Method for identifying targeting ligands based on differential endocytic removal on the vasculature of organs, and ligands identified therewith

Abstract

The present invention relates to a method for identifying ligands for targeting of a molecule and / or a drug delivery system to specific endothelial cells of at least one desired organ based on selective retention of the peptides on the surface of the desired endothelial cells. Ligands and their use for specific targeting of a molecule or a drug delivery system to a desired organ are also described.

Description

[0001] METHOD FOR IDENTIFYING TARGETING LIGANDS BASED ON DIFFERENTIAL ENDOCYTIC REMOVAL ON THE VASCULATURE OF ORGANS, AND LIGANDS IDENTIFIED THEREWITH

[0002] Technical field

[0003] The present invention relates to a method for identifying ligands for targeting of a molecule and / or a drug delivery system to specific endothelial cells of at least one desired organ based on selective retention of the peptides on the surface of the endothelial cells of the desired organ. Ligands and their use for specific targeting of a molecule or a drug delivery system to a desired organ are also described.

[0004] Background of the invention

[0005] Effective and targeted therapy delivery is important for the successful treatment of a large number of diseases. As such, many different drug delivery systems are being designed to increase therapy delivery to target organs. However, off-target delivery has been a major hurdle in designing drug delivery systems with desired efficacy and acceptable toxicity.

[0006] Delivery to brain endothelial cells is especially challenging. Efficient brain delivery will greatly benefit treating neurological disorders such as Alzheimer’s by increasing the effective therapeutic dose reaching the brain and decreasing detrimental side effects. To achieve this, drugs or drug-loaded vehicles, such as nanoparticles, need to selectively interact with and be taken up by the brain vasculature with minimal uptake by the vasculature of peripheral organs.

[0007] With that goal in mind, great efforts have been made to identify ligands promoting interaction with brain endothelial cells (BEG). To date, identification strategies have relied on selecting ligands that bind to target proteins with an elevated expression on the surface of BEG (van Rooy et al., 2010; Li et al., 2012; Zuchero et al., 2016; Tang et al., 2019; Majerova et al., 2020; Cegarra et al., 2022). While such an approach has generated important targeting systems to increase brain delivery of therapies (Chen et al., 2018, Wu et al., 2019; Edavettal et al., 2022), the identified ligands have inherent brain-specificity limitations due to significant expression of target proteins in peripheral tissues, leading to increased off-target delivery of the carriers to peripheral organs (Gaillard et al., 2014; Chen et al.., 2018; Johnsen et al., 2017, 2018; Sonoda et al., 2018; Wu et al., 2019; Aguiar et al., 2021).

[0008] Therefore, novel strategies are required to identify ligands capable of directing therapeutic cargo selectively to the brain without increasing uptake by the peripheral vasculature. While current identification strategies rely on exploiting static differences of cell-membrane composition across phenotypes, the dynamic nature of the cell membranes ( / .e. the endocytic turn-over of cell-membrane components) has so far not been taken into account when it comes to methods for identifying new and improved ligands for drug targeting. As such, the turnover dynamics of cell membrane components can be exploited to generate artificial targets specifically on desired organs by identifying ligands selectively retained on the surface of the corresponding vasculature. Such artificial target generation has thus the potential to overcome the off-target delivery limitations of current ligands.

[0009] The inventors have previously demonstrated that the lower endocytic rate of BEC may be harnessed to selectively retain biotinylated antibodies targeting the protein PECAM-1 on the surface of BEC (Gonzalez-Carter et al., 2020), thereby generating artificial targets to direct avidin-functionalized nanoparticles specifically to the brain. However, due to differences in protein function and membrane turnover, whether this phenomenon would hold for all cell-membrane components of BEC is unknown. Previous protein internalization studies have shown that the cell-membrane proteins transferrin receptor-1 (TfR1) and low- density lipoprotein receptor (LDLR), two of the principal proteins targeted by brain-delivery systems (e.g., Niewoehner et al., 2014; Molino et al., 2017; Zhang et al., 2019; Thomsen et al., 2022), have the highest rate of endocytic internalization in human brain endothelial cells (Ito et al., 2020). Ligands against such targets, therefore, might be removed preferentially from BEC vs. peripheral endothelial cells (PEC), resulting in decreased brain-targeting. Hence, strategies to identify ligands capable of exploiting the dynamic nature of cell membranes to generate artificial brain targets must probe the endocytic internalization of individual cell-membrane components with time across different endothelial phenotypes.

[0010] So far, no method for identifying targeting ligands has been described in the prior art that takes the endocytic internalization rate of the ligand into account with the aim of improving the specificity of the targeting.

[0011] The inventors have therefore herein developed a novel method that provides for these advantages by using the principle of differential endocytic removal of ligands on the endothelial cell surface. As described herein further below such identified ligands can then be used as artificial targets on the cell surface of the desired cell. These ligands can provide for targeted and highly specific drug delivery to any selected organ (“desired organ”) whilst at the same time allowing for a prolonged and high load of drug delivery. Summary of the invention

[0012] The present invention therefore relates in one aspect to a method for identifying ligands suitable for the specific targeting of a molecule and / or a drug delivery system to endothelial cells of at least one desired organ, said method comprising the steps of i. obtaining a pool of endothelial cells from the at least one desired organ (desired endothelial cells) and a pool of endothelial cells from at least one non-desired organ (non-desired endothelial cells); ii. incubating desired and non-desired endothelial cells with a ligand library; iii. selecting ligands based on their differential endocytic removal from the cell surface, wherein said step comprises the steps of separating and collecting the following ligand populations: a. ligands that are bound to the cell surface of the desired endothelial cells (“binding population 1 , BP1”) b. ligands that are bound to the non-desired endothelial cells (“binding population 2, BP2”) c. ligands that are retained on the desired endothelial cell surface (“retained population 1 , RP1”) after 2 hours of incubation, d. ligands that are retained on the non-desired endothelial cell surface (“retained population 2, RP2”) after 2 hours of incubation, and identifying the ligands present in each population (a-d) and selecting the ligands whose ratio of RP1 / BP1 is higher than the ratio of RP2 / BP2 (“selected ligands”).

[0013] In one embodiment of the present invention the said ligands are selected from peptides, aptamers, antibodies or small molecules.

[0014] In a preferred embodiment the said ligands are peptides.

[0015] In one embodiment at least one desired organ is selected from the group consisting of lung, liver, brain, heart, kidney, spleen, stomach, large intestine, small intestine, pancreas and skin. In a preferred embodiment the desired organ is the brain.

[0016] In one embodiment the at least one non-desired organ is selected from the group consisting of lung, liver, brain, heart, kidney, spleen, stomach, large intestine, small intestine, pancreas and skin. In a preferred embodiment the said non-desired organ is lung and / or liver.

[0017] In a preferred embodiment the at least one desired organ is the brain and the at least one non-desired organ is liver and / or lung. In one embodiment the drug delivery system is selected from polymeric nanomicelles, liposomes, inorganic nanoparticles, metal-organic frameworks, dendrimers, functionalized antibodies, or adeno-associated viruses (AAVs).

[0018] In one embodiment the said molecule is selected from therapeutic antibodies, nucleic acids, such as for example DNA, mRNA, siRNA or a chemotherapeutic compound.

[0019] In one embodiment the molecule further comprises a functionalization that recognizes the ligand, preferably the peptide artificial target.

[0020] In one embodiment the molecule may be encapsulated within a drug delivery system, such as for example the drug delivery systems described above.

[0021] In one embodiment the ratio of RP1 / BP1 is more than 1 and the ratio of RP2 / BP2 is less than 1 .

[0022] In a preferred embodiment the ratio of RP1 / BP1 is between 1.5 - 12.5, between 2.0 - 11.5, between 2.5 - 10.0, between 3.0 - 9.5, between 3.5 - 9.0, between 4.0 - 8.5, between 4.5 - 8.0, between 5.0 - 7.5, between 5.5 - 7.0 or between 6.0 and 6.5.

[0023] In one embodiment the ratio of RP1 / BP1 is 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 or 6.0.

[0024] In a preferred embodiment the ratio of RP2 / BP2 is between 0.1 and 0.9, between 0.2 and 0.8, between 0.3 and 0.7 or between 0.4 and 0.6.

[0025] In one preferred embodiment the ratio of RP2 / BP2 is 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9.

[0026] In one embodiment ligands that are retained on the desired endothelial cell surface (“retained population 1 , RP1”) and ligands that are retained on the non-desired endothelial cell surface (“retained population 2, RP2”) are collected after 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h or 24h of incubation following initial ligand binding to the cell surface. Preferred is the collection of RP1 and RP2 after 2h-10h, preferably after 4h-8h of incubation. In one embodiment the identified ligands present in each population are sequenced after collection.

[0027] In a further aspect the present invention relates to a ligand identified with the method as described herein.

[0028] In one embodiment the ligand identified with the method as described herein is a peptide.

[0029] In a further aspect the present invention refers to an isolated peptide comprising the amino acid sequence as depicted in SEQ ID NO.1 or SEQ ID NO.2 and an isolated peptide having an amino acid sequence of at least 80 % identity to SEQ ID NO.1 or SEQ ID NO.2.

[0030] In one embodiment the isolated peptide comprises or consists of the amino acid sequence as depicted in SEQ ID NO.2 and an isolated peptide having an amino acid sequence of at least 95 % identity to SEQ ID NO.2.

