A complex containing cargo and a targeting moiety that binds to intelectin-1

Abstract

The present invention relates to the field of drug delivery for inflammatory diseases, more specifically inflammatory kidney, eye, lung and bowel diseases, as well as kidney, eye, lung and bowel diseases in general.

Description

[Technical Field]

[0001] The present invention relates to the field of drug delivery for inflammatory diseases, more specifically inflammatory kidney, eye, lung and intestinal diseases, and kidney, eye, lung and intestinal diseases in general. [Background technology]

[0002] Chronic kidney disease (CKD), as a class of kidney disease, is estimated to affect 10% of the world's population. CKD involves increasingly destructive disease mechanisms, particularly in the glomeruli, the filtering units of the kidney.

[0003] Despite the increasing prevalence and detrimental social impact of CKD, patient treatment remains limited to slowing the progression of renal deterioration through the administration of systemically acting drugs. Following interventions such as blood pressure reduction and dietary restriction, anti-inflammatory drugs (often at high initial doses) are administered, including steroids (prednisone), mycophenolate mofetil, cyclophosphamide, azathioprine, and monoclonal antibodies such as rituximab (anti-CD20) and belimumab (anti-BLyS). These treatments are often dose-limiting due to systemic suppression of the immune system, which can cause severe side effects such as fatigue, infection, infertility, and cancer. The use of well-tolerated doses has reduced efficacy. As a result, under the current treatment paradigm, CKD ultimately leads to renal failure and the need for regular dialysis and / or kidney transplantation, with significant negative impacts on quality of life and life expectancy. Targeting anti-inflammatory drugs to the kidney is considered a very promising approach to circumvent these problems. However, previously explored targeting strategies, including antibodies, sugars, and liposomes, have not reached clinical practice due to a lack of glomerular targeting, excessive off-target effects on other organs, and / or toxicity issues. Notably, most kidney-targeting strategies are limited to reaching tubular epithelial cells, while glomerular cells often represent the primary (initiation) site of kidney injury and permanent kidney function loss.

[0004] Clearly, current treatments with systemic anti-inflammatory / immunosuppressant drugs can only slow the deterioration of renal function and are associated with debilitating systemic side effects. These challenges could be circumvented through effective targeting of drugs to (glomerular) kidney cells. However, to date, no effective solutions to these challenges have been presented. Summary of the Invention

[0005] The present invention relates to a conjugate comprising a cargo and a targeting moiety that binds to the Intelectin 1 receptor (ITLN-1), for use as an agent for binding to, targeting, purifying, inducing cellular uptake into or transfecting cells expressing ITLN-1, comprising administering the conjugate to a subject, wherein the use is preferably therapeutic, diagnostic, prophylactic and / or therapeutic-diagnostic, more preferably the use is intended for the treatment of kidney disease.

[0006] The present invention further relates to the use of a complex according to the present invention for binding to, targeting, purifying, inducing uptake or transfecting a cell expressing ITLN-1, comprising contacting the complex with said cell in vitro or ex vivo.

[0007] The present invention further relates to an in vitro or ex vivo method for binding to, targeting, purifying, inducing (endosomal) uptake or transfecting cells expressing ITLN-1, which method comprises contacting the cells in vitro or ex vivo with a complex according to the present invention.

[0008] All drug molecules act through interference with molecular processes within the body. In some cases, systemic activity is desirable or necessary, as in the case of anticoagulants and drugs that lower blood pressure by affecting the physiology of capillary endothelial cells. In other cases, activity is limited to specific cells and / or organs, with the targeted process only being observed locally, as opposed to systemic distribution of the drug. An example would be a substance that interferes with the activity of neurotransmitters, such as reuptake inhibitors, which exert their activity only on cells that express the targeted protein. However, in most cases, drugs act on processes or cells that do not play a role in the pathology for which the drug is intended, commonly referred to as side effects. Side effects can occur for several reasons. First, other molecules are affected in addition to the molecule that is the intended target for the relevant pathology. A stereotypical example is acetylsalicylic acid (aspirin), which inhibits both cyclooxygenase 1 and 2 (COX1 / 2), but for anti-inflammatory effects, inhibition of COX2 is primarily desired. Therefore, so-called super-aspirins that inhibit only COX2 were developed, although they later failed due to the more complex biology of COX2 (Colville-Nash and Gilroy Drug News Perspective. 2000;13(10):587-97). Second, cells in tissues other than those involved in the pathology may be sensitive to the drug's effects. A typical example of this is anticancer drugs, which frequently induce molecular processes associated with rapid, uncontrolled cell proliferation. As a result, other rapidly dividing cells, such as cells in the bone marrow, mucosal cells, and hair follicles, are also affected. Third, metabolic products generated through the body's metabolic activity (e.g., detoxification processes in the liver) can inherently have undesirable side effects (Tang and Lu Drug Metab Rev. 2010;42(2):225-49).

[0009] A well-known solution, particularly to the second and third challenges, is targeted delivery of drugs to their site of action. Consequently, such targeting alters biodistribution (through uptake and / or retention). Here, drugs are formulated to increase their concentration at the desired therapeutic target site relative to cells / organs where drug action is undesirable (otherwise, side effects would be induced in response to anticancer drugs) or not needed (because the cells / organs are not associated with pathology). The latter can be economically important, even if it is not primarily related to avoiding side effects. Oligonucleotide drugs, such as small interfering RNA (siRNA), antisense oligonucleotides (ASO), and messenger RNA (mRNA), may not necessarily cause side effects even if they reach cells other than the intended target cells. However, these drugs are expensive to produce, and therefore their combination with a targeting strategy makes treatment more economical. Nevertheless, even with oligonucleotide drugs, there are many scenarios where specific targeting is required to avoid serious side effects.

[0010] Two basic principles for targeting pharmaceuticals can be distinguished: passive and active targeting. Passive targeting is based on tissue characteristics, such as the permeability of vascular endothelium. This principle has also been described as the enhanced permeability and retention (EPR) effect (Wang and Thanou Pharmacol Res. 2010;62(2):90-9). The EPR effect is the concept underlying the targeting of nanoparticle pharmaceuticals to solid tumors. Due to their rapid growth, the vascular endothelium of solid tumors is less organized than that of healthy tissue and is therefore leaky rather than sealed. As a result, nanoparticles of a certain size can allow blood flow to enter solid tumors but not healthy tissues. Once inside the tumor, particles are retained due to incomplete fluid clearance by malformed lymphatic and vascular vessels, thus preferentially releasing drugs at the target site. The first clinically approved formulation utilizing this targeting principle was a liposomal formulation of the cancer drug doxorubicin, called Doxil (Sousa et al. Cancer Chemother Pharmacol. 2018;82(5):741-55).

[0011] A limitation of all passive targeting strategies is the structure of the vascular endothelium in the so-called reticuloendothelial system (RES) organs, of which the liver is the largest and most prominent. RES organs also have large openings between endothelial cells because they filter particulate matter from the blood. Therefore, the physiology of the RES limits the effectiveness and specificity of passive targeting relative to other organs and tissues (Danhier J Control Release. 2016;244(Pt A):108-21).

[0012] More specific and controllable targeting can be achieved through so-called active targeting strategies (Yoo et al. Cancers (Basel) 2019;11(5):640). For active targeting, the drug itself or the drug delivery vehicle (a carrier such as a liposome into which the drug is incorporated) is equipped with molecular entities (targeting ligands) that cause the drug (drug) to be trapped in cellular or extracellular matrix structures that present molecular structures that specifically interact with these molecular entities. Typically, these presented molecular structures are proteins expressed by target cells, but sugars of glycoproteins and glycolipids, lipids of the plasma membrane, or proteins of the extracellular matrix located near or upstream of target cells can also serve as target structures.

[0013] On the other hand, targeting ligands can be derived from natural molecules. Examples that have been explored or are currently being explored in the field of drug targeting are folate, which specifically interacts with the folate receptor overexpressed on the surface of many cancer cells (Martin-Sabroso et al. Pharmaceuticals. 2021;14(1):14), and N-acetylgalactosamine (GalNAc), which specifically binds to the asialoglycoprotein receptor on hepatocytes. Small interfering RNA (siRNA) coupled to GalNAc (Givosiran) has been approved for clinical use, for example, in the treatment of acute intermittent porphyria (Yu and Tu Pharmacol Ther. 2022;230:107967).

[0014] On the other hand, targeting ligands can be created through molecular design. The most well-known example of a designed targeting ligand is an antibody, which can be selected for virtually any cell surface structure. Antibody-drug conjugates are a rapidly growing field of formulations specifically designed for cancer treatment because they reduce / minimize systemic exposure to drugs, thereby enabling the use of highly effective cytotoxic drugs (Beck et al. Nat Rev Drug Discov. 2017; 16(5): 315-37). Because the selection and design of antibodies for human use is a laborious process and antibodies are large proteins that are expensive to produce, small molecular weight protein scaffolds such as darpins, anticalins, and affibodies have also been developed (Gebauer and Skerra Annu Rev Pharmacol Toxicol. 2020; 60: 391-415). In contrast to antibodies, these proteins can be expressed in bacteria, are very stable, and due to their small size, exhibit better tissue penetration. Peptides, which are also used for drug targeting, have received little attention in targeted drug delivery because they exhibit rapid proteolysis and rapid renal excretion in their unmodified forms, even though they are smaller than engineered proteins, as exemplified by peptides targeting the transferrin receptor to achieve transport across the blood-brain barrier (Mojarad-Jabali et al. Int J Pharm. 2022;613;121395). Furthermore, high-affinity peptide ligands are more difficult to generate than high-affinity protein ligands.

[0015] Furthermore, for active targeting strategies, several points require attention to enable highly effective and specific cell targeting: ideally, the targeting structure should be presented exclusively on the target cells. While the antibody-drug conjugates described above are specific to receptors overexpressed on target cells, low expression levels on other cells can cause side effects (Ceci et al. Pharmacol Ther. 2022;236:108106). Furthermore, targeting structures directly accessible from the bloodstream are more easily accessible than structures requiring tissue penetration. As mentioned above, tissue penetration is compromised when the size of the targeting formulation exceeds the size of vascular fenestrae, capillary junctions, pores in the ECM, or other physical structures that separate the formulation from its target cells. In addition, while high affinity binding is advantageous for binding to target structures directly accessible from the bloodstream, it can resist tissue permeability through a phenomenon known as the binding site barrier (Fujimori et al. J Nucl Med. 1990; 31(7): 1191-8). For nanoparticle formulations that penetrate tissues, active targeting does not necessarily enhance tissue enrichment, since accumulation at the target site is dominated by passive targeting and the EPR effect (Kown et al. J Control Release 2012; 164(2) 108-14). Therefore, active targeting of nanoformulations has been shown to be preferentially induced in cells accessible from the bloodstream, such as endothelial cells.

[0016] Finally, once binding occurs, most targeted agents require cellular uptake for their activity. Because targeted agents are directed at cell surface receptors, in almost all cases, this uptake occurs via receptor-mediated endocytosis. Therefore, for effective targeting and intracellular delivery, it is important that (i) a receptor that exhibits effective uptake is targeted, and (ii) the targeting ligand binds to the receptor in a manner that allows endocytosis to occur. Once inside the endosome, the drug typically needs to be released from the carrier. In the case of antibody-drug conjugates, this release occurs through cleavage of the linker between the antibody and the drug, which specifically reacts with the molecular environment inside the endosome or subsequently inside the lysosome (Panowski et al. MAbs 2014;6(1):34-45). Next, drugs conjugated to antibody-drug conjugates are typically sufficiently hydrophobic to cross the endolysosomal membrane and enter the cytosol, where they exert their activity. For oligonucleotides, endolysosomal release must occur in a manner that the carrier further facilitates the transfer of the oligonucleotide through the endolysosomal membrane. Efficient endosomal release is important for the carrier to exhibit cellular uptake and targeting activity itself (Van Asbeck et al. ACS Nano. 2013;7(5):3797-807).

[0017] Oligonucleotides (ONs) are an important group of potential therapeutic molecules that promise both high efficacy and specificity for a wide range of previously undruggable diseases (Smith et al. Annu Rev Pharmacol Toxicol. 2019;59:605-630). The ON class consists of antisense oligonucleotides, small interfering RNAs (siRNAs), microRNAs, messenger RNAs (mRNAs), other small RNAs, and various forms of DNA. ONs are highly negatively charged due to the phosphate groups in their backbone, and the same is true for most ONs with backbone modifications, such as phosphorothioates (Khvorova et al. Nat Biotechnol 2017;35(3):238-248). To exert their biological activity, ONs almost always require uptake into the cytosol or nucleus of cells, even if their transcription or translation products are active extracellularly. Furthermore, for most ONs, cell-specific uptake is a prerequisite for their biological activity due to the presence of corresponding cellular factors, such as specific mRNAs to be suppressed by a given pharmaceutical siRNA, or specific localization in cells that need to express mRNAs for therapeutic benefit in the body due to their physical location, translational ability, exocytotic ability, and / or involvement in specific bodily processes. Both the molecular weight and negative charge of ONs generally limit unassisted uptake by cells and further provide limited options for control over their biodistribution in the absence of targeting ligands. Therefore, ONs are a stereotypical class of drugs that greatly benefit from uptake enhancement and targeting (Hammond et al. EMBO Mol Med. 2021;13(4):e13243).

[0018] Generally, there are two approaches to mediate the targeting of ONs: either through direct covalent conjugation of targeting ligands or packaging into (targeted) nanoparticles (Roberts et al. Nat Rev Drug Discov. 2020;19(10):673-694). Direct conjugation is limited to short ONs (antisense and siRNA) because the size and charge-mediated inhibition of uptake of long ONs cannot be easily overcome by the targeting ligand alone. Furthermore, if the targeting ligand has a positive charge, aggregation into nanoparticles via electrostatic interactions can occur, compromising the specific covalent conjugation. For mRNA, long non-coding RNA, self-amplifying RNA, and large (protein-encoding) DNA, packaging into nanoparticles is often the only viable option due to the need to avoid ON degradation in plasma due to the presence of ribonucleases and deoxyribonucleases, low cytoplasmic uptake by target cells, and the innate immune response to ONs (Dowdy et al. Nat Biotechnol. 2017;35(3):222;229). Such nanoparticles can be formed by electrostatically complexing negatively charged ONs with the targeting ligand itself, if sufficient cationic charge is available in the targeting agent. Alternatively, ONs can be encapsulated / complexed in / on top of (ionizable or cationic) delivery vehicles and further functionalized with (receptor-)targeting ligands.

[0019] The nanoparticle properties currently understood to be necessary for efficient encapsulation / binding of ONs and efficient uptake of nanoparticles in target cells (other than phagocytic immune cells), such as a diameter of 50–250 nm, excess (ionizable) cationic charge over the negatively charged phosphates of ONs, and membrane-interacting properties, overlap with the properties of endogenous systems that remove nanoparticles from the bloodstream. The most suitable of these endogenous systems are the ApoE-mediated transport system, which targets lipid-based nanoparticles to the liver, and the reticuloendothelial system (RES), which actively captures large (>250 nm), cationic nanoparticles via phagocytosis (Francia et al. Bioconjug Chem. 2020;31(9):2046–2059). To achieve targeting of nanoparticles to other tissues, targeting ligands must possess high affinity for target cell surface features, such as receptor proteins, and low affinity for non-target cell surfaces, including low affinity for plasma proteins (most notably IgG and ApoE).

[0020] As a class of renal disease, chronic kidney disease (CKD) affects an estimated 10% of the world's population. CKD involves a progressively destructive disease mechanism in the glomerulus, the kidney's filtering unit. The glomerulus is composed of a tuft of microcapillaries responsible for charge- and size-selective filtration of blood through the glomerular filtration barrier (GFB). The GFB is a three-layered filter composed of fenestrated glomerular endothelial cells lining the glomerular microcapillaries, the glomerular basement membrane (GBM), which consists of a meshwork network of extracellular matrix proteins, and glomerular epithelial cells, also called podocytes, with interdigitating foot processes. Healthy kidneys contain approximately one million of these filtering units, connected to renal tubules where selective reabsorption (and secretion) of solutes and water from the urine occurs. While the glomerulus and renal interstitium are generally considered the more relevant compartments for CKD, the tubular compartment appears to be more relevant for acute kidney injury.

