, Dombrowski Y, McArt DG, Dunne PD, McQuaid S, Gray RT, Murray LJ, Coleman HG, et al. (2017 ) QuPath: Open source software for digital pathology image analysis. Sci Rep 7: 16878. doi: 10.1038 / s41598-017-17204-5

[0128] De Rossi G, Vahatupa M, Cristante E, Arokiasamy S, Liyanage SE, May U, Pellinen L, Uusitalo- Jarvinen H, Bainbridge JW, Jarvinen TAH, et al. (2021 ) Pathological angiogenesis requires syndecan-4 for efficient VEGFA-induced VE-cadherin internalization. Arterioscler Thromb Vasc Biol 41: 1374-1389. doi: 10. 1161 / AT-VBAHA. 121.315941

[0129] Jarvinen TAH (2012 ) Design of target-seeking antifibrotic compounds. Methods Enzymol 509: 243-261. doi: 10. 1016 / B978-0-12-391858-1.00013-7

[0130] Kayushin AL, Korosteleva MD, Miroshnikov AI, Kosch W, Zubov D, Piel N ( 1996) A convenient approach to the synthesis of trinucleotide phosphoramidites-synthons for the generation of oligonucleotide / peptide libraries. Nucleic Acids Res 24: 3748-3755. doi: 10. 1093 / nar / 24. 19. 3748

[0131] Pemmari T, Koho T, Jarvinen TAH (2020) Probing vasculature by in vivo phage display for target organspecific delivery in regenerative medicine. In Vascularization for Tissue Engineering and Regenerative Medicine, pp 1-26. Springer Nature: Wien, Austria, doi: 10. 1007 / 978-3-319-21056- 8_21-1

[0132] Salomaa T, Pemmari T, Maatta J, Kummola L, Salonen N, Gonzalez-Rodriguez M, Parviainen L, Hiihtola L, Vahatupa M, Jarvinen TAH, et al. (2022 ) IL-13Ral suppresses tumor progression in two-stage skin carcinogenesis model by regulating regulatory T cells. J Invest Dermatol 142: 1565-1575. el7. doi: 10. 1016 / j. j id. 2021.11. 013

[0133] Toba M, Alzoubi A, O' Neill K, Abe K, Urakami T, Komatsu M, Alvarez D, Jarvinen TAH, Mann D, Ruoslahti E, et al. (2014 ) A novel vascular homing peptidestrategy to selectively enhance pulmonary drug efficacy in pulmonary arterial hypertension. Am J Pathol 184: 369-375. doi: 10. 1016 / j. a j path. 2013. 10. 008

[0134] Urakami T, Jarvinen TAH, Toba M, Sawada J, Arn-balavanan N, Mann D, McMurtry I, Oka M, Ruoslahti E, Komatsu M (2011 ) Peptide-directed highly selective targeting of pulmonary arterial hypertension. Am J Pathol 178: 2489-2495. doi: 10. 1016 / j. a j path. 2011. 02. 032

[0135] Vahatupa M, Jaaskelainen N, Cerrada-Gimenez M, Thapa R, Jarvinen T, Kalesnykas G, Uusitalo- Jarvinen H (2020) Oxygen-induced retinopathy model for ischemic retinal diseases in rodents. J Vis Exp 163: e61482. doi: 10. 3791 / 61482

[0136] Vahatupa M, Salonen N, Uusitalo- Jarvinen H, Jarvinen TAH (2021 ) Selective targeting and tissue penetration to the retina by a systemically administered vascular homing peptide in oxygen induced retinopathy (OIR). Pharmaceutics 13: 1932. doi: 10. 3390 / pharmaceu-ticsl3111932

Claims

CLAIMS1. An isolated peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12 and conservative sequence variants and pep-tidomimetics thereof.

2. The isolated peptide according to claim 1, wherein the peptide homes and penetrates into a tissue exhibiting angiogenesis.

3. The method according to claim 1 or 2, wherein the target tissue is selected from the group consisting of granulation tissue of injured tissue, ulcers, wounds with angiogenic vessels such as surgical wounds, diabetic wounds and wounds affected by a trauma, tumors, cancer tissues, and tissues affected by a medical condition that exhibits angiogenesis.

4. The method according to claim 3, wherein the medical condition that exhibits angiogenesis is retinopathy, inflammation, an inflammatory condition, a brain condition such as a brain tumor, brain metastasis, a brain disease, or a traumatic brain condition.

5. The isolated peptide according to claim 1, wherein the peptide homes to and penetrates blood-retina barrier in retinopathy or blood-brain barrier in a medical brain condition.

6. A conjugate comprising the peptide according to any one of claims 1-5 conjugated to a cargo moiety.

7. The conjugate according to claim 6, wherein the cargo moiety is a peptide, an oligopeptide, a polypeptide, a protein, a proteoglycan, an oligonucleotide, a polynucleotide, a chemical compound, a small molecule, a lipid, a liposome, a vector, a cell, a ribozyme, a virus, a phage, a microparticle or a nanoparticle.

8. The conjugate according to claim 6 or 7, wherein the cargo moiety is a detectable agent, such as a fluorescent inorganic small molecule, a fluorescent organic small molecule, protein, a fluorescent protein,a phosphorescent agent, a luminescent agent, a chromogenic agent, a radioisotope, a non-radioactive isotope, an ionic contrasting agent, a non-ionic contrasting agent, an electromagnetic agent, an upconverting nanoparticle, a resonance particle, a quantum dot or a gold particle.

9. The conjugate according to claim 6 or 7, wherein the cargo moiety is a therapeutic agent.

10. The conjugate according to claim 9, wherein the therapeutic agent is an anti-inflammatory agent, an anti-angiogenic agent, a regenerative agent, a pro-angiogenic agent, a cytotoxic agent, a pro-apoptotic agent, an anti-apoptotic agent, an antimicrobial agent, an anti-fibrotic agent, an anti-transmitter, a protransmitter, a cytokine, or a cytokine inhibitor.

11. A pharmaceutical composition comprising the conjugate according to any one of claims 6-10 and a pharmaceutically acceptable carrier.

12. A conjugate according to any one of claims 6-10 or the pharmaceutical composition according to claim 11 for use in treating an angiogenesis-related condition.

13. The conjugate or the pharmaceutical composition for use according to claim 12, wherein the angiogenesis-related condition is selected from the group consisting of cancers, retinopathies, tissue injuries and traumas, ulcers, surgical wounds, diabetic wounds, wounds affected by a trauma, inflammation, and brain diseases or pathological conditions of the brain.

14. The conjugate or the pharmaceutical composition for use according to claim 13, wherein the brain diseases or pathological conditions of the brain are selected from brain tumors, brain metastases, brain diseases, or traumatic brain conditions.

15. A conjugate according to any one of claims 6-10 or the pharmaceutical composition according to claim 11 for use in treating retinopathy.

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