[0031] In one embodiment the ligand identified with the method as described herein is a peptide with the proviso that the peptide does not comprise or consist of an amino acid sequence as depicted in SEQ ID NO.1.

[0032] In a preferred embodiment the isolated peptide consists of the amino acid sequence of SEQ ID NO.1 or SEQ ID NO.2.

[0033] In a further aspect the present invention refers to an isolated peptide comprising the amino acid sequence as depicted in SEQ ID NO.6, SEQ ID NO.7, or SEQ ID NO.14 and an isolated peptide having an amino acid sequence of at least 80 % identity to SEQ ID NO.6, SEQ ID NO.7, or SEQ ID NO.14.

[0034] In one embodiment the isolated peptide comprises or consists of the amino acid sequence as depicted in SEQ ID NO.14 or an isolated peptide having an amino acid sequence of at least 80 % identity to SEQ ID NO.14, or an amino acid sequence of at least 85 % identity to SEQ ID NO.6 or SEQ ID NO.7.

[0035] In a preferred embodiment the isolated peptide consists of the amino acid sequence of SEQ ID NO.6, SEQ ID NO.7, or SEQ ID NO.14. In one embodiment the isolated peptide further comprises an N-terminally or C-terminally conjugated moiety / molecule.

[0036] In a further aspect the present invention relates to the peptide as described herein for use in the treatment of a disease, comprising administering said peptide and a drug delivery system or a molecule that binds to the said peptide to a subject in need thereof.

[0037] In a further aspect the present invention relates to the peptide as described herein for use in the treatment of a disease, comprising administering said peptide and a drug delivery system or a molecule that binds to the said peptide to a subject in need thereof, wherein the disease is selected from neurodegenerative diseases, such as Alzheimer’s or Parkinson’s disease, brain cancers, such as glioblastoma, neurological disorders, such as epilepsy, and neuropsychiatric disorders such as schizophrenia and bipolar disorder.

[0038] Brief description of the figures

[0039] Exemplary figure illustrating the principle with a theoretical ligand where Ri> R2

[0040] (at t = 3h). In order to identify ligands capable of increasing targeting to desired organs with time through selective retention on endothelial cell surfaces of the desired organ, the ratio of retained (R, the number of ligands remaining on the cell surface after a certain period of time) to binding (B, the number of ligands initially binding) needs to be higher in desired (R1 / B1) vs. non-desired (R2 / B2) organs (see 2)), as opposed to simply having higher retention on desired vs. non-desired organs ( / .e., Ri> R2) (see 1)). As can be seen in this figure, it is only in 2), where Ri / Bi> R2 / B2, that the targeting to desired vs. non-desired organ increases with time.

[0041] Figure 2 Schematic of avidin-FITC binding assays as further described in the Examples.

[0042] Figure 3: Selective biotin retention on the surface of brain endothelial cells Biotin tags were placed on the surface of primary endothelial cells derived from rat liver, lung or brain through conjugation of biotin-NHS with primary amines of cell-surface proteins. After incubating cells at 37°C for varying time-points to allow for the endocytic removal of the biotin tags, the remaining cell-surface biotin was measured through binding of avidin-FITC (a). Endothelial targeting by differential endocytic removal of cell-surface biotin tags was assessed by the ratio of avidin-FITC binding between different cell types with time (b). Results are displayed as mean + SEM, n = 2 independent experiments (intercalating time-points), triplicate measures. One-phase decay non-linear regression. Figure 4: Binding of avidin-FITC to biotin-labelled extracellular protein domains on endothelial cells. The extracellular domain of cell-membrane proteins on primary rat lung, liver or brain endothelial cells was biotinylated by conjugation with cell-impermeable biotin- NHS (20 mins, 4°C). Biotin labelling was then assessed by quantifying binding of avidin- FITC (30 mins, 4°C) at increasing concentrations. Results are displayed as mean + SEM of triplicate measurements.

[0043] Figure 5: Nanomicelle organ accumulation by TfR1 -targeting ligands. Mice were injected with biotinylated a-TfR1 antibody (25 pg, tail vein injection). After 15 min or 8 hrs, mice (two separate groups, each n = 4) were injected with avidin-functionalized polymeric nanomicelles (200 pg, tail vein injection). After 16hr, mice were perfused with PBS and nanomicelle biodistribution quantified in organ homogenates (a). Brain targeting ratio at each time-point is calculated by the ratio of nanomicelle uptake (b). Results are displayed as mean + SEM, * denotes p < 0.05 as determined by a student’s t-test between respective pairs.

[0044] Figure 6: Schematic of bio-panning procedure to select phage-displayed peptides which bind to (binding population) or are retained on (retained population) the surface of endothelial cells derived from rat lung, liver and brain.

[0045] Figure 7: Identification of phage-displayed peptides selectively retained on the surface of endothelial cells. Sequential bio-panning steps of an M13-phage-bound peptide library were carried out on primary endothelial cells derived from rat lung, liver or brain to identify peptides which either bind to each endothelial cell type (binding population), or which bind and are retained on the cell surface after an 8h time-period (retained population). Peptide recovery after each selection round (bio-panning step) was measured by quantifying phage concentration (PFU) (a). Progressive selection of peptides retained on the cell surface was assessed by the ratio of recovered peptides between binding / retained populations after each selection round (b). The composition of the final peptide population was assessed by sequencing the DNA of individual phage clones after the third bio-panning step (c). Population similarities were assessed by comparing sequences shared between each endothelial phenotype (d). The ratio of retained to binding CFAG peptide on liver, lung and brain endothelial cells was calculated by the percentage enrichment in each population. In populations where CFAG was not present (liver retained, brain binding) the highest theoretical value (4% and 8%, respectively) was used (e). *denotes the VSWP peptide found on brain shared 50% sequence homology with VSVP peptide found on lung and liver. Ratio of phages recovered from the ‘retained population’ to the phages recovered from the ‘binding population’ from the first bio-panning round from each endothelial cell type.

[0046] Figure 9 Delivery of avidin-FITC to endothelial cells by biotin-conjugated peptides. Peptides selectively retained on brain endothelial cells were synthesized with biotin conjugated to their N-terminal. Their ability to increase avidin delivery to primary brain endothelial cells (a) and b.END3 cells (b) was assessed by incubating cells (1 hr, 4°C) with increasing concentrations of peptides followed by incubation (30 min, 4°C) with avidin-FITC (50 pg / mL) (a, b). CFAG-biotin-mediated binding of avidin-FITC was similarly compared in primary brain and liver endothelial cells (c). The specificity of protein binding was assessed by measuring CFAG-mediated binding of avidin-FITC or albumin-FITC (both at equal molar concentrations) to b.END3 cells (d). Internalization rate of CFAG (50 pM) into brain (b.End3) or liver EC was examined by treatment with avidin-FITC following different time-periods of incubation (37°C) after CFAG-biotin binding (e, dotted line indicates plateau of one-phase decay fitting). Endothelial targeting by differential endocytic removal of CFAG on the cell surface was assessed by the ratio of avidin-FITC binding between the two cell types with time (f, error bars determined through error propagation). Results are displayed as mean + SEM, of triplicate separate measurements from 3 (a, b, e, f) or 1 (c, d) independent experiments. Slope statistical significance vs. zero value (c).

[0047] Figure 10 Avidin-FITC binding to 1 °BEC (a c) or b.END3 (b, d) mediated by CFAG-biotin,

[0048] VQNP-biotin or control peptide-biotin (100 pM) (a, b), or the transferrin peptide HAIYPRH- biotin (c, d).

[0049] Figure 11 Avidin-FITC binding to 1 °BEC (a) or C6 astrocytes (b) following incubation with biotinylated peptides (100 uM) in serum containing medium (1 hr, 37°C) (a) or HBSS (1 hr, 4°C) (b), respectively. CFAG-Cy5 binding to b.End3 cells at increasing concentrations (c). Competition of CFAG-Cy5 (3.13 uM) peptide binding to b.End3 cells by unlabelled CFAG (CFAG-biotin) (d).

[0050] Figure 12 Internalization of proteins mediated by cell-surface bound CFAG peptide. The ability of CFAG peptide (conjugated with N-terminal biotin) to bind and promote internalization of avidin-FITC into b.END3 endothelial cells was examined by labelling their cell surface with CFAG (1 hr, 4°C), followed by incubation with avidin-FITC (30 min, 4°C). The cells were then incubated at 37°C for indicated time-points and avidin-FITC localization imaged through confocal microscopy (green signal, FITC; blue signal, Hoechst 33342).

[0051] Scale bar = 50 urn.

[0052] Figure 13: Schematic representation of confocal microscopy visualization of avidin-FITC binding and internalization dynamics into brain endothelial cells.

[0053] Figure 14: Visualization of avidin-FITC internalization into primary brain endothelial cells mediated by CFAG-biotin (50 pM). Cells were treated with CFAG-biotin (1 hr, 4°C), followed by binding of avidin-FITC (30 mins, 4°C). The cells were then incubated at 37°C (4 hrs), fixed, and imaged by confocal microscopy (scale bar = 50 pm).

[0054] Figure 15: UV-vis absorbance spectra of phage library stock and recovered phage population from the first bio-panning step (a). Calibration curve of uv-vis absorbance of phage library (b).