[0021] Because specific renal pathologies are associated with different renal compartments and different cell types, targeted drug delivery to the cell types involved in the specific pathology would be most advantageous. Current treatments are based on systemic interventions to slow the deterioration of renal function. Following interventions such as blood pressure control (preferably blood pressure reduction), fluid balance, and dietary restrictions, high initial doses of anti-inflammatory / immunosuppressant drugs are often administered. Systemic suppression of the immune system often results in severe side effects, such as fatigue, infection, infertility, and cancer, making such treatment regimes dose-limiting. Dose-limiting drugs reduce efficacy. Consequently, many patients ultimately experience renal failure and undergo dialysis and kidney transplantation as their only remaining treatment options. Part of the efficacy challenge arises from the inability to achieve sufficient drug concentrations in the kidney. The above-mentioned challenges associated with current treatments for renal disease could be circumvented through effective targeting of (anti-inflammatory) drugs to kidney cells (in the glomerulus). Such a targeting strategy should increase drug concentrations at the target site while avoiding systemic side effects through a reduction in the total applied dose.

[0022] Due to the severity of systemic side effects, there is an urgent need to target drugs to the kidney in renal pathologies, and various strategies have been explored in animals, primarily rodents. These strategies consist of antibodies specific to antigens on glomerular endothelial cells (ICAM-1, VCAM-1, E-selectin) or podocytes (Ig receptors), glycoconjugated nanoparticles (chitosan), peptides, polymers, and liposomes (van Asbeck et al. J Control Release. 2020;328:762-775). Most renal targeting strategies explored to date reach only tubular epithelial cells, while glomerular cells are often the primary and / or initiating site of kidney injury. To the inventors' knowledge, no targeted drug delivery method exists that can specifically target glomerular cells, the primary cells involved in kidney filter damage.

[0023] The targeting techniques mentioned above have presented limitations, including specificity challenges (e.g., related to targeting to the other (inflamed) endothelium, lack of uptake in glomerular cells (uptake occurs exclusively in the proximal tubule, making them inapplicable for drugs or nanoparticle systems that cannot cross the glomerular filtration barrier), high off-target ratios to the liver or spleen, biodegradation challenges (polymers), and accumulation of materials that may exacerbate inflammation (antibodies). Importantly, all strategies have so far been used only in preclinical studies and have not progressed to the clinical stage.

[0024] While antibodies seem like a natural choice for targeting specific cell surface receptors, they are contraindicated in the case of glomerular targeting. Accumulation of antibodies on the glomerular filtration barrier can exacerbate inflammatory responses through direct interactions with molecules on endothelial cells, the GBM, or the basal side of podocytes, or indirectly through charged-driven deposition of immune complexes on the GBM (Tecklenborg et al. Clin Exp Immunol. 2018;192(2):142-150). Furthermore, the apical / luminal side of tubular epithelial cells is inaccessible to antibodies unless the GFB is intact. Antibody fragments and related scaffolds, such as nanobodies and DARPins, offer alternatives low enough in molecular weight to cross the GFB but have not been explored in the context of renal targeting (Deonarain et al. Drug Discov Today Technol. 2018;30:47-53).

[0025] Polymer conjugates have not undergone further development toward approved drugs. Chitosan is biologically safe and FDA-approved for non-parenteral applications, but to date, its primary application has been in the form of enhanced penetration across epithelial barriers, where chitosan opens tight junctions (Amidi et al. Adv Drug Deliv Rev. 2010;62(1):59-82; Ahmed et al. Drug Des Devel Ther. 2016;10:483-507). Co-formulation with morphine as a nasal uptake enhancer is in phase III clinical trials (Stoker et al. Pain Med. 2008;9(1):3-12). As another example, challenges in the development of polymer-based drugs have been described for N-(2-hydroxypropyl)methacrylamide (HPMA) conjugates. Despite numerous clinical trials, primarily in the area of ​​cancer therapy (Duncan et al. Adv Drug Deliv Rev. 2009;61(13):1131-48), no conjugate has yet been clinically approved.

[0026] Another group of molecules described for accumulation in the kidney are so-called cell-penetrating peptides (CPPs). However, there is no information available regarding the specific renal structures (glomeruli vs. tubules) reached by these peptides, and in most cases, no information regarding the biodistribution of these peptides. Most CPPs are cationic and / or hydrophobic and mediate cellular uptake of themselves and / or the molecules to which they are conjugated. Conjugation can be covalent or non-covalent, such as the charge-driven complexation of cationic CPPs with negatively charged oligonucleotides. A key feature that distinguishes CPPs from other peptides used for cell targeting is the absence of a specific receptor through which cellular interaction is mediated. Instead, it is hypothesized that the interaction occurs through charge- and hydrogen-bond-driven association with the lipid bilayer of the plasma membrane or with sugars in the cell envelope. While these interactions are based on the type of interaction also found in receptor-ligand interactions, they lack structural (shape) complementarity and therefore specificity, making them unlikely candidates for targeting agents.

[0027] Intelectins, such as intelectin 1 (ITLN-1), form a specific class of lectins originally discovered in intestinal cells. Both the amino acid sequence and 3D structure of vertebrate intelectins are highly conserved (Yang et al. Scan J Immunol. 2020; e12882). Among the suggested roles for ITLN-1 is the clathrin-dependent and lactoferrin-mediated uptake of iron in intestinal cells, where the receptor resides as a GPI-anchored protein within lipid rafts (Suzuki et al. Biochem. 2001; 40(51): 15771-15779; Wrackmeyer et al. Biochem. 2006; 45(30): 9188-9197; Akiyama et al. J Biochem. 2013; 154(5): 437-448).

[0028] In lung epithelial cells, ITLN-1 acts in a secreted form to aid in the phagocytic clearance of microorganisms by specifically binding to microbial glycans (Tsuji et al. Glycobiol. 2009;19(5):518-526; Gu et al. Am J Physiol. 2010;298(3):L290-296; Wesener et al. Nat Struct Mol Biol. 2015;22(8):603-610). ITLN-1, also called omentin-1, is secreted by adipocytes to regulate insulin sensitivity, which is associated with metabolic diseases such as diabetes (Jaikanth et al. Exp Clin Endocrin Diabetes 2013;121(7):377-383). Furthermore, ITLN-1 has been found to be overexpressed in multiple tumors (Zhou et al. PLoS ONE 2013;8(12):e81662; Dan et al. Oncotarget 2015;6(18):16168-1682).

[0029] Several ligands for ITLN-1 are relevant to the receptor's aforementioned roles. Regarding bacterial glycan detection, ITLN-1 specifically recognizes terminal acyclic 1,2-diols displayed on galactofuranose and other microbial sugars (Wesener et al. Nat Struct Mol Biol. 2016;22(8):603-610). Binding to bacterial glycans is regulated by bound calcium ions and is similar between human and mouse ITLN-1. Binding to sugar ligands can regulate binding to its protein ligand, i.e., lactoferrin, and vice versa (Sharma et al. Int J Macromol. 2018;108:1010-1016). ITLN-1 is one of many lactoferrin-binding receptors, including CD14, LDL-related protein-1 (LRP-1), TLR-2 and -4, asialoglycoprotein receptor, and cytokine receptor 4 (CXCR4) (Kell et al. Front Immunol. 2020;11:1221). Lactoferrin binding by ITLN-1 has been described primarily in intestinal epithelial cells. Overexpression of ITLN-1 in these cells increases lactoferrin uptake within endosomes (Akiyama et al. J Biochem. 2013;154(5):437-48). The N-lobe domain of human lactoferrin (hLF peptide (hLF)) has been shown to be required for uptake within intestinal epithelial cells, while the C-lobe is involved in bacterial binding (Suzuki et al. Biochem. 2008 47(41):10915-20). Lactoferricin (residues 17-41) and lactoferrampin (residues 268-284) are naturally formed after proteolytic cleavage and possess antimicrobial and cell-penetrating activities. Lactoferricin has been shown to bind to proteoglycans on endothelial and epithelial cells (Mader et al. Am J Pathol. 2006;169(5):1753-1766; Andersen et al. Med Virol 2004;262-271); however, binding to specific receptors, including ITLN-1, has not been demonstrated.Furthermore, lactoferrin-binding domains for unknown (lymphocyte) receptors have been described in loops 28-34 and 38-45 (Legrand et al. Biochem. 1992 31(38):9243-51), residues 4-90 (Rochard et al. FEBS Lett. 1989;255(1):201-4), or residues 39-40 and 20-37 (Mazurier et al. Adv Exp Med Biol. 1994;357:111-9). In contrast, lactoferrin's binding activity for unknown liver receptors has been shown to be located in the C-lobe of lactoferrin (Sitaram et al. Protein Expr Purif. 1998;14(2):2290-36; Sitaram et al. Biochem K. 1997;323:815-22). Omentin-1 shares the same sequence as ITLN-1 but acts as an adipocyte-secreted cytokine that has been implicated in multiple respiratory, neurological, metabolic, and vascular diseases (Zhou et al. Int J Mol Sci. 2018;19(1):73; Niu et al. Front Cell Dev Biol. 2022;9:784035; Watanabe et al. Compr Physiol. 2017;7(3):765-81). To date, omentin-1 has only been described to associate with integrin receptors on macrophages, but its other binding partners remain unknown (Lin et al. Cardiovasc Med. 2021;8:757926).

[0030] To date, the precise tissue distribution of ITLN-1 remains somewhat unclear. In mice, ITLN-1 expression has been observed in various tissues, including intestinal and lung epithelium, a small fraction of cells in the ovary, spleen, thymus, and liver, and specific regions of the brain (Gu et al. Lung Cell Mol Physiol. 2010;298(3); Suzuki et al. Biometals 2004;17(3):301-309). While some staining of the brush border of proximal tubules in the kidney was also observed, mRNA expression in the kidney was found to be extremely low. In humans, significant mRNA expression has been found in the heart, intestine, and thymus, with no detectable expression in other organs (including the kidney) (Suzuki et al. Biochem. 2001;40(51):15771-15779; Tsuji et al. J Biol Chem. 2001;276(26):23456-23463), whereas protein expression has been found predominantly in the intestinal and bladder epithelium and to a lesser extent in some cells of the heart and kidney (Washimi et al. PloS One 2012 7(7):e39889). In another study, mRNA expression was found in the intestine, heart, spleen, and ovary, but in contrast to the above study, protein expression in these tissues was found exclusively in endothelial cells, hence the name endothelial lectin (Lee et al. Glycobiol. 2001 11(1):65-73). It should be noted that in all these studies, different antibodies were used and the localization of ITLN-1 in the glomerulus was not confirmed in the studies. Therefore, the inventors first aimed to confirm the presence of a receptor for ITLN-1 on glomerular endothelial cells.

[0031] Furthermore, the inventors observed that after pro-inflammatory activation of glomerular endothelial cells by TNFα, the expression of ITLN-1 increased, along with an observed increase in the uptake of peptides derived from the N-lobe of human and mouse lactoferrin, making the receptor a novel candidate for active targeting of (anti-inflammatory) drugs to the kidney.

[0032] Active targeting of glomeruli can be uniquely achieved using the peptides of the present invention, as injection of radiolabeled full-length lactoferrin protein has been shown to result in distribution to many organs, consistent with the use of multiple receptors by the full-length protein, including ITLN-1 and LRP-1 (Huang et al. J Biomed Sci. 2007;14(1):121-128).

[0033] As mentioned above, several ligands for ITLN-1 have been described. Currently, none of these ligands have been successfully applied to specifically target ITLN-1-expressing cells in drug delivery, meaning that biodistribution mediated primarily by ITLN-1 has yet to be achieved when adding ITLN-1-selective compounds. While human ITLN-1 specifically binds to bacterial glycans, it does not bind to human glycan epitopes at all. However, the use of bacterial glycans carries the risk of undesired immune activation via ITLN-1 or other pattern recognition receptors. Full-length lactoferrin protein has been used for targeting (Singh et al. J Drug Target. 2016;24(3):212-23; Sabra et al. Int J Biol Macromol. 2020;164:1046-1060), but it contains multiple binding sites in different parts of the protein that bind to various receptors, including heparan sulfate proteoglycans, ITLN-1, LRP-1, CD14, TLR-2 and -4, asialoglycoprotein receptor, and CXCR4. In particular, LRP-1 is highly expressed in different cell types within multiple tissues, including the liver, spleen, and kidney. Therefore, full-length lactoferrin is not suitable for specific targeting of specific receptors and therefore for achieving biodistribution primarily in a single organ / cell type. More specifically, full-length lactoferrin is not suitable for renal targeting.

[0034] Prior art examples demonstrating targeting of non-renal tissues with lactoferrin-drug conjugates / complexes and / or lactoferrin-containing nanoparticles may also suffer from various challenges that reduce or eliminate proper binding to ITLN1, including: 1. Conjugation and / or (electrostatic) interactions of full-length or fragmented lactoferrin with specific types of cargo (e.g., gold nanoparticles) can obscure the ITLN-1 binding domain; 2. The (composite) targeting properties of certain cargo-lactoferrin combinations can favor biodistribution to cells and tissues that do not express ITLN-1; 3. Conjugates or (electrostatically) bound cargoes may prevent access to ITLNs through steric hindrance; 4. Differences in receptor (ITLN-1) density and presentation on cells, for example due to different genetic backgrounds or immunological states in the models used, may affect binding under biologically relevant conditions; and / or 5. Differences in ligand (lactoferrin or lactoferrin-derived peptide) density and presentation on the nanoparticle surface, due to, for example, the formation of a protein corona or different internal / surface structures, can affect binding under biologically relevant conditions.

[0035] Furthermore, full-length lactoferrin protein is known to polymerize into a predominantly tetramer under physiological conditions (Mantel C, Miyazawa K, Broxmeyer HE. Advances in Experimental Medicine and Biology. 1994. 357:121-32; Bagby GC, Bennett RM. Blood 1982. Blood. 60(1):108-12). Finally, full-length lactoferrin is not suitable for RNA delivery because it possesses pyrimidine-specific ribonuclease activity (McCormick JJ, Larson LJ, Rich MA. Nature 1974. 251(5477):737-40).

[0036] The inventors have established that peptides derived from the N-terminal domain of human lactoferrin protein act as targeting peptides for glomerular cells, particularly glomerular endothelial cells and glomerular epithelial cells (podocytes). In contrast to previous findings that peptides from this region act as cell-penetrating peptides (CPPs) (International Application PCT / EP2006 / 010271), the inventors have demonstrated that uptake in glomerular endothelial cells and podocytes is mediated through binding to ITLN-1. Receptor-mediated uptake is fundamentally different from uptake as a CPP. A key assumption in the field of CPPs is that uptake occurs in a receptor-independent manner. The inventors have demonstrated that uptake in glomerular endothelial cells is not attributable to the cell-penetrating activity of the peptides according to the present invention, but is primarily independent of cell surface proteoglycans, as previously described for other cell lines (Figure 1) (Duchardt et al. 2009; J. Biol. Chem. 284:36099-36108).

[0037] Receptor binding was verified by various methods, including reduced uptake after downregulation of receptor expression using siRNA. Importantly, only peptide variants lacking CPP activity demonstrated uptake in ITLN-1-positive cells. Surprisingly, when glomerular endothelial cells were challenged with tumor necrosis factor α (TNFα) to mimic inflammatory conditions, ITLN-1 was upregulated (Figure 2B), consistent with increased uptake of the targeted peptide in inflammatory cells (Figure 1).

[0038] The inventors confirmed that ITLN-1 is expressed in the glomeruli of mouse and human kidneys (previously unknown). Intravenous injection of a fluorescently labeled lactoferrin-derived targeting peptide in mice with glomerular inflammation induced by LPS showed clear distribution in the glomeruli, whereas such distribution was not observed in unloaded control mice (Figure 4C). Additionally, additional targeting peptides were developed through the removal or addition of naturally occurring amino acid residues from the N- and / or C-termini of the original lactoferrin-derived targeting peptide. These modifications further improved the kidney / liver, kidney / lung, and kidney / spleen ratios, as examples of generally improved biodistribution relative to the kidney (i.e., accumulation in other organs was further reduced compared to accumulation in the kidney) (Figure 5). In developing a general targeting strategy, accumulation in these organs (liver, spleen, and lung) is a challenging challenge, even if the target organs are accessible. Distribution to endothelial cells was not observed except in glomerular endothelial cells. As a result, the activity for in vivo delivery is not due to the peptide's activity as a CPP, which requires general uptake in endothelial cells facing the bloodstream, but rather to receptor-mediated activity not recognized by the prior art. Peptide variants also include molecules not encompassed by the prior art. Importantly, upregulation of ITLN-1 expression facilitates more efficient delivery under inflammatory conditions, opening the possibility of targeting inflamed tissues during glomerular disease. Furthermore, it is conceivable that the most inflamed parts of target tissues receive the highest dose, providing a novel mechanism for fine-tuning the dose within an organ or tissue based on need.