[0055] Figure 16: Organ accumulation of nanomicelles binding artificial targets generated by a- TfR1 -antibodies. (a) Brain targeting ratio at each time-point is calculated by the ratio of nanomicelle uptake (b). Results are displayed as mean + SEM, * denotes p < 0.05 as determined by a student’s t-test between respective pairs.

[0056] DETAILED DESCRIPTION

[0057] The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention, and to the examples included therein.

[0058] As mentioned above, effective and targeted therapy delivery is important for the successful treatment of a large number of diseases. As such, many different drug delivery systems are being designed to increase therapy delivery to desired organs. However, off-target effects have been a major hurdle in designing drug delivery systems with desired efficacy and acceptable toxicity.

[0059] The dynamic nature of the cell membranes has so far not been taken into account when it comes to methods for identifying new and improved ligands for drug targeting. Previous strategies have instead focused on the static expression of proteins on the cell surface, leaving the dynamic features of the cell membrane unexamined for this purpose. So far, no method for identifying targeting ligands has been described in the prior art that takes the endocytic internalization rate of the ligands into account with the aim of improving the specificity of the targeting.

[0060] In order to further improve specificity of drug delivery systems there is a need for a novel method for identifying ligands that generate artificial targets on desired cells by being selectively retained on their surface. Ideally such binding shall be independent of single proteins / receptors present on the cell surface as such specific proteins limit the amount of ligands that can bind to the cell surface. At the same time the novel method shall be applicable for the identification of ligands for any endothelial phenotype.

[0061] The inventors have therefore herein developed a novel method that provides for these advantages by using the principle of differential endocytic removal of ligands on the endothelial cell surface.

[0062] The principle of the method of present invention may be more readily understood using the exemplary figure 1. To identify ligands suitable for specific targeting to desired organs but not to non-desired organs, the inventors firstly compared the retention of the ligands on the endothelial cell surfaces of the desired organ (Ri : number of ligands retained on the cell surface of the desired organ after a certain period of time) and on the non-desired organ (R2 : number of ligands retained on the cell surface of the non-desired organ after a certain period of time). As shown in Figure 1 , R1 > R2 means that the retention of the ligand is higher on the desired vs. the non-desired endothelial cells. However, R1 > R2 does not identify per se ligands that will over time enrich on the surface of the endothelial cells of the desired organ. The key aspect of the method of present invention thus lies in the fact that also the ratio of retained ligands to binding ligands (B: the number of ligands initially binding to the desired organ (Bi) or non-desired organ (B2)) are taken into account. This ratio needs to be higher in the desired (R1 / B1) vs. non-desired (R2 / B2) organ(s). Ligands fulfilling these criteria will show an increase on the surface of desired vs. non-desired organ over time, i.e., an enrichment on the target. In Fig.1 Ri> R2 (at t = 3) in both cases, but it is only in the second example (graphic on the right) where Ri / Bi> R2 / B2 that the amount of ligand on the cells surface of the desired vs. non-desired organ increases with time.

[0063] Comparing enrichment is important to ensure that the presence of ligands in the retained population is indeed due to the lower endocytic removal of the ligand, and not simply due to higher initial binding of the ligand, in which case the ratio of ligands present on the surface desired vs. non-desired cells would decrease with time. The ligands identified with this method can therefore provide for highly efficient targeting only to the desired organ by generating artificial targets on desired cells with increasing specificity with time.

[0064] As described herein further below, peptides identified through this method can generate artificial targets on the cell surface of the desired cell, allowing targeted delivery of cargo to these cells with increasing specificity with time.

[0065] METHOD OF THE INVENTION

[0066] In one aspect the present invention therefore relates to a method for identifying ligands suitable for the specific targeting of a molecule and / or a drug delivery system to endothelial cells of at least one desired organ, said method comprising the steps of i. obtaining a pool of endothelial cells from the at least one desired organ (desired endothelial cells) and a pool of endothelial cells from at least one non-desired organ (non-desired endothelial cells); ii. incubating desired and non-desired endothelial cells with a ligand library; iii. selecting ligands based on their differential endocytic removal from the cell surface, wherein said step comprises the steps of separating and collecting the following ligand populations: a. ligands that are bound to the cell surface of the desired endothelial cells (“binding population 1 , BP1”) b. ligands that are bound to the non-desired endothelial cells (“binding population 2, BP2”) c. ligands that are retained on the desired endothelial cell surface (“retained population 1 , RP1”) after 2 hours of incubation, d. ligands that are retained on the non-desired endothelial cell surface (“retained population 2, RP2”) after 2 hours of incubation, and identifying the ligands present in each population (a-d) and selecting the ligands whose ratio of RP1 / BP1 is higher than RP2 / BP2 (“selected ligands”).

[0067] As mentioned above the key aspect of the method of present invention lies in the fact that the ratio of retained ligands to binding ligands is considered. This ratio needs to be higher in the desired (R1 / B1) vs. non-desired (R2 / B2) organ(s). Ligands fulfilling these criteria increase their retention on the desired vs. non-desired cells over time. In one embodiment of the method of present invention the ratio of RP1 / BP1 is therefore more than 1 and the ratio of RP2 / BP2 is less than 1.

[0068] In one embodiment the ratio of RP1 / BP1 is between 1 .5 - 12.5, between 2.0 - 11.5, between 2.5 - 10.0, between 3.0 - 9.5, between 3.5 - 9.0, between 4.0 - 8.5, between 4.5 - 8.0, between 5.0 - 7.5, between 5.5 - 7.0, or between 6.0 and 6.5.

[0069] In one preferred embodiment the ratio of RP1 / BP1 is 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 or 6.0.

[0070] In one embodiment the ratio of RP2 / BP2 is between 0.1 and 0.9, between 0.2 and 0.8, between 0.3 and 0.7, or between 0.4 and 0.6.

[0071] In one embodiment the ratio of RP1 / BP1 is 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9.

[0072] In one embodiment the identified ligands present in each population are sequenced after the collection. Sequencing will be performed using methods as known in the art and suitable for the specific ligand.

[0073] In one embodiment of the present invention the said ligands are selected from peptides, aptamers, antibodies or small molecules. In a preferred embodiment the said ligands are peptides.

[0074] The method of present invention is versatile and can be used to identify ligands that are suitable for specific targeting of endothelial cells from any desired organ. It is to be understood that for putting this method into practice the practitioner will choose the suitable non-desired organs depending on the selected desired organ. It goes without saying that the desired organ cannot be the same as the non-desired organ.

[0075] In one embodiment the at least one desired organ is selected from the group consisting of lung, liver, brain, heart, kidney, spleen, stomach, large intestine, small intestine, pancreas and skin. In a preferred embodiment the desired organ is the brain.

[0076] In one embodiment the at least one non-desired organ is selected from the group consisting of lung, liver, brain, heart, kidney, spleen, stomach, large intestine, small intestine, pancreas and skin. In a preferred embodiment the said non-desired organ is lung and / or liver. In a preferred embodiment the at least one desired organ is the brain and the at least one non-desired organ is liver and / or lung.

[0077] As shown in the schematic drawing of figure 6, the inventors screened a library of phage- displayed peptides against lung, liver, or brain endothelial cells to identify two peptide populations in each endothelial phenotype: either peptides with the highest binding to the cell membrane (i.e. , the ‘binding population’) or peptides with the lowest endocytic removal from the cell surface (i.e., the ‘retained population’). By comparing the composition of these two populations and contrasting them to the populations of each endothelial phenotype, the inventors could identify peptides with a long internalization half-life, specifically in BEC as described in more detail in Example 2.

[0078] In brief, sequencing of the individual phage clones recovered from each population revealed a weak peptide enrichment across all endothelial phenotypes in the binding population, with the most enriched peptides accounting for only 13-17% of the population (fig.7c). In addition, there was a strong overlap in the peptide composition of each endothelial phenotype, with the most enriched peptide being shared by at least two phenotypes. A total of 8 individual sequences were shared between all three phenotypes (fig.7d). This heterogeneous peptide composition of the binding population reflects the large number of cell-surface components available for peptide binding, which are strongly conserved between endothelial phenotypes.

[0079] In the retained population, the inventors found a strong peptide enrichment across all endothelial phenotypes, with the most enriched peptides accounting for 43-50% of the population (fig.7c). In addition, the majority of the enriched peptide sequences were unique to each cell type, with only two shared sequences across all cell phenotypes (fig ,7d). The full peptide sequences as identified in each population are shown in table 1 of Example 2.

[0080] These results indicate that while the cell membrane components are highly conserved between endothelial phenotypes, the endocytic rates of individual components have a strong variability both within and between endothelial phenotypes.

[0081] The inventors focused on sequences enriched in the retained population but not in the binding population of BEC, thereby avoiding retention due to high initial binding (as opposed to low endocytic removal). Furthermore, they focused on sequences enriched in the binding population of peripheral endothelial cells but not present (or with decreased enrichment) in their retained population, thereby ensuring the selection of peptides with low-endocytic rates specifically in BEC ( / .e. RP1 / BP1 > RP2 / BP2).

[0082] For example, peptide sequence CFAGTPSILMLA (hereafter termed “CFAG”) fulfilled these characteristics, as it was enriched in the binding population of liver and lung endothelial cells (7% and 13% population composition, respectively) but disappeared in the liver (was under the detection threshold and thus at around 4% or lower) or was reduced in the lung (9%) in their respective retained population. The ratio of RP2 / BP2 for peptide CFAG in the liver was therefore 0.57 (4% / 7%) and 0.69 in the lung (9% / 13%).

[0083] Conversely, CFAG had the strongest enrichment in the retained population of brain endothelial cells (43%). At the same time, it was not present in the final binding population (under the detection threshold of brain endothelial cells of 8%), though logically, it was present in the initial binding population. The ratio of RP1 / BP1 for peptide CFAG in the brain was therefore 5.38 (43% / 8%). These results suggest that CFAG binds to a cell membrane component present in all three endothelial cell types but has a longer endocytic half-life on the cell membrane of brain endothelial cells.

[0084] The inventors could therefore demonstrate that using the selection criteria of the differential endocytic removal rate allows the identification of highly specific peptides that bind to and remain on the desired cell type in a large amount allowing specific drug targeting in a high amount.

[0085] In one preferred embodiment the desired organ is the brain, and the non-desired organ is the lung and / or the liver. In this embodiment the desired endothelial cells are therefore brain endothelial cells, and the non-desired endothelial cells are liver and / or lung endothelial cells.

[0086] On the other hand, the inventors could also identify peptides that are specific for lung or liver targeting.

[0087] As can be seen from fig.7c certain peptides are enriched only in the retained populations of the lung or the liver at a high percentage, for example peptide “ASHK” with 48% in the lung and “ATVL” with 50% or “GTMP” with 25% in the liver. As explained for the peptide “CFAG” above, also those peptides showed preferential retention on liver or lung endothelial cells. Whilst for these peptides there is no data on their behavior on the brain endothelial cells, as they might be present under the detection threshold of about 8%, it is highly likely that they will constitute suitable peptides for specific targeting to lung and liver.

[0088] In another embodiment of the method of present invention the desired organ is therefore the lung and the non-desired organ is the brain and / or the liver. In this embodiment the desired endothelial cells are therefore lung endothelial cells, and the non-desired endothelial cells are brain and / or liver endothelial cells.

[0089] In a further embodiment the desired organ is the liver, and the non-desired organ is the brain and / or the lung. In this embodiment the desired endothelial cells are therefore liver endothelial cells, and the non-desired endothelial cells are brain and / or lung endothelial cells.

[0090] It is to be understood that the method of present invention can be adapted to identify ligands to specifically target any type of endothelial cells using said cells as desired cells and selecting endothelial cells from different origin as non-desired cells. To adapt the method to other desired and non-desired organs it may be beneficial to modify the incubation time. In general, longer incubation time (i.e., longer time for endocytic removal) will select ligands with lowest endocytic rate, but might lead to the loss of ligands which, though they have a faster endocytic rate, could still be very suitable to generate artificial targets.

[0091] In one embodiment ligands that are retained on the desired endothelial cell surface (“retained population 1 , RP1”) and ligands that are retained on the non-desired endothelial cell surface (“retained population 2, RP2”) are therefore collected after 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11 , 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21 h, 22h, 23h or 24h of incubation. Preferred is the collection of RP1 and RP2 after 2h-10h, preferably 4h-8h of incubation.

[0092] Specifically in the embodiment wherein the desired organ is the brain, and the non-desired organ is the liver and / or lung the collection of RP1 and RP2 is performed after 4h - 8h, preferably after 8h of incubation.

[0093] Specifically interesting desired and non-desired organ combinations according to the present invention are disclosed in the table here below:

[0094] In one embodiment when the desired organ is any of lung, brain, heart, kidney, spleen, stomach, large intestine, small intestine, pancreas or skin, the non-desired organ used is at least the liver, since this organ is one of the most affected organs by off-target effects. Additional non-desired organs can of course be added to the method to achieve more specificity. In a most preferred embodiment, all possible non-desired organs are assessed.

[0095] The ligands, specifically the peptides identified by this method, are suitable to target a molecule and / or a drug delivery system to endothelial cells of a desired organ. As used herein the term “drug delivery system” relates to polymeric or lipid carrier systems that can transport active ingredients to their targets or receptor sites in a subject to be treated in a manner that provides their maximum therapeutic activity, prevent their degradation or inactivation during transit to the target site(s) and protect the subject from adverse reactions due to inappropriate disposition of the active ingredients.

[0096] In one embodiment of present invention the drug delivery system is selected from liposomes, polymeric nanomicelles, dendrimers, metal-organic frameworks, inorganic nanoparticles, lipid nanoparticles, solid lipid nanoparticles (SLN), nanogels, colloidal carrier systems, microparticles of poly (lactide) (PLA), poly (glycolide) (PGA), or poly (lactide-co- glycolide) (PLGA), Adeno Associated Virus (AAV).

[0097] Liposomes refer to spherical vesicles composed of lipid bilayers resulting from emulsification in aqueous medium.

[0098] Polymeric nanomicelles refer to nanoscopic composites of amphiphilic block co-polymers organized into a core / shell structure.

[0099] Dendrimers refer to highly ordered, branched polymeric molecules adopting a symmetric and spherical 3D structure.

[0100] Metal-organic frameworks refer to 3D structures of central metal ions coordinated to organic ligands.

[0101] Inorganic nanoparticles refer to nanoscopic particles composed of spherical structures of inorganic materials such as iron or copper.

[0102] Solid lipid nanoparticles refer to nanoscopic particles composed of solid lipids dispersed as colloids in an aqueous or oily medium.

[0103] Nanogels refer to hydrogel materials formed by 3D crosslinking of polymer networks capable of holding water without dissolving into aqueous medium.

[0104] In one embodiment the said molecule is selected from therapeutic antibodies, nucleic acids (DNA, mRNA, siRNA) or chemotherapeutic compounds.

[0105] In one embodiment of the method, the molecule is functionalized to recognize the ligands that are bound to the cell surface of the desired organ as artificial targets. The functionalization can be for example another peptide that recognizes the ligands. In a further embodiment the molecule is encapsulated within a drug delivery system, such as for example the drug delivery systems described above.

[0106] It is to be understood that in essence any micro / nanoparticle whose surface can be modified with a linker which efficiently recognizes the ligand can be used as a drug delivery system. The essential feature is that the so modified micro / nanoparticle can bind via the linker to the ligands that are provided as artificial targets on the cell surface of the desired organ.

[0107] The method of present invention comprises the step (iii) of selecting ligands based on their differential endocytic removal from the cell surface, wherein said step comprises the steps of separating and collecting, the different ligand populations from the desired and nondesired cell surface. Furthermore, the method comprises a step of identifying and comparing said different ligand populations.

[0108] In one embodiment ligands that are retained on the desired endothelial cell surface (“retained population 1 , RP1”) and ligands that are retained on the non-desired endothelial cell surface (“retained population 2, RP2”) are therefore collected after 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11 h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21 h, 22h, 23h or 24h of incubation, preferably after 2, 3, 4, 5, 6, or after 7h of incubation, preferably at 37°C in the corresponding cell culture medium. In a preferred embodiment the peptides are detected on the cell surface of the desired endothelial cells after 8h of incubation, preferably at 37°C.

[0109] In one embodiment the step (ii) of incubating the pool of desired endothelial cells and the pool of non-desired endothelial cells with a peptide library is performed by treating the endothelial cells with phages at an initial concentration of 1011pfu / mL for 1hr at 4°C, preferably in HBSS. The incubation at 4°C stops the endocytosis of the endothelial cells. After washing and removing unbound peptides, the cells (with cell-surface bound peptides) are placed in 37°C to activate endocytosis and incubated for appropriate time-periods in corresponding cell culture medium.

[0110] In one embodiment the method includes a washing step after step (ii), preferably with HBSS.

[0111] In a further embodiment the method comprises a step of recovering the phages from the cell-surface of the endothelial cells of all populations, i.e., BP1 , BP2, RP1 , RP2 by incubating each pool after step (ii) in acidic buffer of a pH of between 1 to 3, such as for example citric acid, preferably at a concentration of 100 mM citric acid and a pH of 2.2. In one embodiment the steps of incubating (ii), washing and recovering the phages can be repeated several times, preferably at least two, most preferably at least three times. The use of such sequential biopanning steps ensures selection and enrichment of the best candidates.

[0112] As described in Example 2, phage recovery increased in the binding population with each bio-panning step, indicating selection of phages displaying peptides successfully binding to the cell membrane. Importantly however, phage recovery from the retained population increased even more strongly (fig.6a), increasing the retained / binding phage ratio with each bio-panning round (fig.7b), suggesting selection of phages displaying peptides with both successful bindings to and low endocytic removal from the cell surface.

[0113] In a further embodiment a step of amplification of the recovered phages retained on the cell surface can be performed before applying them in a further incubation step as described above.

[0114] It is to be understood that the method of the invention as referred to herein is performed in vitro on isolated endothelial cells of the desired and non-desired organs.

[0115] PEPTIDES OF THE INVENTION

[0116] In a further aspect the present invention relates to the peptides as identified with the method as described above.

[0117] Preferred are the peptides that show low endocytic removal from the desired endothelial cell surface and show high endocytic removal from the non-desired endothelial cell surface.

[0118] The peptides with increasing presence in retained population 1 vs. binding population 1 but decreasing presence in retained population 2 vs. binding population 2 (R1 / B1 > R2 / B2) are therefore preferred and are selected (“selected peptides”).