[0039] Intravenous injection into mice showed that lactoferrin-derived peptides were distributed primarily to the kidney (Figures 3 and 4), and glomerular staining was more pronounced in a mouse model of glomerular inflammation (Figure 4). Endothelial cell distribution was not observed except in glomerular endothelial cells. Consequently, activity for in vivo delivery is due to receptor-mediated activity rather than to the peptide's activity as a CPP, which would require general uptake in endothelial cells facing the bloodstream. Rather than acting as a CPP, the inventors demonstrated that uptake in glomerular endothelial cells and podocytes is mediated through binding to ITLN-1. Receptor-mediated uptake is fundamentally different from receptor-independent uptake of CPPs. While all cells have a cell envelope, receptor expression is highly dependent on the specific cell type and the cell's functional state (cell cycle, response to inflammation, etc.). Thus, receptor binding creates the molecular basis for cell-type-specific targeting, whereas activity as a CPP does not. Our conclusion about receptor dependence, rather than CPP activity, was based on the fact that the mouse homolog of a lactoferrin-derived peptide (mLF) was internalized by glomerular endothelial cells with the same activity as hLF (Figure 1A and C). However, in HeLa cells, a cell line frequently used to test CPP activity, this peptide showed little internalization (Figure 1B). Second, treatment of glomerular endothelial cells with tumor necrosis factor α (TNFα), which mimics an inflammatory environment, increased receptor expression (Figure 2B), and this increased expression was accompanied by increased peptide internalization (Figure 1E). Third, both the receptor and peptide were internalized by clathrin-mediated endocytosis, as indicated by colocalization with the protein transferrin, a well-established marker for clathrin-mediated endocytosis (Figure 2C and D). Finally, suppression of ITLN-1 expression by siRNA resulted in a significant decrease in peptide internalization (Figure 2E). It has previously been reported that ITLN-1 acts as a receptor for full-length lactoferrin.However, the finding that uptake of these peptides is receptor-mediated was unexpected for several reasons, including the fact that both linear and cyclized peptides bind to receptors other than the structural constraints of the full-length lactoferrin protein (via the two cysteine ​​residues present), and the fact that endocytosis of the receptor-peptide complex is substantially more rapid than the off-rate of the peptides of the present invention from the receptor. The increased expression of ITLN-1 during inflammation was even more surprising, especially since little was known about the biology of this receptor, especially outside of glomerular endothelial cells. In summary, it was also completely unknown that this receptor could be used for specific drug delivery to the kidney under inflammatory conditions, which is of particular interest and significance for the application of therapeutic agents to CKD affecting the glomeruli. Therefore, effective glomerular targeting during inflammation, when it is most needed for localized anti-inflammatory treatment, offers unique features not yet demonstrated by any other glomerular-targeting molecules, to the extent that they exist.

[0040] Alternative peptide sequences, created by truncating either or both the N- and C-termini of the starting peptide, retained the ability to target the kidney while exhibiting improved kidney / liver ratios (Figure 5). Furthermore, the kidney / lung and kidney / spleen ratios were also improved, demonstrating that some peptides substantially avoid all major off-target organs while maintaining sufficient affinity for the intended receptors. Peptide variants also encompass molecules not encompassed by the prior art. These variants include a reduction in overall positive charge, which reduces liver targeting, through the addition and / or removal of naturally occurring amino acid residues at the N- and / or C-termini of the original targeting peptide. In one or more peptide variants, this overall positive charge was reduced by the introduction of naturally occurring negatively charged residues upstream or downstream in the polypeptide sequence of wild-type human lactoferrin protein. In a patent application (International Application No. PCT / EP2006 / 010271) on the use of hLF peptides as cell-penetrating peptides, the importance of disulfide bridges for activity was emphasized. However, the data herein on peptide variants show that peptides without disulfide bridges exhibit an even more favorable kidney / liver ratio, and unexpectedly, complexation of the peptide with mRNA showed enhanced kidney targeting (Figure 3A / B).

[0041] Based on previous findings, it was completely unexpected that uptake by glomerular cells in vitro and in vivo occurred via specific receptors and was not solely attributable to the CPP activity of these peptides. Furthermore, these alternative peptides also demonstrated that positively charged residues within peptides typically involved in CPP uptake are not only unimportant but also a source of off-target effects, thus distinguishing these peptides from prior art peptides for use as CPPs. Importantly, upregulation of ITLN-1 expression facilitates more efficient delivery in inflammatory settings, opening the possibility of targeting inflamed tissues during glomerular disease.

[0042] Our experiments demonstrated that glomerular endothelial cells express ITLN-1 (Figure 2A). The observed localization of ITLN-1 occurred, at least in part, on the plasma membrane of glomerular endothelial cells. However, since ITLN-1 is a GPI-anchored protein, questions remain as to whether uptake is the result of constitutive protein turnover or whether binding of the hLF peptide enhances internalization. Receptor binding was verified by several methods, including reduced uptake after downregulating receptor expression with siRNA (Figure 2E). Importantly, only peptide variants lacking CPP activity exhibited uptake in ITLN-1-positive cells. Surprisingly, when glomerular endothelial cells were challenged with TNFα to mimic inflammatory conditions, ITLN-1 was upregulated, consistent with increased uptake of the targeted peptide in inflammatory cells. We demonstrated the presence of ITLN-1 in the glomeruli of both unaffected and mice with glomerular inflammation (Figure 2F). This demonstrated that ITLN-1 is expressed in glomerular cells and small blood vessels in the mouse kidney. Intravenous injection of a fluorescently labeled targeting peptide in mice in which glomerular inflammation had been induced by LPS (lipopolysaccharide) showed clear distribution in the glomeruli, whereas such distribution was not observed in unloaded control mice (Figure 4C). Importantly, the presence of ITLN-1 on glomerular cells and its upregulation during (glomerular) inflammation were previously unknown.

[0043] As mentioned above, ITLN-1 expression has been described in multiple tissues and cell types. The inventors have established that targeting ITLN-1 using lactoferrin-derived peptides according to the present invention results in surprisingly significant targeting of the kidney, while other organs, such as the lung, spleen, and intestine, contain only trace amounts of the peptide (Figures 3-4), which is contrary to what would be expected based on the expression pattern of ITLN-1. Experiments by the inventors have also shown that intravenous injection of a hLF peptide variant with a reduced amount of positively charged residues further enhances targeting to the kidney, while also resulting in less enrichment in the liver, lung, and spleen (Figure 5). Combined with the finding that ITLN-1 expression was not detected in the liver and spleen, this suggests that enrichment in the kidney is primarily due to ITLN-1 binding, while enrichment in the liver and spleen is due to charge-dependent endocytosis and is independent of ITLN-1.

[0044] Intravenous injection of free peptides (i.e., peptides of the present invention that are not part of a supramolecular nanoparticle structure and are not conjugated to cargo molecules that would limit glomerular filtration based on size and charge) resulted in localization in glomerular and tubular cells. The inventors demonstrated that coupling hLF peptides, or their derivatives, with PEG resulted in exclusive localization in glomeruli (Figure 6). Glomerular endothelial cells within the GFB are fenestrated with 50-100 nm pores, while the slit pores of podocyte foot processes have gaps of approximately 30 nm (Wartiovaara et al. J Clin Invest. 2004;114(10):1475-83). Therefore, particles smaller than 30 nm will pass through the filtration barrier and reach the proximal tubule, which are expert cells in the reabsorption of different types of molecules. This cellular characteristic is demonstrated by the fact that unconjugated hLF peptides showed tubular uptake, whereas ITLN-1 was not expressed in these cells. Thus, conjugation of hLF to delivery vehicles larger than 30-50 nm results in ITLN-1-dependent targeting that is exclusive to the glomerulus via the blood.

[0045] Thus, in a first aspect, there is provided a conjugate comprising a cargo and a targeting moiety that binds to intelectin 1 (ITLN-1), intended for use as an agent to bind to, target, purify, induce cellular uptake (endocytosis, e.g., clathrin-mediated or macropinocytosis) or transfect cells expressing ITLN-1, comprising administering the conjugate to a subject. Preferably, the affinity of binding of ITLN-1 by said moiety is greater than or equal to 10 -4 Super~10 -6 (micromolar sensitivity), more preferably 10 -7 Super~10 -9 (nanomolar sensitivity) and even more preferably 10 -10 Super~10 -12 Equilibrium dissociation constant (K) (picomolar sensitivity) D )

[0046] In embodiments herein, uptake into a cell can occur by processes known to those skilled in the art, such as endocytosis into the cell, for example, clathrin-mediated or macropinocytotic uptake.

[0047] The complex is referred to herein as a complex in accordance with the invention. The cargo is referred to herein as a cargo in accordance with the invention. A targeting moiety that binds to ITLN-1 is referred to herein as a targeting moiety in accordance with the invention. Intelectin 1 is referred to interchangeably as ITLN-1. ITLN-1 is known to those skilled in the art, as described extensively above.

[0048] In embodiments herein, ITLN-1 may be any intelectin 1 that binds to lactoferrin, such as intelectin 1 that binds to human, bovine, murine, caprine, ovine, or non-human primate lactoferrin. ITLN-1 may or may not be a mutant lactoferrin receptor intelectin 1 that retains its endogenous function. ITLN1 may be human intelectin 1 or a mutant thereof that retains its endogenous function.

[0049] In the embodiments herein, the targeting moiety that binds to ITLN-1 may be any targeting moiety known to those skilled in the art that has the ability to bind to ITLN-1.

[0050] In embodiments herein, the term "cell" is used interchangeably with the term "target cell."

[0051] In the embodiments herein, the term "transfected cell" has its general meaning known to those skilled in the art, i.e., the process of deliberately introducing a nucleic acid into a cell, preferably into an intracellular compartment (e.g., the cytosol for mRNA, the nucleus for most DNA vectors), where said nucleic acid is in an active form.

[0052] In embodiments herein, the targeting moiety may be selected from the group consisting of proteins, peptides, peptidomimetics, DNA, RNA, carbohydrates, polymers, heterocycles, and lipids. The proteins, peptides, peptidomimetics, DNA, RNA, carbohydrates, polymers, heterocycles, and lipids are known to those skilled in the art and may be any proteins, peptides, peptidomimetics, DNA, RNA, carbohydrates, polymers, heterocycles, and lipids that are capable of binding to ITLN-1.

[0053] In the embodiments herein, the targeting moiety may be a peptide. Such a peptide may be any peptide capable of binding to ITLN-1. The peptide is referred to herein as a peptide according to the present invention.

[0054] In embodiments herein, the peptide may be a fragment from a full-length lactoferrin protein, or a variant thereof, that has the intrinsic function of the lactoferrin protein, or a protein functionally and / or structurally related to lactoferrin, having at least one region capable of binding to ITLN-1.

[0055] In embodiments herein, the peptide may be a fragment from lactoferrin protein or a variant thereof that has an intrinsic function of lactoferrin protein, wherein the peptide comprises or consists of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or at least 30 consecutive amino acids of lactoferrin protein or a variant thereof.

[0056] In embodiments herein, the lactoferrin protein may be human, bovine, murine, caprine, ovine, or non-human primate lactoferrin or a variant thereof that has the intrinsic function of lactoferrin or a protein functionally and / or structurally related to lactoferrin and has at least one region capable of binding to ITLN-1.

[0057] In embodiments herein, the lactoferrin protein may be human lactoferrin or a variant thereof that has the intrinsic function of human lactoferrin or a protein functionally and / or structurally related to human lactoferrin, and that has at least one region capable of binding to human ITLN-1.

[0058] In embodiments herein, a human lactoferrin protein or variant thereof having the intrinsic function of human lactoferrin may comprise or consist of an amino acid sequence having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In embodiments herein, a human lactoferrin protein may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 1.

[0059] In embodiments herein, a bovine lactoferrin protein or variant thereof having the intrinsic function of bovine lactoferrin may comprise or consist of an amino acid sequence having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In embodiments herein, a bovine lactoferrin protein may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 2.

[0060] In embodiments herein, the peptides may have a length of about 6 amino acids to about 30 amino acids, and thus may have a length of at least 6 amino acids to a maximum of 30 amino acids. In embodiments herein, the peptides may have a length of 6 to 30 amino acids, 7 to 28 amino acids, 8 to 26 amino acids, 9 to 24 amino acids, 10 to 22 amino acids, 12 to 20 amino acids, or 14 to 18 amino acids. The peptides may have a length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. The peptides may or may not contain non-naturally occurring amino acids that maintain targeting moiety capability. If the peptide contains a cystine amino acid, such cystine amino acid may be or may be substituted with a non-naturally cyclizing amino acid-like moiety. The peptide may contain one or more natural or non-natural unpaired or reduced cysteine ​​residues that may be used for conjugation to other cysteines, maleimides, thiols, etc. The peptide may also contain a natural unpaired cysteine ​​residue that can be coupled to a lipid-PEG with a terminal maleimide. Such lipid-PEG conjugates can be incorporated into the complex before or after coupling with the targeting moiety. The peptide may also contain multiple natural or non-natural cysteines or non-natural variants thereof, such as homocysteine, with at least one of these cysteine ​​residues available for conjugation to a cargo. The remaining cysteines may be available for modifying and / or stabilizing the structure of the targeting moiety.

[0061] In embodiments herein, the peptides may have a net charge of at most +7, more preferably +6, more preferably +5, even more preferably +4 or +3. In embodiments herein, the peptides in the complexes used may have a net charge of at most +3, e.g., +4, +5, +6 or +7, at physiological pH, i.e., pH 7.4, or the peptides may have a net charge of at most +3, e.g., +4, +5, +6 or +7, at endosomal pH, i.e., pH 5.0-7.4.