[0119] In one aspect the present invention relates to an isolated peptide comprising the amino acid sequence as depicted in SEQ ID NO.1 or SEQ ID NO.2.

[0120] SEQ ID NO.1 (CFAGTPSILMLA) is also referred to as peptide “CFAG” herein.

[0121] SEQ ID NO.2 (VQNPQRPSLMSY) is also referred to as peptide “VQNP” herein. In one embodiment the isolated peptide has an amino acid sequence of at least 80 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence depicted in SEQ ID NO.1 or SEQ ID NO.2.

[0122] In one embodiment the isolated peptide consists of the amino acid sequence of SEQ ID NO.1 or SEQ ID NO.2.

[0123] The isolated peptides comprising or consisting of the amino acid sequence depicted in SEQ ID NO.1 or SEQ ID NO.2 are particularly suitable for the specific targeting of brain endothelial cells.

[0124] Further particularly suitable isolated peptides for the specific targeting of brain endothelial cells comprise or consist of the amino acid sequence depicted in SEQ ID NO.3, 4 or 5. In one embodiment the present invention therefore relates to an isolated peptide comprising or consisting of the amino acid sequence as depicted in SEQ ID NO.3, 4 or 5, or an isolated peptide having an amino acid sequence of at least 80 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence depicted in SEQ ID NO.3, 4 or 5.

[0125] In a further embodiment the isolated peptide comprises the amino acid sequence as depicted in SEQ ID NO.6 or SEQ ID NO.7.

[0126] SEQ ID NO.6 (ATVLKPITRSLD) is also referred to as peptide “ATVL” herein.

[0127] SEQ ID NO.7 (GTMPHVSSHASG) is also referred to as peptide “GTMP” herein.

[0128] In one embodiment the isolated peptide has an amino acid sequence of at least 80 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence depicted in SEQ ID NO.6 or SEQ ID NO.7.

[0129] In one embodiment the isolated peptide consists of the amino acid sequence of SEQ ID NO.6 or SEQ ID NO.7.

[0130] The isolated peptides comprising or consisting of the amino acid sequence depicted in SEQ ID NO.6 or SEQ ID NO.7 are particularly suitable for the specific targeting of liver endothelial cells. Further particularly suitable isolated peptides for the specific targeting of liver endothelial cells comprise or consist of the amino acid sequence depicted in SEQ ID NO.8, 9 or 10. In one embodiment the present invention therefore relates to an isolated peptide comprising or consisting of the amino acid sequence as depicted in SEQ I D NO.8, 9 or 10, or an isolated peptide having an amino acid sequence of at least 80 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence depicted in SEQ ID NO.8, 9 or 10.

[0131] In a further embodiment the isolated peptide comprises the amino acid sequence as depicted in SEQ ID NO.14.

[0132] SEQ ID NO.14 (ASHKSDPKSNLF) is also referred to as peptide “ASHK” herein.

[0133] In one embodiment the isolated peptide has an amino acid sequence of at least 80 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence depicted in SEQ ID NO.14.

[0134] In one embodiment the isolated peptide consists of the amino acid sequence of SEQ ID NO.14.

[0135] The isolated peptides comprising or consisting of the amino acid sequence depicted in SEQ ID NO.14 are particularly suitable for the specific targeting of lung endothelial cells.

[0136] Further particularly suitable isolated peptides for the specific targeting of lung endothelial cells comprise or consist of any one of the amino acid sequences depicted in SEQ ID NO.1 , 4, 11-13 or 15-17. In one embodiment the present invention therefore relates to an isolated peptide comprising or consisting of the amino acid sequence as depicted in SEQ ID NO.1 , 4, 11-13 or 15-17, or an isolated peptide having an amino acid sequence of at least 80 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence depicted in SEQ ID NO.1 , 4, 11-13 or 15-17.

[0137] The peptides of present invention can be modified. In one embodiment the peptides comprise an N-terminally or C-terminally conjugated moiety / molecule. The moiety can be biotin, a biotin derivative {e.g., 2-iminobiotin), the ‘SpyTag’ peptide (designed to specifically and covalently attach to the ‘SpyCatcher’ protein) or ferrocene (efficiently recognized by cucurbit[7]uril). In another embodiment, the peptides may be detected directly by antibodies that bind to the peptide sequence.

[0138] USES AND METHODS OF TREATMENT

[0139] In a further aspect the present invention relates to the use of the ligands, preferably peptides, as described herein for specific targeting of a molecule / delivery system to a desired organ.

[0140] In one embodiment the invention relates to the ligand as identified with the method described herein for use in the treatment of a disease, comprising administering the said ligand to generate artificial targets on the surface of the endothelial cells of the desired organ and administering a molecule / drug delivery system that binds to the said artificial targets to a subject in need thereof. In a preferred embodiment the administration is performed sequentially, the ligands are administered to a subject in need thereof first and the molecule / drug delivery system second.

[0141] In one embodiment the invention relates to any of the peptides as described herein for use in the treatment of a disease, comprising administering the said peptides and a molecule / drug delivery system that binds to the said peptide to a subject in need thereof.

[0142] In one embodiment the invention relates to an isolated peptide comprising or consisting of the amino acid sequence as depicted in SEQ ID NO.1 or SEQ ID NO.2, or an isolated peptide having an amino acid sequence of at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO.1 or SEQ ID NO.2 for use in the treatment of a disease, comprising administering said peptide and a drug delivery system, or a molecule, that binds to the said peptide to a subject in need thereof.

[0143] In one embodiment the invention relates an isolated peptide comprising or consisting of the amino acid sequence as depicted in SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5, or an isolated peptide having an amino acid sequence of at least 80 %, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identity to SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5 for use in the treatment of a disease, comprising administering said peptide and a drug delivery system or a molecule that binds to the said peptide to a subject in need thereof. In one embodiment the invention relates to an isolated peptide comprising or consisting of any one of the amino acid sequences as depicted in SEQ ID NOs.6 to 10, or an isolated peptide having an amino acid sequence of at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to any one of the amino acid sequences depicted in SEQ ID NOs.6 to 10 for use in the treatment of a disease, comprising administering said peptide and a drug delivery system, or a molecule, that binds to the said peptide to a subject in need thereof.

[0144] In one embodiment the invention relates to an isolated peptide comprising or consisting of any one of the amino acid sequence as depicted in SEQ ID NO.1 , 4, or 11-17, or an isolated peptide having an amino acid sequence of at least 80 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to any one of the amino acid sequences depicted in SEQ ID NO.1 , 4, or 11-17 for use in the treatment of a disease, comprising administering said peptide and a drug delivery system, or a molecule, that binds to the said peptide to a subject in need thereof.

[0145] In one embodiment the diseases are selected from neurodegenerative diseases, such as for example Alzheimer’s disease or Parkinson’s disease, and brain cancers, such as for example glioblastoma. In one embodiment the diseases are selected from neurological disorders including but not limited to epilepsy and neuropsychiatric disorders such as schizophrenia and bipolar disorder.

[0146] In a preferred embodiment, wherein the disease is a neurodegenerative disease, the molecule can be a monoclonal antibody, such as for example aducanumab or lecanemab, capable of preventing the formation of toxic protein aggregates in the brain, or growth factors capable of supporting neuronal survival, such as for example GDNF or BDNF.

[0147] In a preferred embodiment, wherein the disease is a brain cancer, the molecule can be a chemotherapeutic drug, such as for example temozolomide.

[0148] In a further embodiment the ligands identified with the method of present invention can be used to selectively modulate the function of brain endothelial cells. This can be advantageous to for example selectively open the blood-brain barrier to allow therapy entry into the brain. A further option is to use brain endothelial cells as drug reservoirs.

[0149] In one embodiment the invention relates to the use of the ligand, preferably the peptide, as identified with the method described herein for generating artificial targets on the surface of the endothelial cells of the desired organ. In a preferred embodiment of the use, the ligand is administered to the subject leading to the formation of the artificial target on the surface of the endothelial cells of the desired organ.

[0150] In one embodiment the invention relates to the use of an isolated peptide comprising or consisting of the amino acid sequence as depicted in SEQ ID NO.1 or SEQ ID NO.2, or an isolated peptide having an amino acid sequence of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO.1 or SEQ ID NO.2 for generating artificial targets on the surface of the endothelial cells of the desired organ. In a preferred embodiment of the use, the ligand is administered to the subject leading to the formation of the artificial target on the surface of the endothelial cells of the desired organ.

[0151] In one embodiment the invention relates to the use of an isolated peptide comprising or consisting of the amino acid sequence as depicted in SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5, or an isolated peptide having an amino acid sequence of at least 80 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identity to SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5 for generating artificial targets on the surface of the endothelial cells of the desired organ. In a preferred embodiment of the use, the ligand is administered to the subject leading to the formation of the artificial target on the surface of the endothelial cells of the desired organ.

[0152] In one embodiment the invention relates to the use of an isolated peptide comprising or consisting of the amino acid sequence as depicted in SEQ ID NOs.6 to 10, or an isolated peptide having an amino acid sequence of at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to any one of the amino acid sequences depicted in SEQ ID NOs.6 to 10 for generating artificial targets on the surface of the endothelial cells of the desired organ. In a preferred embodiment of the use the ligand is administered to the subject leading to the formation of the artificial target on the surface of the endothelial cells of the desired organ.

[0153] In one embodiment the invention relates to the use of an isolated peptide comprising or consisting of the amino acid sequence as depicted in SEQ ID NO.1 , 4, or 11-17, or an isolated peptide having an amino acid sequence of at least 80 %, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to any one of the amino acid sequences depicted in SEQ ID NO.1 , 4, or 11-17 for generating artificial targets on the surface of the endothelial cells of the desired organ. In a preferred embodiment of the use, the ligand is administered to the subject leading to the formation of the artificial target on the surface of the endothelial cells of the desired organ.

[0154] The term “treatment” as used in the present application, refer to a set of hygienic, pharmacological, surgical and / or physical means used with the intent to cure and / or alleviate a disease and / or symptoms with the goal of remediating the health problem. The term “treatment” includes preventive and curative methods, since both are directed to the maintenance and / or reestablishment of the health of an individual or animal. Regardless of the origin of the symptoms, disease and disability, the administration of a suitable medicament to alleviate and / or cure a health problem should be interpreted as a form of treatment or therapy within the context of this application.

[0155] The use of the word "a" or "an" may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one". The use of the term “another” may also refer to one or more. The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0156] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. The term “comprises” also encompasses and expressly discloses the terms “consists of’ and “consists essentially of”. As used herein, the phrase "consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. As used herein, the phrase "consisting of” excludes any element, step, or ingredient not specified in the claim except for, e.g., impurities ordinarily associated with the element or limitation.

[0157] The term "or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0158] As used herein, words of approximation such as, without limitation, "about", "around”, “approximately” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as "about" may vary from the stated value by ±1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10%. Accordingly, the term “about” may mean the indicated value ± 5% of its value, preferably the indicated value ± 2% of its value, most preferably the term “about” means exactly the indicated value (± 0%).

[0159] The term “sequence identity” refers to a percentage value obtained when two sequences are compared using a pairwise sequence alignment tool. In the present case, the sequence identity is obtained using the global alignment tool “EMBOSS Needle” using the default settings (Rice et al., 2000. Trends Genet. 16(6):276-7; Li et al., 2015. Nucleic Acids Res. 43(W1):W580-4). The global alignment tool is available at: https: / / www.ebi.ac.uk / Tools / psa / .

[0160] The following examples and figures are provided by way of illustration and are not intended to limit the scope of the present invention.

[0161] EXAMPLES

[0162] Example 1 : Determination of endocytic rates in lung, liver, and brain endothelial cells

[0163] The endocytic rates in primary endothelial cells extracted from rat lungs, liver, or brain were determined by measuring the internalization of cell-membrane proteins with time. To this end, the extracellular domains of cell-surface proteins were labeled with biotin, and their retention on the cell surface after various incubation (37°C) time intervals was monitored by quantifying avidin (avidin-FITC) binding (fig.3a). The concentration of biotin targets (i.e., biotin-labeled proteins) generated on each endothelial monolayer was comparable between all EC phenotypes, as determined by the similar levels of avidin binding at the initial timepoint (t = Ohr, fig.3a). Saturation of biotin targets did not account for the comparable levels of avidin binding, since avidin binding increased comparably in all cell types with increasing avidin concentrations (fig.4). However, the retention of biotinylated proteins on the cell membrane differed markedly between brain EC and peripheral EC. While there was a sharp and comparable decrease in avidin binding to lung and liver EC with increasing incubation times, binding of avidin to brain EC was maintained for a longer period (fig.3a). One-phase decay fitting determined the internalization half-life of biotinylated cell-surface proteins on lung, liver and brain EC to be 2.09, 2.41 and 5.49 hours, respectively.

[0164] These results indicate that while there is a comparable concentration of cell-membrane proteins in all three EC phenotypes, their rate of endocytic removal from the cell surface is markedly lower in BEC vs. peripheral EC. Considering the avidin-biotin interaction as a delivery system, the targeting of each endothelial phenotype can be assessed by their respective avidin-binding ratios (fig.3b). While the similar endocytic rates between liver and lung EC resulted in a constant endothelial targeting ratio, the targeting to BEC increased markedly with time against both lung and liver. Brain targeting increased nearly three-fold at the final time-point examined (20h).

[0165] Example 2: Identification of ligands selectively retained on the surface of BEC

[0166] The above results demonstrate that the lower rate of endocytosis of cell-surface proteins may be harnessed to increase the targeting of BEC against peripheral EC. To examine whether this effect held for individual cell-surface proteins, we conducted preliminary in vivo experiments to test whether the brain-targeting of nanoparticles by TfR ligands could be enhanced by increasing the time interval between ligand and ligand and ligand and nanoparticle injection (fig.5). To this end, mice were intravenously (i.v.) injected with avidin- functionalized nano micelles at various time points after i.v. Injection of biotinylated anti-TfR antibodies. While there was stronger targeting of the nanoparticles to the brain compared to the lung, the brain-to-lung targeting ratio decreased with increasing time intervals, indicating the rate of endocytosis of TfR ligands was higher in BEC compared to lung EC. Therefore, to harness differential endocytic profiles of EC to target the brain, we identified novel ligands that could be selectively retained on the surface of BEC by unbiasedly probing the endocytic rate of individual cell-membrane components. To this end, we screened a library of phage-displayed peptides against lung, liver, or brain endothelial cells to identify two peptide populations in each endothelial phenotype: either peptide with the highest binding to the cell membrane (i.e., the ‘binding population’) or peptides with the lowest endocytic removal from the cell surface (i.e., the ‘retained population’) (fig.6). By comparing these two populations' peptide compositions and contrasting them to the populations of each endothelial phenotype, we aimed to identify peptides with a long internalization halflife, specifically in BEC.

[0167] To assess whether the screening procedure enriched phage-displayed peptides preferentially retained on cell surfaces, we first quantified the number of phages recovered from the cell surface after each bio-panning round (fig.7a). Phage recovery was 10-20-fold lower in the retained population compared to the binding population for all cell types after the first bio-panning round, indicating strong endocytic removal of phages from the cell surface. Interestingly, the first rounds retained / binding phage ratio (fig.8) mirrored the endocytic half-lives of biotinylated cell-surface proteins, with the highest proportion in BEC, followed by liver and lung EC, respectively. Phage recovery increased in the binding population with each bio-panning step, indicating enrichment of phages displaying peptides successfully binding to the cell membrane. Importantly however, phage recovery from the retained population increased even more strongly (fig.7a), increasing the retained / binding phage ratio with each bio-panning round (fig.7b), suggesting enrichment of phages displaying peptides with both successful bindings to and low endocytic removal from the cell surface.

[0168] To verify the enrichment of phage-displayed peptides by the selection paradigm, we sequenced individual phage clones recovered from the third bio-panning step of each population. In the binding population, there was weak peptide enrichment across all endothelial phenotypes, with the most enriched peptides accounting for only 13-17% of the population (fig.7c). In addition, there was a strong overlap in the peptide composition of each endothelial phenotype, with the most enriched peptide being shared by at least two phenotypes. A total of 8 individual sequences were shared between all three phenotypes (fig.7d). The heterogeneous peptide composition of the binding population reflects the large number of cell-surface components available for peptide binding, which are strongly conserved between endothelial phenotypes.

[0169] In contrast, in the retained population, there was a strong peptide enrichment across all endothelial phenotypes, with the most enriched peptides accounting for 43-50% of the population (fig.7c). In addition, the majority of the enriched peptide sequences were unique to each cell type, with only two shared sequences across all cell phenotypes (fig.7d) (full sequences see Tablel). These results indicate that while the cell membrane components are highly conserved between endothelial phenotypes, the endocytic rates of individual components have a strong variability both within and between endothelial phenotypes. Next, we compared individual peptide sequences of each cell type's binding and retained populations to identify candidates selectively had on the brain endothelial cells. To this end, we focused on sequences enriched in the controlled population but not in the binding population of BEC, thereby avoiding retention due to high initial binding (as opposed to low endocytic removal). Furthermore, we focused on sequences enriched in the binding population of peripheral endothelial cells but not present (or with decreased enrichment) in their retained population, thereby ensuring the selection of peptides with low-endocytic rates, specifically BEC. One such peptide sequence fulfilled these characteristics: CFAGTPSILMLA (hereafter termed CFAG) was enriched in the binding population of liver and lung endothelial cells (7 and 13% population composition, respectively) but disappeared from the former. At the same time, it reduced to 9% in the latter in their respective retained populations.

[0170] Conversely, CFAG had the strongest enrichment in the retained population of brain endothelial cells. At the same time, it was not present in the final binding population, though logically, it was present in the initial binding population. These results suggest that CFAG binds to a cell membrane component present in all three endothelial cell types, but which has a longer endocytic half-life, specifically in the cell membrane of brain endothelial cells.

[0171] The most enriched peptides in the retained population on BECs were peptides CFAGTPSILMLA (hereafter termed “CFAG”) and VQNPQRPSLMSY (hereafter termed “VQNP”) as can be seen in table 1 here below:

[0172] Table 1 : Full amino acid sequence and frequency of analyzed individual clones from the third bio-panning round for each endothelial cell type and selection regime. a) Brain retained population b) Liver retained population c) Lung retained population d) Liver binding population e) Lung binding populations f) Brain binding populations

[0173] On the other hand, also for example peptides “ASHK”, “GTMP” and “ATVL” have a longer half-life specifically on the cell membrane of lung endothelial cells and liver endothelial cells, respectively.