[0062] In embodiments herein, the peptide may comprise or consist of a peptide selected from the group consisting of: SEQ ID NO:3 (KCFQWQRNMRKVRGPPVSCIKR); SEQ ID NO:4 (KCLRWQNEMRKVGGPPLSCIKR); SEQ ID NO:5 (KCFQWQRNMRKVRGPPVSCIKRDS); SEQ ID NO:6 (CFQWQRNMRKVRGPPVSCIKR); SEQ ID NO:7 (KCFQWQRNMRKVRGPPVSC); SEQ ID NO:8 (RKVRGPPVSCIKR); SEQ ID NO:9 (RKVRGPPVSCIKRDS); SEQ ID NO: 10 (RKVRGPP); SEQ ID NO: 11 (KCFQWQRNMRKVRGPPVSCIKRD); SEQ ID NO: 12 (KCFQWQRNMRKVRGPPVSCIK); SEQ ID NO: 13 (KCFQWQRNMRKVRGPPVSCI); SEQ ID NO: 14 (KCFQWQRNMRKVRGPPVSC); SEQ ID NO: 15 (KCFQWQRNMRKVRGPPVS); SEQ ID NO: 16 (KCFQWQRNMRKVRGPPV); SEQ ID NO: 17 (KCFQWQRNMRKVRGPP); SEQ ID NO: 18 (CFQWQRNMRKVRGPPVSCIKR); SEQ ID NO: 19 (FQWQRNMRKVRGPPVSCIKR); SEQ ID NO:20 (QWQRNMRKVRGPPVSCIKR); SEQ ID NO:21 (WQRNMRKVRGPPVSCIKR); SEQ ID NO: 22 (QRNMRKVRGPPVSCIKR); SEQ ID NO:23 (RNMRKVRGPPVSCIKR); SEQ ID NO:24 (NMRKVRGPPVSCIKR); SEQ ID NO:25 (MRKVRGPPVSCIKR); SEQ ID NO:26 (RKVRGPPVSCIKR); SEQ ID NO:27 (CFQWQRNMRKVRGPPVSCIKRD); SEQ ID NO:28(FQWQRNMRKVRGPPVSCIKRD); SEQ ID NO:29 (QWQRNMRKVRGPPVSCIKRD); SEQ ID NO: 30 (WQRNMRKVRGPPVSCIKRD); SEQ ID NO: 31 (QRNMRKVRGPPVSCIKRD); SEQ ID NO: 32 (RNMRKVRGPPVSCIKRD); SEQ ID NO: 33 (NMRKVRGPPVSCIKRD); SEQ ID NO: 34 (MRKVRGPPVSCIKRD); SEQ ID NO: 35 (RKVRGPPVSCIKRD); SEQ ID NO: 36 (FQWQRNMRKVRGPPVSCIKRDS); SEQ ID NO: 37 (FQWQRNMRKVRGPPVSCIKRDS); SEQ ID NO: 38 (QWQRNMRKVRGPPVSCIKRDS); SEQ ID NO: 39 (WQRNMRKVRGPPVSCIKRDS); SEQ ID NO: 40 (QRNMRKVRGPPVSCIKRDS); SEQ ID NO: 41 (RNMRKVRGPPVSCIKRDS); SEQ ID NO: 42 (NMRKVRGPPVSCIKRDS); SEQ ID NO: 43 (MRKVRGPPVSCIKRDS); SEQ ID NO: 44 (RKVRGPPVSCIKRDS); SEQ ID NO: 45 (CFQWQRNMRKVRGPPVSC); SEQ ID NO: 46 (FQWQRNMRKVRGPPVSC); SEQ ID NO: 47 (QWQRNMRKVRGPPVSC); SEQ ID NO: 48 (WQRNMRKVRGPPVSC); SEQ ID NO: 49 (QRNMRKVRGPPVSC); SEQ ID NO:50 (RNMRKVRGPPVSC); SEQ ID NO:51 (NMRKVRGPPVSC); SEQ ID NO:52 (MRKVRGPPVSC); SEQ ID NO:53 (RKVRGPPVSC); SEQ ID NO:54 (CFQWQRNMRKVRGPPVS); SEQ ID NO:55(FQWQRNMRKVRGPPVS); SEQ ID NO:56 (QWQRNMRKVRGPPVS); SEQ ID NO:57 (WQRNMRKVRGPPVS); SEQ ID NO:58 (QRNMRKVRGPPVS); SEQ ID NO:59 (RNMRKVRGPPVS); SEQ ID NO: 60 (NMRKVRGPPVS); SEQ ID NO: 61 (MRKVRGPPVS); SEQ ID NO: 62 (RKVRGPPVS); SEQ ID NO: 63 (CFQWQRNMRKVRGPPV); SEQ ID NO: 64 (FQWQRNMRKVRGPPV); SEQ ID NO: 65 (QWQRNMRKVRGPPV); SEQ ID NO: 66 (WQRNMRKVRGPPV); SEQ ID NO: 67 (QRNMRKVRGPPV); SEQ ID NO: 68 (RNMRKVRGPPV); SEQ ID NO: 69 (NMRKVRGPPV); SEQ ID NO:70 (MRKVRGPPV); SEQ ID NO:71(RKVRGPPV); SEQ ID NO:72 (CFQWQRNMRKVRGPP); SEQ ID NO:73 (FQWQRNMRKVRGPP); SEQ ID NO:74 (QWQRNMRKVRGPP); SEQ ID NO: 75 (WQRNMRKVRGPP); SEQ ID NO:76 (QRNMRKVRGPP); SEQ ID NO:77 (RNMRKVRGPP); SEQ ID NO:78 (NMRKVRGPP); SEQ ID NO:79 (MRKVRGPP); Sequence number 80 (KC X3 X4 WQ X7 X8 M X10 X11 X12 X13 X14 P X16 X17 X18 C X20 X21 X22 X23) (wherein X3 is F, R, or Y, X4 is R or Q, X7 is any natural or unnatural amino acid, X8 is any natural or unnatural amino acid, X10 is R, K, or ornithine, X11 is R, K, or ornithine, X12 is V or L, X13 is R or G, X14 is G or A, X16 is P or S, X17 is V, L, I, X18 is S, T, C, X20 is I or V, X21 is R, K, or ornithine, X22 is R, K, or ornithine, and X23 is D, S, A, or T); SEQ ID NO: 81 (X1, X2, X3, X4, X5 P X7) (wherein X1 is R or K or ornithine, X2 is R, K or ornithine, X3 is V or L, X4 is R or G, X5 is G or A, and X7 is P or S); SEQ ID NO: 82 (M X2, X3, X4, X5, X6 P X8) (wherein X2 is R, K or ornithine, X3 is R, K or ornithine, X4 is V or L, X5 is R or G, X6 is G or A, and X8 is P or S); SEQ ID NO: 83 (X1 X2 X3 X4 X5 P X7 X8 X9 C X11 X12 X13 X14) (wherein X1 is R, K or ornithine, X2 is R, K or ornithine, X3 is V or L, X4 is R or G, X5 is G or A, X7 is P or S, X8 is V, L or I, X9 is S or T, X11 is I or V, X12 is R, K or ornithine, X13 is R, K or ornithine, and X14 is D, S, A or T); SEQ ID NO: 84 (X1 X2 X3 X4 X5 P X7 X8 X9 C X11 X12 X13 X14 S) (wherein X1 is R, K or ornithine, X2 is R, K or ornithine, X3 is V or L, X4 is R or G, X5 is G or A, X7 is P or S, X8 is V, L or I, X9 is S or T, X11 is I or V, X12 is R, K or ornithine, X13 is R, K or ornithine, and X14 is D, S, A or T).

[0063] In embodiments herein, the targeting moiety, preferably a peptide, may include a protecting moiety and / or a labeling moiety, which may be any protecting moiety and labeling moiety known to those skilled in the art.

[0064] In embodiments herein, targeting moieties, preferably peptides, can be terminally protected to increase stability in biological solutions against degradative enzymes (such as exonucleases, exopeptidases, and exoglycosidases) and against chemical reactions that affect the termini of the targeting moiety. Such terminal protection modifications may include C-terminal modifications with terminal D-amino acids, N-terminal acetylation, C-terminal amidation, N-alkylamides, aldehydes, esters, p-nitroanilide, 7-amino-4-methylcoumarin, polyethylene glycol (PEG), PAS (an oligopeptide consisting of proline, alanine, and serine residues), poly(glycerol) (PG), polyvinylpyrrolidone (PVP), poly(N(2-hydroxypropyl)methacrylamide) (PHPMA), and N-terminal modifications with formyl, pyroglutamyl, fatty acids, urea, carbamates, sulfonamides, alkylamines, PEG, PAS, PG, PVP, or PHPMA. Both C-terminal amidation and N-terminal acetylation remove charge from the termini of the peptide, thereby altering solubility, and possibly biodistribution and biological activity, since they reliably reproduce the native peptide bonds when the peptide is selected from the internal sequence of a protein and are preferred embodiments of the present invention.

[0065] Thus, in embodiments herein, the protecting moiety may be selected from the group consisting of a terminal D-amino acid, a D-amino acid, an unnatural amino acid, N-terminal acetylation, N-terminal acylation, C-terminal amidation, C-terminal modification or substitution with N-alkylamide, aldehyde, ester, p-nitroanilide, 7-amino-4-methylcoumarin, formyl, pyroglutamyl, fatty acid, urea, carbamate, sulfonamide or alkylamine, glycosylation, PEGylation, PAS, a suitable fluorescent group, a chelator for ions, preferably a radioisotope for ions, e.g., a suitable fluorescent label and / or N-terminal modification or substitution with diethylenetriaminepentaacetic acid (DTPA) or an equivalent substance.

[0066] In embodiments herein, the targeting moiety, preferably a peptide, can be stabilized in biological solutions by, for example, D-amino acids, retro-inverso sequences, modified backbone structures, such as peptidomimetic, peptoid, or pseudopeptide bonds, and by cyclization, for example, by the formation of inter- or intramolecular cystine bonds.

[0067] In embodiments herein, the labeling moiety may be any labeling moiety known to those of skill in the art, such as one or more selected from the group consisting of a fluorophore, a hapten (such as biotin or digoxigenin), a DNA barcode, a PNA barcode, an XNA barcode, or an RNA barcode, a stable isotope, a substrate for click chemistry, and a radioactive label.

[0068] In embodiments herein, the cargo may be modified to increase its stability in biological solutions, for example, in the presence of degradative enzymes such as exonucleases, exopeptidases, and exoglycosidases, and in the presence of chemical reactions that affect the termini of the targeting moiety. Such modifications of the cargo may include modifications of the nucleic acid (NA) backbone (phosphorothioate linkages, locked nucleic acids (LNA), etc.), modifications of the NA bases, modifications of the NA ends (e.g., capping), sugar modifications (e.g., 2'O-methylation of RNA), hybridization with other (more stable) NAs, (stabilizing) polymers (poly(ethylene glycol) (PEG), polyamino acids such as poly(proline-alanine-serine) (PAS), poly(glycerol) (PG), poly(N-vinylpyrrolidone) PVP, poly(carboxybetaine) (pCB), poly(sulfobetaine) (pSB), phosphobetaine-based polymers, carbohydrates (e.g., heparin), or poly(N-(2-hydroxybenzoyl)-1, ... These modifications may include the use of terminal protection modifications of peptides and proteins such as conjugation to (e.g., hydroxypropyl, methacrylamide, PHPMA), terminal D-amino acids, N-terminal acetylation, C-terminal amidation, C-terminal modifications with N-alkylamides, aldehydes, esters, p-nitroanilide, 7-amino-4-methylcoumarin, poly-ethylene glycol (PEG), PAS, PG, PVP, pCB, pSB, carbohydrates, or PHPMA, and / or N-terminal modifications with formyl, pyroglutamyl, fatty acids, urea, carbamates, sulfonamides, alkylamines, PEG, PAS, PG, PVP, pCB, pSB, carbohydrates, or PHPMA.

[0069] Other modifications may include shielding the cargo from nonspecific interactions with off-target receptors and other body components with low-affinity polymers or molecules, such as PEG, resulting in a net increase in targeting efficiency by the targeting moiety. The relative amount and length of such shielding polymers must be sufficient to reduce the available interaction surface and thus reduce undesired molecular interactions. In such cases, the shielding polymer may also partially or completely cover the targeting moiety or reduce its accessibility. Therefore, it is preferable to conjugate the targeting moiety directly or indirectly to the freely available end of the shielding moiety rather than directly to the cargo. Such freely available end can be modified with any suitable click chemistry or conjugation chemistry, such as maleimide coupling to (natural) cysteine ​​residues, vinyl reagents for tetrazines, methylcyclopropene reagents, TCO reagents for tetrazines, norbornene for tetrazines, dibenzocyclooctyne (DBCO) reagents for azides, and alkyne reagents for azides. The conjugation may be performed before or after coupling to or insertion into the cargo. If the conjugation is performed prior to formation or association with the cargo, e.g., conjugation of a targeting peptide to a PEGylated lipid to form a peptide-PEG-lipid conjugate, the overall characteristics must be compatible with the conditions used for formulation. For example, the peptide-PEG-lipid conjugate, when used as part of a lipid nanoparticle composition, is ideally soluble in organic solvents such as ethanol at appropriate concentrations.

[0070] In the embodiments herein, cargo can be covalently or non-covalently bound to targeting moiety.Cargo can be covalently bound to targeting moiety using one or more naturally occurring functional groups in the targeting moiety, such as amino terminus, carboxy terminus, side chain amino group, side chain carboxy group, or preferably cysteine, or using the terminal or side chain functionalities of unnatural amino acids, such as azide, aldehyde, alkene, alkyne, isocyanate, or isothiocyanate.The bond depends on the functionality in the cargo to form a covalent bond with the targeting moiety, and can be an amide bond, an ester bond, a thioester bond, a disulfide bond, a thioalkane formed through Michael addition, or another type of chemical functionality resulting from the conjugation of a binding partner.

[0071] In embodiments herein, multiple targeting moieties in a conjugate, for example, 2, 3, 4, 5, 6 or more targeting moieties or dendrimer structures of targeting moieties, can be covalently attached to the cargo.

[0072] In embodiments herein, the affinity of the conjugate for ITLN-1 and / or ITLN-1-expressing cells can be enhanced by the multivalent use of targeting moieties, and affinity can be enhanced by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or at least 1 log.

[0073] In embodiments herein, the cargo may be any useful moiety intended for use in a complex to bind to, target, purify, induce cellular uptake of, or transfect cells expressing ITLN-1. The cargo may be a nucleic acid (e.g., small interfering RNA (siRNA), antisense oligonucleotide (AON or asRNA), microRNA (miRNA), RNA aptamer, messenger RNA, trans-amplifying RNA, self-amplifying RNA, long non-coding RNA, split-replicon RNA, DNA aptamer, plasmid DNA, viral DNA, viral RNA, doggybone vector, circular RNA), peptide (e.g., hormone-mimetic peptide, protein-binding peptide, RNA- or DNA-binding peptide, cytokine, growth factor), polypeptide or protein (e.g., anti-inflammatory protein, immunomodulatory protein, pro-resolving protein, protein that affects cellular redox state and antioxidant response, ligand-binding receptor, signaling protein, transcription factor), carbohydrate (e.g., monosaccharide, disaccharide, oligosaccharide, and multi-head), lipid, polymer, small molecule, or combination or mixture thereof. The cargo may be a particle, for example a microparticle or nanoparticle, a liposome, a lipid nanoparticle, a polymer particle, a silica particle, a carbon nanotube, a gold particle, or a vehicle such as a lipid or polymer micelle.

[0074] In embodiments herein, when the cargo is a vehicle, the cargo may comprise any useful compound. The particles may contain pharmaceutically acceptable compounds, for example, pharmaceutically active compounds (e.g., anti-inflammatory compounds (e.g., non-steroidal anti-inflammatory drugs such as aspirin, ibuprofen, naproxen, celecoxib, diclofenac, indomethacin, oxaprozin, and / or piroxicam, more preferably steroidal anti-inflammatory drugs such as prednisone, cortisone, and methylprednisone, or anti-rejection drugs such as tacrolimus, cyclosporine, mycophenolate mofetil, azathioprine, rapamycin, sirolimus), anti-cancer drugs (e.g., chemotherapy, for example, alkylating agents (e.g., altretamine, bendamustine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, mechlorethamine, melphalan, oxaliplatin, temozolomide, thiotepa, and trabectedin), nitrosoureas (e.g., carmustine, lomustine, and streptozocin), and antimetabolites (e.g., azacitidine, 5-fluorouracil (5-FU), 6 -mercaptopurine (6-MP), capecitabine, cladribine, clofarabine, cytarabine (Ara-C), decitabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, nelarabine, pemetrexed, pentostatin, pralatrexate, thioguanine, trifluridine / tipiracil combination), antitumor antibiotics (e.g., anthracyclines daunorubicin, doxorubicin (adriamycin), liposomal doxorubicin, epirubicin, idarubicin), anti-cancer drugs (e.g., valrubicin, and the antitumor antibiotics bleomycin, dactinomycin, mitomycin C, and mitoxantrone); topoisomerase inhibitors irinotecan, irinotecan liposomal, topotecan, etoposide (VP-16), mitoxantrone, and teniposide); mitotic inhibitors (e.g., taxanes cabazitaxel, docetaxel, Nab-paclitaxel, and paclitaxel; and the vinca alkaloids vinblastine, vincristine, vincristine liposomal, and vinorelbine);and other chemotherapy drugs (e.g., all-trans retinoic acid, arsenic trioxide, asparaginase, eribulin, hydroxyurea, ixabepilone, mitotane, omacetaxine, pegaspargase, procarbazine, romidepsin, and vorinostat), cytokine drugs (e.g., cytokines (e.g., IL-1, IL-2, TNF-α, IL-6, IL-7, IL-10, IL-12, IL-17, IL-21, IL-22, IL-23, IFN-α ... These include recombinant versions of IFN-β, IFN-γ, IFN-λ1, IFN-λ2, IFN-λ3, IFN-ω, IP-10, MIP-1α, and TGF-β(1-3)), as well as anti-cytokines (e.g., cytokine-binding antibodies, decoy receptors, and IL-1R antagonists), growth factors (e.g., bone morphogenetic proteins (BMPs), vascular endothelial growth factors (VEGFs), granulocyte-macrophage colony-stimulating factors (GM-CSFs), epidermal growth factors (EGFs), and erythropoietin (ERPs)). erythropoeitin (EPO), insulin-like growth factor (IGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF), thrombopoietin (TPO)), hormones (e.g., aldosterone, vasopressin, adrenocorticotropic hormone (ACTH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), oxytocin, prolactin, thyroid-stimulating hormone (TSH), renin, angiotensin, glucagon, insulin, estrogen, progesterone, parathyroid hormone (PTH), thyroid hormones, epinephrine, norepinephrine, testosterone, melatonin, growth hormone-releasing hormone (GHRH), thyrotropin-releasing hormone (TRH), gonadotropin-releasing hormone (GnRH), corticotropin-releasing hormone (CRH), and humoral factors), cardiovascular medications medication) (e.g., anticoagulants (examples include apivaxane, dabigatran, edoxaban, heparin, rivaroxaban, and warfarin), antiplatelet agents (examples include aspirin, clopidogrel, dipyridamole, prasugrel, and ticagrelor),Angiotensin-converting enzyme (ACE) inhibitors (examples include benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, and trandolapril), angiotensin II receptor blockers (azilsartan, candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan, and valsartan), beta-adrenergic blockers (acebutolol, azithromycin, azithromycin), tenolol, betaxolol, bisoprolol, metoprolol, nadolol, propranolol, and sotalol), calcium channel blockers (e.g., amlodipine, diltiazem, felodipine, nifedipine, nimodipine, nisoldipine, and verapamil), cholesterol-lowering drugs (e.g., the statins atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin), nicotinic acid (Nitotinic These drugs include niacin, a diuretic (e.g., niacin, a diuretic acid), and ezetimibe, a cholesterol absorption inhibitor), digitalis preparations (digoxin), diuretics (e.g., acetazolamide, amiloride, bumetanide, chlorothiazide, chlorthalidone, furosemide, hydrochlorothiazide, indapamide, metolazone, spironolactone, and torsemide), and vasodilators (isosorbide dinitrate, isosorbide mononitrate, hydralazine, nitroglycerin, and minoxidil), intestinal regulators (e.g., proton pump inhibitors (e.g., omeprazole, lansoprazole, rabeprazole), benzodiazepine, benzocaine, benzodiazepine ...Gatifloxacin, tobramycin, gentamicin, polymyxin D, neomycin, bacitracin, azithromycin, and erythomycin), lipid-based artificial tears (examples include castor oil, glycerol, and mineral oil), NSAIDS and corticosteroids, glaucoma medications (examples include levobunolol, timolol, betaxolol, bimatoprost, travoprost, latanoprost, tafluprost, brimonidine, brinzolamide, and dorzolamide). and antiviral treatments (e.g., acyclovir, valacyclovir, and famciclovir), pulmonary medications (e.g., antiasthmatics (e.g., dyphylline, guaifenesin, albuterol, levalbuterol), antihistamines (e.g., brompheniramine, carbinoxamine, chlorpheniramine, clemastine, diphenhydramine, hydroxyzine, tripolidine, azelastine, cetirizine, desloratadine, fexofenadine, levocetirizine), lysine, loratadine, olopatadine), antitussives (examples include dextromethorphan and benzonatate), bronchodilators (ipratropium, theophylline, albuterol, epinephrine, levalbuterol, arformoterol, formoterol, olodaterol, terbutaline, pirbuterol, metaproterenol, salmeterol, isoproterenol, indacaterol, tiotropium, umeclidinium, aclidinium, ipratropium, rebefenacin, glycopyrrolate, ipratropium, Theophylline, Aminophylline, and Dyphylline), decongestants (examples include levmetamphetamine, naphazoline, oxymetazoline, phenylephrine, propylhexedrine, pseudoephedrine, and xylometazoline), expectorants (guaifenesin), leukotriene modifiers (examples include montelukast, zafirlukast, zileutron), pulmonary surfactants (examples include beractant, lucinactant, calfactant, and boractant),Mucolytics (acetylcysteine), anti-infectives (examples include zanamivir, ribavirin, tobramycin, pentamidine, and colistimethate), inhaled corticosteroids (examples include fluticasone, budesonide, mometasone, beclomethasone, and ciclesonide), mast cell stabilizers (examples include cromolyn and nedocormil), and phosphodiesterase 4 inhibitors (including roflumilast), antimicrobials (e.g., antibiotics (e.g., aminoglycosides (e.g., Examples of antihistamines include amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, and spectinomycin, ansamycins (examples include geldanamycin, herbimycin, and rifaximin), carbacephems (e.g., laracarbef), carbapenems (e.g., ertapenem, doripenem, imipenem, and meropenem), cephalosporins (e.g., cefadroxil, cefazolidin, cephradine, cephradine, cephapirin, cephalothin, cephalexin, cefaclor, cefoxitin, cefotetan, cefamandole, cefmetazole, cefonicid, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, moxalactam (maxalactam), ceftriaxone, cefepime, ceftaroline fosamil, and ceftobiprole), glycopeptides (e.g., teicoplanin, vancomycin, telavancin, dalbavancin, and oritavancin), lincosamides (clindamycin and lincomycin), lipopeptides (e.g., daptomycin), macrolides (azithromycin, clarithromycin, erythromycin, roxithromycin, telithromycin, spiramycin, and fidaxomicin), monobactams (e.g., aztreonam), nitrofurans (e.g., furazolidone and nitrofurantoin), oxazolidinones (examples include linezolid, pocizolid, radezolid, and torezolid),Penicillins (e.g., amoxicillin, ampicillin, azlocillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, temocillin, and ticarcillin), polypeptides (e.g., bacitracin, cilistin, and and polymyxin B), quinolones (examples include ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nadifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, and temafloxacin), sulfonamides (examples include mafenide, sulfacetamide, sulfadiazine, sulfadimethoxine, sulfamethizole, sulfamethoxazole, sulfanilimide , sulfasalazine, sulfisoxazole, and sulfonamide chrysoidine), tetracyclines (e.g., demeclocycline, doxycycline, methacycline, minocycline, oxytetracycline, and tetracycline), mycobacterium-specific antibiotics (e.g., clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, ioazid), pyrazinamide, rifampicin, rifabutin, rifapentine, and streptomycin), antifungal agents (e.g., polyene antifungals (e.g., amphotericin B, candicidin, filipin, hamycin, natamycin, nystatin, and rimocidin), azoles (e.g., imidazoles bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, isoconazole, ketoconazole, luliconazole, miconazole, omoconazole, oxiconazole, sertaconazole, The antibacterial agents include the triazoles albaconazole, efinaconazole, epoxiconazole, fluconazole, isavuconazole, itraconazole, posaconazole, propiconazole, ravuconazole, terconazole, and voriconazole, and the thiazoles (e.g., abafungin), allylamines (e.g., butenafine, naftifine, and terbinafine), echinocandins (anidulafungin, caspofungin, and micafungin), and triterpenoids (e.g.,Antiparasitic drugs (e.g., broad-spectrum nitazoxanide, antiprotozoal drugs (e.g., melarsoprol, eflornithine, metronidazole, tinidazole, and miltefosine), antinematodes (e.g., mebendazole, pyrantel pamoate, thiabendazole, diethylcarbamazine, and ivermectin), antitasteworm drugs (e.g., niclosamide, praziquantel, and albendazole), antitrematodes (e.g., praziquantel), and antiamoebics (e.g., rifampicin and amphotericin B)), antidiabetic drugs (e.g., insulin (analogs), amylinomimetics drugs) (e.g., pramlintide), α-glucosidase inhibitors (e.g., acarbose and miglitol), biguanides (e.g., metformin (analogs and combinations), dopamine agonists (e.g., bromocriptine), dipeptidyl peptidase-4 (DDP-4) inhibitors (e.g., alogliptin, linagliptin, saxagliptin, and sitagliptin), glucagon-like peptide-1 receptor agonists (e.g., albiglutide, dulaglutide (Dualglutaide), exenatide, liraglutide, and semaglutide), meglitinides (e.g., nateglinide and repaglinide), sodium glucose transaminase inhibitors (e.g., metformin (analogs and combinations) ... SGLT-2 inhibitors (examples include dapagliflozin, canagliflozin, ertugliflozin, and empagliflozin), sulfonylureas (examples include glimepiride, gliclazide, glipizide, glyburide, chlorpropamide, tolazamide, and tolbutamide), thiazolidinediones (e.g., rosiglitazone and pioglitazone), antiviral drugs (e.g., abacavir, acyclovir, adefovir, amantadine, ampligen, amprenavir, umifenovir, atazanavir, atripla, oseltamivir, zanamivir, peramivir, baloxavir, bictegravir, emtricitabine, tenofovir,Boceprevir, Brevirtide, Cidofovir, Cobicistat, Daclatasvir, Darunavir, Delavirdine, Didanosine, Docosanol, Dolutegravir, Doravirine, Edoxudine, Efavirenz, Emtricitabine, Enfuvirtide, Ensivirtide, Encitrervir, Entecavir, Entravirine, Famciclovir, Fomivirsen, Fosamprenavir, Foscarnet, Ganiciclovir, Ibacitabine, Ibalizumab, Idoxuridine, Imiquimod, Inosinate pranobex pranobex), indinavir, lamivudine, letermovir, lopinavir, loviride, maraviroc, methisazone, moroxydine, nelfinavir, nevirapine, nitazoxanide, norvir, penciclovir, pleconaril, podophyllotoxin, raltegravir, remdesivir, ribavirin, rilpivirine, rimantadine, ritonavir, saquinavir, simeprevir, sofosbuvir, stavudine, talibavirin, telaprevir, telbivudine, tenofovir, tiprenavir, trifluridine, trizivir, tromantadine, truvada, umifenovir, valacyclovir, valganciclovir, vicriviroc, vidarabine, zalcitabine, zanamivir, and zidovudine), or structural or functional analogs thereof) or diagnostic compounds.

[0075] In embodiments herein, the peptide is linked to the cargo covalently, electrostatically, hydrophobically, or via an intermediate molecule, or through a combination thereof.

[0076] In embodiments herein, the cell may be a eukaryotic cell. The eukaryotic cell may be a mammalian cell, such as a human, bovine, murine, caprine, ovine, or non-human primate cell. In embodiments herein, the cell may be a human cell. In embodiments herein, the cell may be an ITLN-1-expressing kidney cell, such as a glomerular endothelial cell or a podocyte. In yet other embodiments, the cell may be an ITLN-1-expressing ocular cell (such as a corneal epithelial cell), an ITLN-1-expressing intestinal cell, or an ITLN-1-expressing lung cell.

[0077] In embodiments herein, administration of the conjugates can be by any means known to those skilled in the art. Since many of the applications will be pharmaceutical applications, the conjugates may be present in pharmaceutical compositions that include additional pharmaceutical compounds, such as a pharmaceutically active compound and a pharmaceutically acceptable excipient. Such compositions are also provided as part of the present invention.

[0078] In embodiments herein, administration may be in the form of a spray (pulmonary, skin, eye, nose, oropharynx, ear), creme (skin), lotion (skin), drug depot / sustained release formulation, shampoo (skin / hair), pill (intestinal), eye drops (eye), microneedle (skin), wash / drip (intraperitoneal, transmucosal, vaginal, oral), biomaterial for regenerative medicine (bone, cartilage, connective tissue, skin, vasculature), coating on an (implantable) medical device, or any other suitable form that can be administered to a subject in need of treatment. In embodiments herein, administration to a subject can be by injection, preferably by intravenous (IV) injection.

[0079] In embodiments herein, administration of the conjugate to a subject can result in increased intracellular concentrations of the cargo in ITLN-1-expressing cells. In embodiments herein, administration of the conjugate to a subject can result in biodistribution of the conjugate primarily to ITLN-1-expressing cells such that the cargo accumulates at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, and preferably at least 10-fold more in organs containing ITLN-1-expressing cells than in off-target organs, which refer to organs that do not contain ITLN-1-expressing cells.

[0080] In embodiments herein, administration of the conjugate to a subject results in a biodistribution that is primarily kidney-specific, such that the cargo accumulates in the kidney at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, and preferably at least 10-fold more in the kidney than in any of the off-target organs.

[0081] In embodiments herein, the use as an agent to bind to, target, purify, induce cellular uptake into, or transfect cells expressing ITLN-1, including administration of the conjugate to a subject, may be therapeutic, diagnostic, prophylactic, and / or therapeutic-diagnostic use.

[0082] In embodiments herein, the use of the conjugate as an agent to bind to, target, purify, induce cellular uptake into, or transfect cells expressing ITLN-1, including administration to a subject, may be intended for use in the treatment of kidney disease, e.g., chronic kidney disease.

[0083] In embodiments herein, the use of the conjugate as an agent for binding to, targeting, purifying, inducing cellular uptake into, or transfecting cells expressing ITLN-1, including administration to a subject, may be intended for use in the treatment of inflammatory diseases, such as inflammatory kidney diseases, e.g., inflammatory indications that cause chronic kidney disease.

[0084] In embodiments herein, the use of a conjugate as an agent to bind to, target, purify, induce cellular uptake into, or transfect cells expressing ITLN-1, including administration to a subject, may be intended for use in treating pathologies associated with upregulation, overexpression, and / or increased efficacy of the ITLN1 receptor.

[0085] In embodiments herein, the use of a conjugate as an agent to bind to, target, purify, induce cellular uptake into, or transfect cells expressing ITLN-1, including administration to a subject, may be intended for use in the treatment of acute kidney injury.

[0086] In embodiments herein, the use of a conjugate as an agent for binding to, targeting, purifying, inducing cellular uptake, or transfecting cells expressing ITLN-1, including administration of the conjugate, may be intended for use as a conditioning treatment for kidney transplantation, in which the conjugate is administered intracorporeally and / or extracorporeally as a conditioning treatment for kidney transplantation. Extracorporeal means that the conjugate is administered to the kidney of the transplant recipient after it has been removed from the donor. In embodiments herein, administration of the conjugate to a subject may result in more rapid clearance of the cargo from the system (blood) compared to clearance of a cargo without a targeting moiety. In embodiments herein, clearance of the cargo may be more than 95% complete within less than one month, preferably less than one week, less than one day, less than one hour, less than 30 minutes, less than 20 minutes, less than 15 minutes, less than 10 minutes, less than 7 minutes, and more preferably less than 5 minutes.

[0087] The complexes defined in the embodiments herein can be conveniently used to bind to, target, purify, induce cellular uptake, or transfect cells expressing ITLN-1, including contacting the complex with cells expressing ITLN-1 in vitro or ex vivo. The features of this second aspect are preferably the features of the first aspect herein. In this aspect, the cells may or may not be part of an organ when the complex is administered extracorporeally.