[0174] Example 3: Generation of artificial targets for protein delivery, specifically on brain endothelial cells

[0175] Following identifying phage-displayed peptides selectively retained on the surface of brain endothelial cells, we investigated whether the peptides in their free-form (i.e., synthesized peptides not bound to phage particles) could be employed to generate artificial cell-surface targets for the selective delivery of proteins into brain endothelial cells. To this end, the two most enriched peptide sequences on the retained population of BEC (i.e., CFAG and VQNP) were synthesized with a biotin conjugate on their N-terminal to allow delivery of avidin as a model protein. As controls, we synthesized a reverse sequence of VQNP and a commercially available peptide targeting TfR1 (Tf peptide HAIYPRH), as can be seen in table 2: Table 2: Synthesized peptide characteristics

[0176] Both CFAG and VQNP allowed efficient delivery of avidin to primary brain endothelial cells (fig.9a, 10a), demonstrating that the peptides retained their ability to bind to the endothelial cell surface in their free form and were able to display the biotin conjugate for engaging avidin. Importantly, no avidin delivery was seen for the control peptide (fig.9a, 10a). Furthermore, the identified peptides allowed higher avidin delivery than the Tf peptide (fig. 9c, 10c). The delivery of avidin was also examined in brain endothelial cells derived from mice (b.End3 cells). Interestingly, CFAG had a stronger effect on avidin delivery to b.End3, while VQNP failed to increase avidin delivery (fig.9b, 10b). Similarly, to primary BEC, the control peptide did not affect avidin delivery, while the Tf peptide induced lower avidin delivery than CFAG (fig.9b, 10b, 10d).

[0177] The specificity of avidin delivery by CFAG was examined by measuring albumin delivery as a control protein. No increase in albumin delivery was seen with increasing CFAG concentrations (fig.9d), demonstrating that the delivery of avidin was specifically due to the protein-biotinylated peptide interaction. In addition, CFAG increased avidin binding to brain endothelial cells in the presence of serum proteins (fig.11a) and had selectivity for brain endothelial cells over astrocytes (fig.10b). To test the saturation of peptide binding, b.End3 cells were treated with fluorescently-tagged CFAG (Cy5) in the presence of increasing concentrations of unlabelled CFAG (CFAG-biotin). CFAG-Cy5 is efficiently bound to the cell surface, with a lower concentration limit of 20nM. CFAG-Cy5 binding commenced to saturate at 25 mM, but a binding plateau was not reached with the highest concentration tested (100 M) (fig.11c). Co-treatment with unlabelled CFAG reduced CFAG-Cy5 binding at the highest concentrations tested (fig.11d).

[0178] Next, we examined whether the free CFAG peptide could still exploit endothelial cells' differential endocytic internalization rate to generate artificial targets selectively on BEC. CFAG binding was comparable in both brain and liver endothelial cells, resulting in a lack of avidin targeting (fig.9d). However, CFAG-mediated delivery to liver endothelial cells decayed at a significantly faster rate than delivery to brain endothelial cells (fig.9e). The differential endocytic removal of CFAG on the surface of endothelial cells allowed the peptide to generate artificial targets preferentially on the surface of brain endothelial cells, leading to an increase in the targeting of avidin to brain endothelial cells with time (fig ,9f).

[0179] Example 4: Intracellular protein delivery into brain endothelial cells by identified ligands The ability of CFAG peptide to generate artificial targets for selective protein delivery to BEC relies on its low endocytic internalization rate. Therefore, we examined whether CFAG could induce avidin internalization into brain endothelial cells following protein binding despite its retention on the cell surface. To this end, BEC was decorated with CFAG peptide and immediately treated with avidin at 4°C to avoid endocytosis. Protein localization was then visualized at various time points following incubation at 37°C (fig.2c) (fig.12). At the initial time-point (Oh), the FITC signal was evenly distributed on the cell surface (as determined by the homogenous distribution in the cell-body areas distant from the nucleus) (closed arrows) without penetrating the cytosol (as determined by the strong signal in the cell-membrane adjacent to the nucleus and lack of signal in the perinuclear space) (open arrows) (fig.12), indicating avidin bound to the cell-surface CFAG peptide. Within one hour, the even membrane distribution was disrupted, and a punctate pattern began to appear, indicating endocytic internalization of the avidin-CFAG complex. By 4 hours, the cell membrane localization had completely disappeared, with all the FITC signal present in a granular pattern surrounding the nucleus (arrowheads), indicating successful internalization of most peptide-bound avidin into endocytic vesicles. A similar way of avidin-FITC internalization by cell-surface-bound CFAG was seen for primary brain endothelial cells (fig.14).

[0180] Example 5: Organ accumulation of nanomicelles binding artificial targets generated by a- TfR1 -antibodies.

[0181] Mice were injected with biotinylated a-TfR1 antibody (25 ug, tail vein injection). After 15 min or 8 hrs, mice (two separate groups, each n = 4) were injected with avidin-functionalized polymeric nanomicelles (200 ug, tail vein injection). After 16hr, mice were perfused with PBS and nanomicelle biodistribution quantified in organ homogenates.

[0182] The results can be seen in Figure 16: (a) Brain targeting ratio at each time-point is calculated by the ratio of nanomicelle uptake (b). In this example, it was demonstrated that antibodies against the cell-surface protein TfR do not increase brain targeting with time, i.e., do not generate brain-specific targets despite strong binding to the brain. This clearly demonstrates the need to carefully measure the binding and retention of molecular tags to successfully generate brain-specific artificial targets. Example 6: In vivo assessment of the CFAG peptide for generating artificial brain targets The inventors are currently examining the ability of the CFAG peptide to generate artificial brain targets in mice. To this end, mice are intravenously injected (tail-vein injection) with biotinylated CFAG peptides. The generation of brain-specific artificial targets will be assessed by the retention of the peptide on the endothelial cell surface of different organs (namely brain, liver, lung, kidney, pancreas and heart). Peptide retention will be assessed by guantifying the accumulation of fluorescently labelled avidin (injected intravenously) at various time-points (15min, 1 h, 2h, 4h, 8h) following CFAG peptide administration. These experiments will allow the inventors to corroborate the in vitro findings showing the in vivo utility of CFAG. Furthermore, the peptide dose and administration time-interval to maximally generate artificial brain targets for therapeutic delivery will be assessed.

[0183] Materials and Methods

[0184] Materials and reagents

[0185] The phage-displayed peptide library was purchased from New England Biolabs (Ipswich, MA). IPTG, PEG8000, LB broth, tetracycline, agar, fibronectin, collagen, gelatine, bovine fluorescent albumin (albumin-FITC), bovine pancreas trypsin, collagenase type-l, dispase I, and puromycin were purchased from Sigma-Merck (St. Louis, MO). Cell-impermeable biotin-NHS (sulfo-NHS-biotin), avidin (neutravidin)-FITC, and X-gal were purchased from ThermoFisher Scientific (Waltham, MA). Rat primary liver, lung endothelial cells, and peripheral endothelial basal cell culture medium (ECM) were purchased from Cell Biologies (Chicago, IL). Rat primary brain endothelial cells were isolated in-house. Mouse brain endothelial cells b.End3 (CRL-2299) was purchased from the American Type Culture Collection. Microvascular endothelial cell growth medium (EGM-2 plus growth factor supplements) was purchased from Lonza (Basel, CH). All peptides were custom ordered from GenScript Biotech (Piscataway, NJ).

[0186] Cell culturing

[0187] Rat primary lung, liver, and brain endothelial cells were extracted from the homogenized organs of 6-8 week-old Sprague Dawley rats. Peripheral (lung and liver) endothelial cells were purified by pre-coating cells with anti-PECAM1 (CD31) antibody and separated by secondary antibody-coated magnetic beads. Peripheral endothelial cells were plated on gelatin (0.5% w / v)-coated flasks and cultured in endothelial basal medium supplemented with 10% v / v FBS, 1 % v / v endothelial cell growth supplements, and penicillin (100IU) / streptomycin (100 ug / mL). Cells were detached by trypsinization and plated on gelatin-coated culture wells. Once monolayers reached 100% confluency (2-3 days following plating), experimentation was carried out.

[0188] Rat primary brain endothelial cells were extracted as described previously (Gonzalez-Carter et al., 2019, 2020). Briefly, rat brain cortices (cleaned of meninges and visible blood vessels) were homogenized and digested with an enzyme mixture (trypsin, collagenase, dispase). Microvessels were separated from the digested tissue homogenate by centrifugation in a separation gradient buffer (25% v / v BSA). The resulting microvessel pellet was further digested with an enzyme mixture and plated in culture flasks coated with collagen / fibronectin. Culturing was done in EGM-2 endothelial cell culture medium (with FBS, VEGF, FGF, IGF, EGF, ascorbic acid, hydrocortisone, and gentamycin) (termed full- EGM), supplemented with puromycin to eliminate contaminating cells. Once a pure culture was obtained, cells were detached by trypsinization and plated on collagen / fibronectin- coated wells in full EGM. Once cells reached 90% confluency, VEGF was removed from the culturing medium to promote a BBB phenotype. Experimentation was carried out once monolayers reached 100% confluency (2-3 days following removal of VEGF).