[0088] In a third aspect, there is provided an in vitro or ex vivo method for binding to, targeting, purifying, inducing cellular uptake, or transfecting cells expressing ITLN-1, comprising contacting a complex with a cell expressing ITLN-1 in vitro or ex vivo. The features of this second aspect are preferably the features of the first aspect herein. In this aspect, the cell may or may not be part of an organ when the complex is administered extracorporeally. [Brief explanation of the drawings]

[0089] [Figure 1A] Lactoferrin-derived peptides are preferentially taken up by mouse glomerular endothelial cells (mGEnCs). Flow cytometry analysis of mGEnCs incubated for 30 min with different concentrations of fluorescein-labeled peptides, namely, human lactoferrin-derived peptide (hLF), mouse lactoferrin-derived peptide (mLF, lacking CPP activity), and the known cell-penetrating peptides R9 and penetratin, shows increased uptake of lactoferrin-derived peptides. [Figure 1B] HeLa cells show uptake of hLF, R9 and penetratin, but not mLF. [Figure 1C] Confocal microscopy of mGEnCs after 30 min of incubation with 5 μM of each peptide shows localization in endosome-like structures. [Figure 1D]Digestion of heparan sulfate from the cell surface has a limited effect on the uptake of lactoferrin-derived peptides (5 μM). The percentage of uptake for cleaved heparan sulfate is shown relative to undigested cells. [Figure 1E] Treatment of mGEnCs with tumor necrosis factor alpha (TNFα) increases the uptake of 5 μM hLF (30 min). [Figure 2A] Uptake of glomerular-targeting peptides occurs via receptor-mediated endocytosis, involving ITLN-1 as a receptor. Non-permeabilized (flow cytometry) and permeabilized (confocal microscopy) cells were stained with antibodies against ITLN-1 and low-density lipoprotein receptor-related protein 1 (LRP-1), both of which have been described as potential receptors for lactoferrin, demonstrating the presence of ITLN-1 in / on mGEnCs. [Figure 2B] Treatment of mGEnCs with TNFα increases ITLN-1 protein expression. [Figure 2C] hLF colocalized with rhodamine-labeled transferrin, indicating uptake via clathrin-coated pits. [Figure 2D] ITLN-1 also colocalizes with transferrin. [Figure 2E] Downregulation of ITLN-1 in mGEnCs using siRNA reduces the uptake of lactoferrin-derived peptides, but not the CPPs R9 and penetratin, and the transferrin receptor ligand transferrin. [Figure 2F] Frozen mouse kidney sections from normal BALB / c and MRL / MpJ mice (background strains), as well as MRL / MpJ-Faslpr mice expressing lupus-associated glomerular inflammation, were stained for ITLN-1, demonstrating its expression in the glomeruli. [Figure 3A]Biodistribution of In-DOTA-hLF after intravenous injection in female and male mice. The peptide as free peptide (dark gray male and female symbols) or as particles with mRNA (light gray male and female symbols) shows high concentrations in the kidney and urine, slightly lower concentrations in the liver, and at least 10-fold lower concentrations in any other organ. Incorporation of hLF into nanoparticles shows improved targeting to the kidney. Gender is indicated by male / female symbols. [Figure 3B] Normal BALB / c mice were intravenously injected with In-labeled hLF either as naked peptide or complexed with mRNA to 80 nm nanoparticles (14 mice / group). Radioactivity was measured in the designated organs, demonstrating increased distribution ratios between the kidney and liver, and between the spleen and lung. Statistical tests and differences are shown in the graphs. [Figure 3C] Blood is collected from mice at several time points after peptide or polyplex injection and analyzed for the amount of radiolabeled peptide. Blood concentrations of peptide are plotted as a percentage of the injected dose and decline over time, with clearance being more rapid than the theoretical glomerular filtration rate (GFR), suggesting active binding / uptake. [Figure 4A] In a mouse model of LPS-induced inflammation, hLF is distributed to the glomeruli. Analysis of organs extracted after intravenous injection of Cy5.5-hLF in LPS-treated (left) or untreated (right) mice shows a predominant distribution in the kidney and liver. [Figure 4B] Confocal microscopy of organ sections shows renal localization of Cy5.5-hLF in the (proximal) tubules and exclusively in the glomeruli in LPS-treated mice. Glomeruli are indicated by arrows. Liver, lung, and spleen show much lower fluorescence. [Figure 4C] Quantification of confocal images from designated areas of the kidney and other organs. [Figure 5A]The hLF variants demonstrate improved targeting to the kidney. The percentage of total absorbed peptide detected in the kidney was calculated for each peptide variant and presented as a violin plot (top left). The peptide sequences with each charge are shown in Table 1 as SEQ ID NOS: 90-97. The percentage of injected dose per gram of tissue was presented for the kidney (bottom left), liver (bottom right), and spleen (top right). [Figure 5B] Additionally, kidney / liver ratios were determined for In-labeled hLF mutants with modifications to both the N- and C-termini of the peptide. Box plots of the mutants correspond to the sequences listed in Table 1. [Figure 6] Analysis of mouse kidney sections after intravenous injection of PEG-coupled hLF mutants into LPS-challenged C57B1 / 6 mice. Note that due to quenching and coupling efficiency, the signal is expected to be lower compared to injection of unconjugated peptide. Cy5.5 signal is pseudocolored, and glomeruli are circled. DETAILED DESCRIPTION OF THE INVENTION

[0090] [Definition] "Sequence identity" is defined herein as a relationship between two or more amino acid (peptide, polypeptide, or protein) sequences or two or more nucleic acid (nucleotide, polynucleotide) sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between amino acid sequences or nucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one peptide or polypeptide to the sequence of a second peptide or polypeptide. In a preferred embodiment, identity or similarity is calculated across all SEQ ID NOs identified herein. "Identity" and "similarity" can be readily calculated by known methods, including, but not limited to, those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988).

[0091] Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods for determining identity and similarity between two sequences include, for example, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387(1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S.F. et al., J. Mol. Biol. 215:403-410(1990)). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410(1990)). The well-known Smith Waterman algorithm may also be used to determine identity.

[0092] Preferred parameters for polypeptide sequence comparison include the following: algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); comparison matrix: BLOSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992); gap penalty: 12; and gap length penalty: 4. A program useful with these parameters is publicly available as the "Ogap" program from the Genetics Computer Group, Madison, WI. The aforementioned parameters are the default parameters for amino acid comparisons (with no penalty for end gaps).

[0093] Preferred parameters for nucleic acid comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: Match = +10, Mismatch = 0; Gap penalty: 50; Gap length penalty: 3. Available as the Gap program from the Genetics Computer Group, Madison, Wis. Shown above are the default parameters for nucleic acid comparison.

[0094] Optionally, when determining the degree of amino acid similarity, those skilled in the art may also consider so-called "conservative" amino acid substitutions, as will be apparent to those skilled in the art. Conservative amino acid substitutions refer to the interchangeability of residues with similar side chains. For example, the group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic hydroxyl side chains is serine and threonine; the group of amino acids with amide-containing side chains is asparagine and glutamine; the group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains is lysine, arginine, and histidine; and the group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitutional variants of the amino acid sequences disclosed herein are those in which at least one residue in the disclosed sequence has been removed and a different residue inserted in its place. Preferably, the amino acid changes are conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to ser; Arg to lys; Asn to gln or his; Asp to glu; Cys to ser or ala; Gln to asn; Glu to asp; Gly to pro; His to asn or gln; Ile to leu or val; Leu to ile or val; Lys to arg, gln, or glu; Met to leu or ile; Phe to met, leu, or tyr; Ser to thr; Thr to ser; Trp to tyr; Tyr to trp or phe; and Val to ile or leu.

[0095] A "nucleic acid molecule" or "polynucleotide" (the terms are used interchangeably herein) is represented by a nucleotide sequence. A "polypeptide" is represented by an amino acid sequence. A "nucleic acid construct" is defined as a nucleic acid molecule that has been isolated from a naturally occurring gene or has been modified to contain segments of nucleic acid that are joined or juxtaposed in a manner that does not appear to occur in nature. A nucleic acid molecule is represented by a nucleotide sequence. Optionally, the nucleotide sequence present in the nucleic acid construct is operably linked to one or more control sequences that direct the production or expression of said peptide or polypeptide in a cell or in a subject.

[0096] "Operably linked" is defined herein as an arrangement where a control sequence is suitably positioned relative to a nucleotide sequence encoding a polypeptide of the invention such that the control sequence directs the production / expression of the peptide or polypeptide of the invention in a cell and / or in a subject. "Operably linked" may also be used to define an arrangement where a sequence is suitably positioned relative to another sequence encoding a functional domain such that a chimeric polypeptide is encoded in a cell and / or in a subject.

[0097] "Expression" is interpreted accordingly to include any step involved in the production of a peptide or polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification and secretion.

[0098] "Control sequences" is defined herein to include all components necessary or advantageous for the expression of a polypeptide. At a minimum, control sequences include a promoter and transcriptional and translational stop signals. Optionally, a promoter represented by a nucleotide sequence present in the nucleic acid construct is operably linked to another nucleotide sequence encoding a peptide or polypeptide identified herein.

[0099] The term "transformation" refers to a permanent or transient genetic change induced in a cell following the incorporation of new DNA (i.e., DNA exogenous to the cell). As intended in the present invention, when the cell is a bacterial cell, the term refers to an extrachromosomal, self-replicating vector that typically harbors a selectable antibiotic resistance.

[0100] An "expression vector" may be any vector that can be conveniently subjected to recombinant DNA procedures and can result in the expression of a nucleotide sequence encoding a polypeptide of the present invention in a cell and / or a subject. As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes or nucleic acids, located upstream in the transcription direction of the gene's transcription start site. It is associated with binding sites for DNA-dependent RNA polymerase, a transcription start site, and any other DNA sequence, including, but not limited to, binding sites identified by the presence of transcription factor binding sites, repressor and activator protein binding sites, and any other sequence of nucleotides known to those skilled in the art, that act directly or indirectly to regulate the amount of transcription from the promoter. Within the context of the present invention, a promoter preferably ends at nucleotide -1 of the transcription start site (TSS).

[0101] "Polypeptide," as used herein, refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. A polypeptide consists of consecutive amino acids. The term "polypeptide" encompasses naturally occurring or synthetic molecules.

[0102] Sequence identity is preferably determined over the entire length of the subject sequence.

[0103] The sequence information provided herein should not be construed too narrowly with respect to requiring the inclusion of misidentified bases, as those skilled in the art are capable of identifying such misidentified bases and know how to correct for such errors.

[0104] The term "active pharmaceutical compound / ingredient" (API) for the purposes of this invention includes (messenger) RNA, oligonucleotides and DNA, even if the proposed activity is derived from a protein, polypeptide or peptide produced or controlled by the RNA or DNA.

[0105] Within this document and its claims, the verb "to comprise" and its conjugations are used in their open-ended sense to mean that the items following the term are included, but not that items not recited are excluded. Furthermore, the verb "to consist" may be replaced with "to consist essentially of," meaning that the nucleic acid construct or vector or cell product or composition or nucleic acid molecule or peptide or polypeptide defined herein may contain additional components other than those specifically identified; said additional components do not alter the inherent characteristics of the invention. Furthermore, reference to an element with the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that one and only one element be present. Thus, the indefinite article "a" or "an" typically means "at least one." The terms "about" or "approximately," when used in connection with a numerical value (e.g., about 10), preferably mean that the value can be ±10% of the given value (of 10).

[0106] All patents and references cited herein are incorporated by reference in their entirety.

[0107] Unless otherwise indicated, each embodiment described herein may be combined with any other embodiment described herein.

[0108] Further embodiments Further embodiments of the present invention are now listed below.

[0109] 1. A conjugate comprising a cargo and a targeting moiety that binds to intelectin 1 (ITLN-1), the conjugate intended for use as an agent to bind to, target, purify, induce cellular uptake into, or transfect cells expressing ITLN-1, comprising administering the conjugate to a subject.

[0110] 1a. The targeting moiety is a. specifically binds to Intelectin 1 (ITLN-1); and / or b. has sufficient affinity for ITLN-1 for stable binding; and / or c. does not contain domains capable of binding to other cell surface receptors under biologically relevant conditions; and / or d. accessible to binding by ITLN-1; and / or e. is not a full-length lactoferrin protein; A conjugate for use according to embodiment 1.

[0111] 2. A conjugate for use according to embodiment 1 or 1a, wherein the targeting moiety is selected from the group consisting of proteins, peptides, peptidomimetics, DNA, RNA, carbohydrates, polymers, heterocycles, and lipids.

[0112] 3. The conjugate for use according to embodiment 2, wherein the targeting moiety is a peptide.

[0113] 4. A conjugate for use according to embodiment 3, wherein the peptide comprises or consists of at least 6 consecutive amino acids of lactoferrin protein or a variant thereof, preferably exclusively an ITLN-1 binding domain, a combination of ITLN-1 binding domains, or a composite ITLN-1 binding domain, preferably up to 300 consecutive amino acids, more preferably up to 100 consecutive amino acids, even more preferably up to 50 consecutive amino acids, and most preferably up to 30 consecutive amino acids.

[0114] 4a. A conjugate for use according to embodiment 4, wherein the peptide is not a full-length and / or wild-type lactoferrin protein.

[0115] 5. A conjugate for use according to embodiment 4 or 4a, wherein the lactoferrin protein is human, bovine, murine, caprine, ovine, or non-human primate lactoferrin.

[0116] 6. A complex for use according to embodiment 5, wherein the human lactoferrin protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1 and the bovine lactoferrin protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2.

[0117] 7. A conjugate for use according to any of embodiments 3 to 6, wherein the peptide has a length of about 6 amino acids to about 30 amino acids, preferably at least 6 and up to 30 amino acids.

[0118] 8. A conjugate for use according to any of embodiments 3 to 7, wherein the peptide has a net charge of at most +7.

[0119] 9. The conjugate for use according to any of embodiments 3 to 8, wherein the peptide comprises or consists of a peptide selected from the group consisting of SEQ ID NOs: 3 to 84.

[0120] 10. A conjugate for use according to any of embodiments 1 to 9, wherein the targeting moiety, preferably a peptide, comprises a protecting moiety and / or a labeling moiety.

[0121] 11. A conjugate for use according to any of embodiments 1 to 10, wherein the targeting moiety, preferably a peptide, is stabilized in biological solutions.

[0122] 12. A conjugate for use according to embodiment 10 or 11, wherein the protecting moiety is selected from the group consisting of a terminal D-amino acid, a D-amino acid, an unnatural amino acid, N-terminal acetylation, N-terminal acylation, C-terminal amidation, C-terminal modification or substitution with N-alkylamide, aldehyde, ester, p-nitroanilide, 7-amino-4-methylcoumarin, formyl, pyroglutamyl, a fatty acid, urea, carbamate, sulfonamide or alkylamine, glycosylation, PEGylation, PAS, a suitable fluorescent group, a chelator for ions, preferably a radioisotope for ions, such as, for example, a suitable fluorescent label and / or N-terminal modification or substitution with diethylenetriaminepentaacetic acid (DTPA) or an equivalent.

[0123] 13. The conjugate for use according to embodiment 10, wherein the labeling moiety is selected from the group consisting of a fluorophore, a hapten, a DNA barcode, a PNA barcode, an XNA barcode, or an RNA barcode, a stable isotope, a substrate for click chemistry, and a radioactive label.

[0124] 14. A conjugate for use according to any of embodiments 1 to 13, wherein the cargo is covalently or non-covalently bound to the targeting moiety.

[0125] 15. A conjugate for use according to embodiment 14, wherein the cargo is covalently attached to the targeting moiety using one or more naturally occurring functional groups within said targeting moiety, such as the amino terminus, the carboxy terminus, a side chain amino group, a side chain carboxy group, or preferably cysteine.

[0126] 16. The conjugate for use according to embodiment 14 or 15, wherein the multiple targeting moieties are covalently linked to the cargo, for example by means of a dendrimeric structure of the targeting moieties.

[0127] 17. A conjugate for use according to any of embodiments 1 to 16, wherein the affinity of the conjugate for ITLN-1 is enhanced by the multivalent use of the targeting moiety, preferably by at least two-fold, more preferably by at least three-fold.

[0128] 17a. The conjugate for use according to embodiment 17, wherein the affinity of the conjugate for ITLN-1 is the affinity for ITLN-1-expressing cells.

[0129] 18. The conjugate for use according to any one of embodiments 1 to 17a, wherein the cargo is a nucleic acid, peptide, polypeptide or protein, carbohydrate, lipid, polymer, small molecule or mixture thereof.

[0130] 19. The conjugate for use according to any one of embodiments 1 to 18, wherein the cargo is a particle, for example a microparticle or nanoparticle, a liposome, a lipid nanoparticle, a polymer particle, a silica particle, a carbon nanotube, a gold particle, or a vehicle such as a lipid micelle or a polymer micelle.

[0131] 20. The conjugate for use according to any one of embodiments 1 to 19, wherein the particle comprises a pharmaceutically acceptable compound, such as a pharmaceutically active compound or a diagnostic compound.

[0132] 21. The conjugate for use according to any one of embodiments 1 to 20, wherein the peptide is linked to the cargo covalently, electrostatically, hydrophobically, or via an intermediate molecule, or through a combination thereof.

[0133] 22. The conjugate for use according to any one of embodiments 1 to 21, wherein the binding is directed to a cell, and the cell is an ITLN1-expressing kidney cell, such as a glomerular endothelial cell or a podocyte.

[0134] 23. The conjugate for use according to any one of embodiments 1 to 22, wherein administration to the subject is carried out by injection, preferably by intravenous (IV) injection.

[0135] 24. The conjugate for use according to any one of embodiments 1 to 23, wherein administration of the conjugate to a subject results in an increase in the intracellular concentration of the cargo in ITLN1-expressing cells.

[0136] 25. A conjugate for use according to embodiment 24, wherein administration of the conjugate to a subject results in biodistribution of the conjugate primarily to ITLN1-expressing cells such that the cargo accumulates at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, preferably at least 10-fold more in organs containing ITLN1-expressing cells than in off-target organs, which refer to organs that do not contain ITLN1-expressing cells.

[0137] 26. The conjugate for use according to embodiment 24 or 25, wherein administration of the conjugate to a subject results in a biodistribution that is primarily specific to the kidney, such that the cargo accumulates in the kidney at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, preferably at least 10-fold more than in any of the off-target organs.

[0138] 27. The conjugate for use according to any one of embodiments 1 to 26, wherein the use is therapeutic, diagnostic, prophylactic and / or therapeutic-diagnostic use.

[0139] 27a. The conjugate for use according to embodiment 27a, wherein the therapeutic-diagnostic use is a combination of therapeutic and diagnostic use.

[0140] 28. A conjugate for use according to any one of embodiments 1 to 27a, intended for use in the treatment of kidney diseases.

[0141] 28a. The conjugate for use according to embodiment 28, wherein the renal disease is a chronic renal disease.

[0142] 28b. The conjugate for use according to embodiment 28 or 28a, wherein the cargo is a steroid, an anti-inflammatory drug, an antifibrotic drug, a steroidal anti-inflammatory drug, a protein kinase inhibitor, a gene-correcting agent, or an oligonucleotide, or the cargo is messenger RNA, circular RNA, trans-amplifying RNA, self-amplifying RNA, or DNA, wherein the cargo is preferably an anti-inflammatory drug or an antifibrotic drug, and the cargo is preferably a nucleic acid.

[0143] 29. A conjugate for use according to any one of embodiments 1 to 28b, intended for use in the treatment of inflammatory diseases, preferably inflammatory (chronic) kidney diseases.

[0144] 30. A conjugate for use according to any one of embodiments 1 to 29, intended for use in the treatment of pathologies associated with upregulation, overexpression and / or increased effectiveness of ITLN-1.

[0145] 30a. The conjugate for use according to embodiment 30, wherein the pathology is inflammation, such as nephritis or ulcerative colitis, or the pathology is cancer, such as prostate cancer.

[0146] 31. A conjugate for use according to any one of embodiments 1 to 30a, intended for use in the treatment of acute kidney injury.

[0147] 32. The conjugate according to any one of embodiments 1 to 31 for use as a conditioning treatment for kidney transplantation, administered internally and / or externally.

[0148] 33. A conjugate for use according to any one of embodiments 1 to 32, wherein clearance of the cargo from the system is promoted compared to a cargo without a targeting moiety, and clearance from the system is preferably clearance from the blood circulation system.

[0149] 34. The conjugate for use according to embodiment 33, wherein clearance of the cargo is more than 95% complete within less than 1 month, preferably less than 1 week, less than 1 day, less than 1 hour, less than 30 minutes, less than 20 minutes, less than 15 minutes, less than 10 minutes, less than 7 minutes, more preferably less than 5 minutes.

[0150] 35. Use of a conjugate as defined in any one of claims 1 to 32 to bind to, target, purify, induce uptake of, or transfect cells expressing ITLN-1, comprising contacting the conjugate with cells expressing ITLN-1 in vitro or ex vivo.

[0151] 35a. The use according to embodiment 35, wherein the contacting is under conditions that allow specific binding and / or uptake by the receptor, comprising a concentration of less than 5 μM, a pH of 6.0 to 7.5, a duration of contact of at least 2 minutes and up to 72 hours, preferably 2 to 20 minutes, and a temperature of 15 to 37° C.

[0152] 36. An in vitro or ex vivo method for binding to, targeting to, purifying, inducing (endosomal) uptake of or transfecting cells expressing ITLN-1, comprising contacting the cells in vitro or ex vivo with a complex as defined in any of embodiments 1 to 22.

[0153] 36a. The method according to embodiment 36, wherein the contacting is under conditions that allow receptor-specific binding and / or uptake, comprising a concentration of less than 5 μM, a pH of 6.0 to 7.5, a duration of contact of at least 2 minutes and up to 72 hours, preferably 2 to 20 minutes, and a temperature of 15 to 37° C. [Example]

[0154] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

[0155] [Example 1] This example illustrates the specific uptake of human lactoferrin (hLF) peptide into glomerular endothelial cells, independent of its activity as a cell-penetrating peptide (CPP) as previously described (International Application PCT / EP2006 / 010271).

[0156] [method] Conditionally immortalized mouse glomerular endothelial cells (mGEnCs) were cultured as previously described (Rops et al. Kidney Int. 2004;66(6):2193-201). HeLa cells, a frequently used model cell line, were cultured in DMEM / F12 medium containing 10% fetal calf serum (FCS). To test uptake, cells were incubated for 20 minutes at 37°C in their respective medium with 10% FCS containing fluorescein-labeled peptides at the indicated concentrations. In some cases, cells were activated with 10 ng / ml mouse TNFα (Sigma) for 16 hours or co-incubated with Alexa Fluor 633-labeled transferrin (Invitrogen Molecular Probes). Cells were then washed with PBS and analyzed using confocal laser scanning microscopy on a TCS SP5 confocal microscope (Leica Microsystems) or detached with trypsin / EDTA and analyzed by counting 20,000 cells on a FACSCalibur flow cytometer (BD Biosciences).

[0157] [result] Incubation of mGEnCs with multiple cell-penetrating peptides demonstrated that the hLF peptide exhibited much more efficient dose-dependent uptake than two well-known cell-penetrating peptides, nona-arginine (R9) and penetratin (Figure 1A). In contrast, in HeLa cells, all peptides showed equivalent uptake efficiency at lower concentrations (Figure 1B), while 20 μM R9 demonstrated highly efficient uptake, as previously observed (Duchardt et al. J Biol Chem. 2009;284(52):36099-108). The preferential uptake of hLF by mGEnCs was not due to differences in proteolysis of the peptide prior to uptake or in binding to serum proteins. We also tested the uptake of a mouse variant of hLF (termed mLF), which has a lower net positive charge (Table 1). Uptake in mGEnCs occurred similarly to hLF (Figure 1A). In contrast, HeLa cells showed only minimal uptake of mLF (Fig. 1B). Mutants of the hLF peptide that lacked disulfide bridges or consisted of D-amino acids both showed significantly reduced uptake in mGEnCs.

[0158] To further demonstrate that hLF uptake occurred via a mechanism distinct from that typically observed for CPPs, we addressed the role of heparan sulfate proteoglycans (HSPGs). We previously demonstrated that hLF uptake in HeLa cells, following prototypic CPPs, depends on the presence of HSPGs (Duchardt et al. J Biol Chem. 2009;284(52):36099-108; Wallbrecher et al. Cell Mol Life Sci. 2014;71(14):2717-2729). In glomerular endothelial cells, removal of heparan sulfate by heparinase treatment resulted in only a slight decrease (approximately 20%) in the uptake of hLF and mLF, whereas R9 uptake was reduced by approximately 60% (Figure 1D).

[0159] Within mGEnCs, lactoferrin-derived peptides showed punctate staining indicative of endocytic uptake (Fig. 1C). The fluorescence was almost perfectly colocalized with fluorescently labeled transferrin, a marker of clathrin-dependent endocytosis.

[0160] Interestingly, treatment of mGEnCs with TNFα, which results in proinflammatory activation of the cells, enhanced hLF and penetratin uptake by 50–100%, although penetratin uptake was still barely detectable by confocal microscopy (Figure 1E). Furthermore, transferrin uptake was not significantly affected by TNFα treatment, indicating no general effect on clathrin-mediated endocytosis.

[0161] [interpretation] Here, we demonstrate that a 22-amino acid peptide derived from lactoferrin, previously identified as a CPP, exhibits uptake significantly exceeding that of other cell-penetrating peptides. Its uptake activity in HeLa cells was similar to that of the prototype CPPs nona-arginine and penetratin. The fact that the mLF peptide, which has only minimal activity as a CPP in HeLa cells, also exhibited potent uptake suggests a receptor-dependent uptake mechanism. This is reinforced by the peptide's exclusive colocalization with transferrin, providing evidence for clathrin-mediated endocytosis as an uptake pathway, in contrast to the prototype CPP, which simultaneously associates with different endocytic uptake pathways (Duchardt et al. Traffic 2007;8(7):848-866). Finally, the limited role of heparan sulfate in the uptake of lactoferrin-derived peptides by glomerular endothelial cells contradicted the heparan sulfate-dependent uptake of CPPs in HeLa cells (Favretto et al. J Contr Rel. 2014;180:81-90, Duchardt et al. J Biol Chem. 2009;284(52):36099-108).

[0162] [Table 1]

[0163] [Example 2] This example illustrates the presence of ITLN-1 on glomerular endothelial cells and its involvement in the uptake of hLF peptides.

[0164] [method] Conditionally immortalized mouse glomerular endothelial cells (mGEnCs) were cultured as previously described (Rops et al. Kidney Int. 2004;66(6):2193-201). Cells were incubated in their respective medium with 10% FCS containing 5 μM fluorescein-labeled peptides for 20 minutes at 37°C. In some cases, cells were activated with 10 ng / ml mouse TNFα (Sigma) for 16 hours or co-incubated with 100 μg / ml Alexa Fluor 633-labeled transferrin (Invitrogen Molecular Probes). Pretreatment with ITLN-1 or control siPool siRNA (siTools Biotech) was performed according to the manufacturer's protocol. Cells were then detached with trypsin / EDTA and analyzed by flow cytometry on a FACSCalibur flow cytometer (BD Biosciences). For immunostaining, mouse kidney cells or 5 μm cryosections were fixed with 4% paraformaldehyde for 10 minutes and, where indicated, permeabilized with 0.3% Triton X-100 in PBS. Cells and cryosections were then stained with anti-inteletin 1 (R&D Systems) or anti-LRP-1 (Abcam) antibodies for 1 hour, washed, and then incubated with the appropriate Alexa Fluor-conjugated secondary antibody (Invitrogen Molecular Probes) for 1 hour. Cells and cryosections were analyzed by confocal laser scanning microscopy using a TCS SP5 confocal microscope (Leica Microsystems).

[0165] [result] Glomerular endothelial cells were examined for the expression of two previously described lactoferrin receptors, ITLN-1 (ITLN-1) and low-density lipoprotein receptor-related protein 1 (LRP-1) (Suzuki et al. Biochemistry 2001;40(51):15771-15779; Willnow et al. J|BiolChem. 1992;267(36):26172-26180). We found that ITLN-1 was expressed on the membrane of these cells (Figure 2A) and was strongly present in the cytoplasm of these cells (Figure 2A). In contrast, we did not observe significant expression of LRP-1 by flow cytometry or confocal laser scanning microscopy. Interestingly, while ITLN-1 expression coincided with the uptake of non-CPP mLF in multiple endothelial cell lines, including ciGEnC, HUVEC, and EOMA, HeLa cells lacked ITLN-1 expression (Table 2). Furthermore, activation of mGEnCs with TNFα enhanced ITLN-1 expression, consistent with increased uptake of hLFs and mLFs after activation (Figure 2B). We were unable to directly visualize the colocalization of ITLN-1 with fluorescein-labeled hLFs after uptake because we were unable to immobilize the peptide. Therefore, we co-incubated mGEnCs with hLFs and fluorescently labeled transferrin, which resulted in complete colocalization with hLFs (Figure 2C), and stained for ITLN-1. This demonstrated significant intracellular colocalization of ITLN-1 with transferrin-containing vesicles (Figure 2D). To directly demonstrate the involvement of ITLN-1 in the uptake of lactoferrin-derived peptides, we pretreated mGEnCs with ITLN-1 siRNA, resulting in almost complete downregulation of the protein (Figure 2E, upper right panel). Cells treated with ITLN-1 siRNA showed reduced uptake of hLF and mLF compared with control siRNA, whereas uptake of R9, penetratin, and transferrin was unaffected (Fig. 2E, bar graph panel). Finally, we stained kidney cryosections from BALB / c and MRL / MpJ mice (healthy (middle panel) and diseased (right panel)) for ITLN-1.ILTN-1 protein expression could be observed in glomeruli and peritubular capillaries (Fig. 2F).

[0166] [interpretation] The characteristics of hLF uptake in glomerular endothelial cells strongly suggested that the uptake of lactoferrin-derived peptides may involve specific receptors. We confirmed the involvement of ITLN-1 in the uptake of lactoferrin-derived peptides by downregulating ITLN-1 with siRNA, which reduced the uptake of lactoferrin-derived peptides but not prototype CPPs. We were able to show that while glomerular endothelial cells express ITLN-1, this receptor is not expressed in cells that do not exhibit specific uptake of hLF. In contrast, we did not find expression of LRP-1, another described receptor for hLF. Furthermore, we examined the expression of ITLN-1 in normal BALB / c and MRL / MpJ mice (background strains), as well as in MRL / MpJ-Fas mice that develop lupus-associated glomerular inflammation. lpr We were able to demonstrate the presence of ITLN-1 in mouse glomeruli. Collectively, these data indicate that ITLN-1 contributes to the uptake of lactoferrin-derived peptides in glomerular endothelial cells.

[0167] [Table 2]

[0168] [Example 3] This example illustrates that after intravenous injection, the hLF peptide is distributed primarily to the kidneys of normal, healthy mice.

[0169] [method] Female and male 10-12 week old C57Bl / 6 mice (Charles River) were treated with 200 μM 11150 μl of In-DOTA-labeled hLF peptide was injected via the tail vein. Assuming a blood volume of 1.2 ml, this would theoretically result in a circulating concentration of approximately 8.5 μM. Urine, blood, and organs were collected 4 hours after injection, and radioactivity was measured in a gamma counter. The percentage injected dose per gram of tissue (%ID / gram) was calculated, along with the % absorbed dose for each tissue.

[0170] [result] The biodistribution of hLF peptide was primarily located in the kidneys of both female and male mice (Figure 3A). Distribution to the liver was approximately twofold lower, while the spleen and lungs had 5- to 10-fold lower peptide concentrations. Other organs, including the intestine, skin, and heart, showed over 20-fold lower distribution of hLF peptide. The combined percentage of absorbed dose in the kidney and liver exceeded 90% of the total dose in all organs. After 4 hours, blood concentrations were <1%, and hLF peptide was either taken up by cells or excreted in the urine.

[0171] [interpretation] The surprising primary distribution of the hLF peptide in the kidney suggests a specific uptake mechanism in renal cells. Based on the fact that the hLF peptide has activity as a CPP, rapid uptake in most well-perfused organs was expected, as previously shown for a selection of different CPPs (Sarko et al. Mol Pharmaceut. 2010;7(6):2224-2231). Furthermore, the biodistribution of radiolabeled full-length human lactoferrin protein was previously shown to involve multiple organs, primarily excluding the kidney (Kanoun et al. J Radioanalytical Nuclear Chem. 2018;317:177-185). The use of full-length lactoferrin protein to target nanoparticles resulted in enhanced uptake in the liver, spleen, and brain, while uptake in the kidney was reduced (Qi et al. J Nanobiotechnology. 2021;19:446; Kaili et al. Int J Pharmceutics. 2011;415:273-283; Farhan et al. Environmental Sci Pollution Res. 2018;9:1-17). In contrast, in our case, organs such as the liver, spleen, lung, and intestine, which have described other promising receptors for full-length lactoferrin, absorbed less than 5% of the lactoferrin-derived peptides. The predominant abundance in the kidney confirms the ITLN-1-mediated uptake we identified in glomerular cells. This distribution also suggests that ITLN-1-mediated uptake of hLF exceeds that via mechanisms associated with prototypic CPPs, and therefore suggests that hLF acts by targeting ITLN-1 and not like the CPPs described in the prior art. Furthermore, the observed biodistribution pattern is indicative of the absence of binding to other receptors, proteins, sugars, and / or cellular components that would lead to uptake in other organs. No significant differences in kidney targeting were observed between female and male mice.

[0172] [Example 4] This example demonstrates that complexation of hLF peptide with mRNA results in enhanced distribution to the kidney.

[0173] [method] Polyplexes of hLF peptide and mRNA were formed by rapid fluid mixing using an N / P ratio of 3. The complexes were then measured for their size using dynamic light scattering (DLS). Female and male 10-12 week old C57Bl / 6 mice (Charles River) were injected with 200 μM of hLF peptide, either free or complexed with mRNA. 111 50 μl of In-DOTA-labeled hLF peptide was injected via the tail vein. Assuming a blood volume of 1.2 ml, this would theoretically result in a circulating concentration of approximately 8.5 μM. Urine, blood, and organs were collected 4 h after injection, and radioactivity was measured using a gamma counter. The percentage injected dose per gram of tissue (%ID / gram) was calculated for each tissue. Blood was collected from mice at several time points (1 min to 240 min) to measure the blood concentration of the peptide.