[0189] Mouse b.End3 cells were detached by trypsinization and plated in collagen-coated cell culture wells in DMEM (10% v / v FBS, penicillin (100IU) / streptomycin (100 ug / mL)) until reaching 100% confluency (4-5 days).

[0190] All cells were maintained at 37°C in a humidified atmosphere with 5% CO2.

[0191] Measurement ofendocytic internalization of biotinylated cell-surface proteins

[0192] To determine endocytic internalization rates, confluent monolayers of primary rat liver, lung, or brain endothelial cells were treated with the cell-membrane impermeable biotinylation reagent sulfo-biotin-NHS (20 mins, 4°C, 500 ug / mL), thereby attaching a biotin molecule to primary amines of the extracellular domain of cell-membrane proteins. Following thorough washing, the cells were incubated in their respective culture medium (37°C) for appropriate periods, after which the cells were washed (HBSS) and treated with neutravidin-FITC (30 mins, 4°C, 50 ug / mL). Cells were thoroughly washed (HBSS) to remove unbound neutravidin-FITC, and fluorescence (490 / 525 em / ex) was read with a Spark multimode microplate reader (Tecan).

[0193] Phage-displayed peptide selection

[0194] Bio-panning was selected on the M13-phage combinatorial library displaying 109unique dodecamer peptide sequences. Confluent primary rat lung, liver, or brain endothelial cells were treated with phages at an initial concentration of 1011pfu / mL ( / .e., 100 copies of each peptide sequence) in HBSS (1 hr, 4°C). After thoroughly washing with HBSS, cells were either incubated in 100 mM citric acid (pH 2.2) to recover cell-surface phages (recovered phages were then neutralized with equal volumes of 1 M Tris-HCI, pH 7.5) or incubated in corresponding cell culture medium (at 37°C) for 8 hrs to allow phage endocytic internalization. After this time, phages retained on the cell surface were recovered as above. Recovered phages were amplified in E. Coli per the manufacturer’s instructions and employed for the subsequent bio-panning steps. The phage purity / concentration of the amplified phage eluate recovered after each bio-panning step was assessed through UV- vis (fig.15).

[0195] Unamplified phages from the final bio-panning step were titered in LB-agar plates supplemented with IPTG / X-gal. The DNA from individual plaques derived from library phages ( / .e., blue-colored plaques) was sequenced to determine their peptide composition, per the manufacturer’s instructions. In brief, the individual phage plaque was amplified in E.Coli and isolated through PEG / NaCI precipitation. DNA was isolated by incubating phages in Tris-HCl / EDTA / Nal buffer, followed by precipitation in ethanol. The DNA pellet was resuspended in MilliQ H2O. Purified ssDNA was quantified by fluorimetry (Qubit ssDNA assay with a Qubit 4 fluorometer) (Invitrogen) and sequenced through Sanger sequencing using the -96 gill sequencing primer (5’-CCCTCATAGTTAGCGTAACG-3’) performed with BigDye™ Terminator v3.1 Cycle Sequencing Kit and electrophoresed on an Applied Biosystems Automated 3730x1 DNA analyzer.

[0196] Peptide synthesis

[0197] All peptides were custom synthesized by GenScript Biotech. Either biotin or Cy5 was conjugated onto the peptide N-terminal. To recreate the peptide conformation adopted when bound to the phage, the amino acid linker sequence Gly-Gly-Gly-Ser found between the pill phage protein and the peptide sequence was added to the C-terminal of the 12- amino acid sequence (X12GGGS). In addition, the C-terminal was amidated to avoid a negatively charged carboxylate group not found in the phage-bound form. Peptide purity was > 98%.

[0198] Peptide cell-binding

[0199] Confluent endothelial monolayers were treated with increasing CFAG-Cy5 concentrations in HBSS (1 hr, 4°C). Cells were thoroughly washed with HBSS and fluorescence (649 / 667 em / ex) quantified with a Spark multimode microplate reader (Tecan). The same protocol was followed for competition assays, except cells were incubated with CFAG-Cy5 in the presence of increasing concentrations of unlabelled CFAG (CFAG-biotin). Peptide-mediated protein cell delivery

[0200] Confluent endothelial monolayers were treated with increasing peptide concentrations in HBSS (1 hr at 4°C, unless otherwise stated). After removing the unbound peptide, cells were either incubated in an appropriate cell culture medium (37°C) for varying time points or directly treated with neutravidin-FITC or albumin-FITC (0.83 mM, in HBSS, 30 mins at4°C). After removing unbound protein with thorough HBSS washes, fluorescence (490 / 525 em / ex) was quantified with a Spark multimode microplate reader (Tecan) or imaged through confocal microscopy (fig.2a-c).

[0201] Statistical analysis

[0202] Statistical analysis was carried out through linear and non-linear (one-phase decay) regression analysis, one-way ANOVA (with Tukey’s post-hoc tests), and f-tests (unpaired, two-way) for indicated experiments with the use of GraphPad Prism software.

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Claims

CLAIMS1 . A method for identifying ligands suitable for the specific targeting of a molecule and / or a drug delivery system to endothelial cells of at least one desired organ, said method comprising the steps of i. obtaining a pool of endothelial cells from the at least one desired organ (desired endothelial cells) and a pool of endothelial cells from at least one non-desired organ (non-desired endothelial cells); ii. incubating desired and non-desired endothelial cells with a ligand library; iii. selecting ligands based on their differential endocytic removal from the cell surface, wherein said step comprises the steps of separating and collecting the following ligand populations: a. ligands that are bound to the cell surface of the desired endothelial cells (“binding population 1 , BP1”) b. ligands that are bound to the non-desired endothelial cells (“binding population 2, BP2”) c. ligands that are retained on the desired endothelial cell surface (“retained population 1 , RP1”) after 2 hours of incubation, d. ligands that are retained on the non-desired endothelial cell surface (“retained population 2, RP2”) after 2 hours of incubation, and identifying the ligands present in each population (a-d), and selecting the ligands whose ratio of RP1 / BP1 is higher than RP2 / BP2 (“selected ligands”).

2. The method of claim 1 , wherein the said ligands are selected from peptides, aptamers, antibodies or small molecules, preferably peptides.

3. The method of claims 1 or 2, wherein the at least one desired organ is selected from the group consisting of lung, liver, brain, heart, kidney, spleen, stomach, large intestine, small intestine, pancreas and skin, preferably wherein the desired organ is the brain.

4. The method of claim 3, wherein the at least one desired organ is the brain and the at least one non-desired organ is lung and / or liver.

5. The method of any one of the preceding claims, wherein the drug delivery system is selected from polymeric nanomicelles, liposomes, nanoparticles, inorganicnanoparticles, metal-organic frameworks, dendrimers, functionalized antibodies, or adeno-associated viruses (AAVs).

6. The method of any one of the preceding claims, wherein the said molecule is selected from therapeutic antibodies, nucleic acids, such as for example DNA, mRNA, siRNA or a chemotherapeutic compound.

7. The method of any one of the preceding claims, wherein the ratio of RP1 / BP1 is more than 1 and the ratio of RP2 / BP2 is less than 1.

8. The method of any one of the preceding claims, wherein the ratio of RP1 / BP1 is more than 1 , preferably between 1.5 - 12.5, between 2.0 - 11.5, between 2.5 - 10.0, between 3.0 - 9.5, between 3.5 - 9.0, between 4.0 - 8.5, between 4.5 - 8.0, between 5.0 - 7.5, between 5.5 - 7.0 or between 6.0 and 6.5.

9. The method of any one of the preceding claims, wherein the ratio of RP2 / BP2 is between 0.1 and 0.9, between 0.2 and 0.8, between 0.3 and 0.7, or between 0.4 and 0.6.

10. The method of any one of the preceding claims, wherein ligands that are retained on the desired endothelial cell surface (“retained population 1 , RP1”) and ligands that are retained on the non-desired endothelial cell surface (“retained population 2, RP2”) are collected after 2h-1 Oh, preferably after 4h-8h of incubation.11 . A ligand, preferably a peptide, identified and selected with the method of any one of the preceding claims.

12. An isolated peptide comprising or consisting of the amino acid sequence as depicted in SEQ ID NO.2, or the amino acid sequence having at least 95 % identity to SEQ ID NO.2.

13. An isolated peptide comprising or consisting of the amino acid sequence as depicted in SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.14, or an amino acid sequence having at least 80 % identity to SEQ ID NO.14, or an amino acid sequence having at least 85 % identity to SEQ ID NO.6 or SEQ ID NO.7.

14. The peptide of any one of claims 11 to 13 further comprising an N-terminally or C- terminally conjugated moiety or molecule.

15. The peptide according to any one of claims 11 to 14, or the peptide comprising or consisting of the amino acid sequence as depicted in SEQ ID NO:1 , or an amino acid sequence having at least 80% identity to the amino acid sequence as depicted in SEQ ID NO:1 , for use in the treatment of a disease, comprising administering said peptide and a drug delivery system or a molecule that binds to the said peptide to a subject in need thereof, wherein the disease is selected from neurodegenerative diseases, such as Alzheimer’s or Parkinson’s disease, brain cancers, such as glioblastoma, neurological disorders, such as epilepsy, and neuropsychiatric disorders such as schizophrenia or bipolar disorder.

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