[0174] [result] Intravenous injection of hLF / mRNA polyplexes, approximately 80 nm in size, resulted in similar peptide concentrations in the kidney compared with the free peptide (Figure 3A). Surprisingly, we observed a decrease in distribution to the liver, spleen, and lung (Figure 3B). While the kidney / liver ratio for polyplexes increased approximately 2.5-fold compared with the free peptide, even greater significant increases were observed in the kidney / spleen (3-fold) and kidney / lung (6-fold) ratios. Differences in other organs were due to low amounts of peptide in each organ, with minimal peripheral effects. Additionally, we observed rapid blood clearance of the free or mRNA-complexed peptide, which was faster than the theoretical glomerular filtration rate (GFR) (Figure 3C).

[0175] [interpretation] The relative increase in renal biodistribution after complexation of hLF suggests the role of multivalency in the uptake mechanism. The presence of multiple hLF peptides on the surface of polyplexes, and therefore multiple available ITLN-1 binding epitopes, would favor receptor-mediated uptake. The reduced uptake in other organs, including the liver, spleen, and lung, could be explained by their dependence on positively charged residues, which are partially encompassed by the mRNA. The rapid blood clearance is consistent with binding and / or uptake of the active peptide.

[0176] [Example 5] This example illustrates the enhanced biodistribution of intravenously injected hLF peptide to glomerular cells in the kidneys of mice with LPS-induced renal inflammation.

[0177] [method] At the start of the study, 8-10 week-old C57Bl / 6 mice (Charles River) received an intraperitoneal injection of either 100 μl of 2 mg / kg LPS in a 5% glucose solution or 5% glucose solution alone. 24 hours later, mice were injected with 50 μl of 200 μM Cy5.5-labeled peptide. Assuming a blood volume of 1.2 ml, this would theoretically result in a concentration of approximately 8.5 μM in the circulation. After 4 hours, organs were perfused with phosphate buffer, followed by 4% PFA. Organs were removed, and peptide distribution was analyzed using an in vivo imaging system (IVIS; Perkin Elmer). Next, organs were fixed by overnight incubation in 4% PFA, followed by overnight incubation in 30% sucrose. Organs were then frozen for analysis by confocal laser scanning microscopy using a TCS SP5 confocal microscope (Leica Microsystems). Glomerular and tubular regions were selected within the microscopic images, where the mean fluorescence intensity was determined by ImageJ software.

[0178] [result] Analysis of removed organs by IVIS showed increased biodistribution to the kidneys of mice with LPS-induced inflammation (Figure 4A). For both LPS-treated and untreated mice, the highest signal for the peptide was observed in the kidneys, followed by the liver, while the hLF peptide was virtually undetectable in the spleen, heart, eyes, brain, and lymph nodes. Urine was stained by excreted Cy5.5-labeled peptide in all mice except uninjected mice. Histological analysis of frozen sections showed that glomerular localization was evident in the kidneys of LPS-treated mice, while tubular localization was similar (Figure 4B). Furthermore, histological analysis confirmed that Cy5.5-labeled hLF was barely detectable in the spleen, while specific cells were weakly positive in the liver. Software analysis of multiple images of kidney frozen sections confirmed increased glomerular localization (Figure 4C).

[0179] [interpretation] Induction of renal inflammation with LPS, a well-established model of acute renal inflammation, resulted in increased biodistribution of Cy5.5-labeled hLF peptide to the glomerulus. Glomerular targeting is consistent with the ITLN-1-dependent uptake observed in vitro, the inflammation-induced increase in receptor expression, and the exclusive glomerular localization of ITLN-1 in the kidney. Importantly, this implies that pathophysiologically relevant glomerular cell types relevant to CKD are reached. Tubular uptake of the peptide is likely due to endocytic clearance via megalin or cubilin receptors, which are involved in the reabsorption of various proteins.

[0180] [Example 6] This example illustrates the development of hLF peptide derivatives that demonstrate enhanced targeting to the kidney.

[0181] [method] Female and male 10-12 week old C57Bl / 6 mice (Charles River) were treated with 200 μM 11150 μl of In-DOTA-labeled hLF peptide was injected via the tail vein. hLF peptide variants had different total charges, where the charge was reduced at either the N-terminal and / or C-terminal moieties (Table 1, SEQ ID NOs: 90-97). Four hours after injection, organs were harvested and radioactivity was measured in a gamma counter. The percentage injected dose per gram of tissue (%ID / gram) was determined for each tissue, and the ratio between organs was calculated.

[0182] [result] Intravenous injection of radiolabeled hLF variants with N- and / or C-terminal modifications resulting in a lower net charge showed increased localization to the kidney, while localization to the liver and spleen was decreased (Figure 5). This resulted in kidney absorption of up to 90% of the total amount of absorbed peptide in mice. Not surprisingly, the kidney / liver ratio increased more than 30-fold, from approximately 2.5 to 80. The exception was the shortest hLF peptide variant, consisting of seven amino acids and with a net charge of 3+ (see Table 1, SEQ ID NO: 96), which showed reduced distribution to the kidney as well as to the liver and spleen. However, the kidney / liver ratio increased further for peptides with lower net charges.

[0183] [interpretation] Altering the net charge of the hLF peptide by adding or removing naturally occurring amino acids significantly improved its kidney targeting efficacy. Previous studies have shown that kidney and liver targeting of CPPs is essentially independent of the net charge of the CPP (Sarko et al. Mol Pharmaceut. 2010;7(6):2224-2231). Therefore, it was surprising that reducing the net charge of the hLF peptide reduced uptake in the liver, spleen, and lungs, but not in the kidneys. Furthermore, shortening the peptide, although it may affect receptor binding, resulted in increased uptake in the kidneys. Furthermore, reducing the length by seven amino acids not only reliably reduced off-target effects in other organs, but also negatively affected uptake in the kidneys, resulting in increased urinary peptide excretion.

[0184] [Example 7] This example illustrates that conjugation of improved hLF variants to larger molecules that cannot pass through the renal filter results in exclusive renal distribution to the glomerulus while avoiding tubular uptake.

[0185] [method] Cy5.5-labeled hLF-6 (Table 1) was dissolved in 4 mM citrate buffer at pH 5 to block cysteine ​​reactivity. Conjugation was performed in 40 mM phosphate buffer at pH 7 in the dark for 2 hours by adding 40 kDa PEG-maleimide to achieve a 1:10 PEG:peptide ratio. At the start of the experiment, 8-10 week-old C57Bl / 6 mice (Charles River) received an intraperitoneal injection of either 100 μl of 2 mg / kg LPS in 5% glucose solution or 5% glucose solution alone. Twenty-four hours later, mice were injected with 50 μl of 200 μM Cy5.5-labeled PEG-hLF-6. After 4 hours, organs were perfused with phosphate buffer, followed by 4% PFA. Organs were removed, and peptide distribution was analyzed using an in vivo imaging system (IVIS; Perkin Elmer). Organs were then fixed by overnight incubation in 4% PFA followed by 30% sucrose, and then frozen for analysis by confocal laser scanning microscopy using a TCS SP5 confocal microscope (Leica Microsystems).

[0186] [result] Confocal microscopy analysis of kidney cryosections showed that PEG-hLF-6 was localized exclusively in the glomeruli, with no localization in the proximal tubules (Figure 6). PEG-hLF-6 was observed as multiple larger and smaller dots throughout the interior of the glomeruli. Urine from these mice was not stained (fluorescently).

[0187] [interpretation] Conjugation of an improved variant of hLF to 40 kDa PEG resulted in altered biodistribution in the kidney after intravenous injection in mice. When free peptide was injected into LPS-challenged mice, strong tubular localization was observed, with weaker staining in the glomeruli. Conjugation resulted in exclusive localization in the glomerulus. This suggests that attaching the peptide to nanoparticles that cannot pass the glomerular filter (>30-50 nm) prevents leakage into the tubular compartment and uptake by tubular cells via scavenger receptors such as megalin and cubulin. This is also supported by the fact that we failed to observe urinary excretion of the peptide.

[0188] [Example 8] This example illustrates that the full-length lactoferrin protein and its natural derivative lactoferricin do not have the same (renal-specific) biodistribution as the lactoferrin-derived peptides of the present invention.

[0189] [method] Lactoferrin and lactoferricin are cloned into expression plasmids containing the C-terminal sortase sequence protein-LPETG-HHHHHH. LPETGHHHHHH is SEQ ID NO: 98. The proteins are then expressed overnight in E. coli and purified from sonicated bacterial cell lysates with Ni-NTA beads using 400 mM imidazole to elute the proteins. After overnight dialysis, full-length lactoferrin and hLF-6 are labeled using a sortase exchange reaction with a fluorescent dye (Alexa647 or Cy5.5) on the C-terminus using a fluorescently labeled donor peptide (formula: biotin-GSSG-LPETG-labeled or biotin-GSSG-LPETG-PEG40k-labeled) as a substrate for 2 hours at 4°C. GSSGLPETG is SEQ ID NO: 99. Excess donor peptide is removed using streptavidin binding of the biotin tag on the N-terminus of the donor peptide. The purified, labeled protein / peptide is then injected into LPS-challenged mice via intravenous injection into the tail vein. Two hours later, the mice are sacrificed, and organs are collected and prepared for imaging by IVIS. Biodistribution is quantified for each organ, demonstrating relative distribution.

[0190] [result] Biodistribution imaging of full-length lactoferrin and lactoferricin shows accumulation primarily in the liver and spleen, with lesser accumulation in the kidney, brain, and intestine, whereas the hLF-6-peptide shows significantly greater accumulation in the kidney in the presence and absence of the 40 kDa PEG linker.

[0191] [interpretation] The different biodistribution of hLF-6, an exemplary peptide of the present invention, from full-length lactoferrin and lactoferricin (see Table 1) indicates that transferring the binding domain to another receptor results in more specific targeting. This also distinguishes this approach from the prior art, where biodistribution to multiple organs and very limited biodistribution to specific cell types have also been demonstrated. Differences in biodistribution are most likely due to differences in co-receptor binding and / or other factors, known or unknown.

Claims

1. A conjugate comprising a cargo and a targeting moiety that binds to Intelectin 1 (ITLN-1), intended for use as an agent for binding to, targeting to, purifying, inducing cellular uptake into, or transfecting a cell that expresses ITLN-1, comprising administering said conjugate to a subject; the targeting moiety is a peptide; The peptide has a length of at least 6 and at most 30 amino acids; and The peptide comprises or consists of a peptide selected from the group consisting of SEQ ID NOs: 3-84. Complex.

2. The conjugate for use according to claim 1, wherein the peptide has a length of at least 7 and at most 24 amino acids.

3. 3. The conjugate for use according to claim 1 or 2, wherein the peptide comprises or consists of a continuous sequence of at least 6 amino acids selected from the sequences represented by any one of SEQ ID NOs: 80 to 84.

4. 4. The conjugate for use according to any one of claims 1 to 3, wherein the affinity of said conjugate to ITLN-1 is enhanced, preferably at least 2-fold, more preferably at least 3-fold, by the multivalent use of said targeting moiety.

5. The complex for use according to claim 4, wherein the ITLN-1 is presented on an ITLN-1-expressing cell.

6. 6. The conjugate for use according to any one of claims 1 to 5, wherein the cargo is a nucleic acid, a peptide, a polypeptide or a protein, a carbohydrate, a lipid, a polymer, a small molecule, or a mixture thereof, and the cargo preferably comprises a pharmaceutically acceptable compound, such as a pharmaceutically active compound or a diagnostic compound.

7. 7. The conjugate for use according to any one of claims 1 to 6, wherein the cargo is a particle, such as a microparticle or nanoparticle, a liposome, a lipid nanoparticle, a polymer particle, a silica particle, a carbon nanotube, a gold particle, or a vehicle such as a lipid or polymer micelle.

8. The conjugate for use according to any one of claims 1 to 7, for targeting ITLN1-expressing kidney cells, such as cells that are glomerular endothelial cells or podocytes.

9. 9. The conjugate for use according to any one of claims 1 to 8, wherein administration of the conjugate to a subject results in a biodistribution of the conjugate primarily to ITLN1-expressing cells, such that the cargo accumulates at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, and preferably at least 10-fold more in organs containing ITLN1-expressing cells than in off-target organs, wherein off-target organs refer to organs that do not contain ITLN1-expressing cells, and preferably results in a biodistribution that is primarily kidney-specific, such that the cargo accumulates at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, and preferably at least 10-fold more in the kidney than in any of the off-target organs.

10. A conjugate for use according to any one of claims 1 to 9, intended for use as a medicament.

11. The conjugate for use according to claim 10, wherein the use is therapeutic, diagnostic, prophylactic and / or combined therapeutic and diagnostic.

12. 12. A conjugate for use according to claim 10 or 11 for use in the treatment of kidney disease.

13. The conjugate for use according to claim 12, wherein the kidney disease is a chronic kidney disease.

14. 14. The conjugate for use according to any one of claims 10 to 13, wherein the cargo is an anti-inflammatory drug, an anti-fibrotic drug, a steroid, a protein kinase inhibitor, a gene correcting drug, an oligonucleotide, or wherein the cargo is messenger RNA, circular RNA, trans-amplifying RNA, self-amplifying RNA, or DNA.

15. The conjugate for use according to any one of claims 10 to 14, wherein the cargo is an anti-inflammatory or anti-fibrotic drug, and the cargo is preferably a nucleic acid.

16. A conjugate for use according to any one of claims 10 to 15, intended for use in the treatment of pathologies associated with upregulation, overexpression and / or increased effectiveness of ITLN-1.

17. 17. The conjugate for use according to claim 16, wherein the pathology is inflammation such as nephritis and ulcerative colitis, or the pathology is cancer, for example prostate cancer.

18. A conjugate for use according to any one of claims 1 to 17, wherein clearance of said cargo from the system is enhanced compared to cargo without a targeting moiety.

19. 19. The conjugate for use according to claim 18, wherein said clearance from the system is clearance from the blood circulation system.

20. 20. Use of a complex as defined in any one of claims 1 to 19, comprising contacting said complex with a cell expressing ITLN-1 in vitro or ex vivo to bind to, target, purify, induce uptake of, or transfect said cell.

21. 21. A method for binding to, targeting, purifying, inducing uptake or transfecting a cell expressing ITLN-1, comprising contacting said cell with a complex as defined in any one of claims 1 to 20.

22. 22. The method of claim 21, which is in vitro or ex vivo.

23. 23. The use according to claim 20 or the method according to claim 21 or 22, wherein the uptake is endosomal uptake.

24. 24. The use according to claim 20 or the method according to claim 21 or 22 or 23, wherein the contacting is done under conditions that allow specific binding and / or uptake by the receptor, preferably comprising a concentration of complex less than 5 μM, preferably a pH of 6.0 to 7.5, a duration of contact preferably of at least 2 minutes and up to 72 hours, more preferably 2 to 20 minutes, and a temperature preferably of 15 to 37°C.

25. 1. A conjugate comprising a cargo and a targeting moiety that binds to Intelectin 1 (ITLN-1), the conjugate being intended for use as an agent to bind to, target, purify, induce cellular uptake into, or transfect cells that express ITLN-1, comprising administering the conjugate to a subject.

26. 26. The conjugate for use according to claim 25, wherein the targeting moiety is selected from the group consisting of proteins, peptides, peptidomimetics, DNA, RNA, carbohydrates, polymers, heterocycles, and lipids, wherein the targeting moiety is preferably a peptide, and the peptide preferably comprises or consists of at least 6 and up to 300 consecutive amino acids of lactoferrin protein, or a variant thereof, and the lactoferrin protein is preferably human, non-human primate, murine, bovine, caprine, or caprine lactoferrin.

27. 27. The conjugate for use according to claim 25 or 26, wherein the affinity of said conjugate for ITLN-1 is enhanced, preferably at least 2-fold, more preferably at least 3-fold, by the multivalent use of said targeting moiety.

28. 28. The conjugate for use according to any one of claims 25 to 27, wherein the cargo is a nucleic acid, a peptide, a polypeptide or a protein, a carbohydrate, a lipid, a polymer, a small molecule or a mixture thereof, and the cargo preferably comprises a pharmaceutically acceptable compound such as a pharmaceutically active compound or a diagnostic compound.

29. 29. The conjugate for use according to any one of claims 25 to 28, wherein the cargo is a particle, such as a microparticle or nanoparticle, a liposome, a lipid nanoparticle, a polymer particle, a silica particle, a carbon nanotube, a gold particle, or a vehicle such as a lipid or polymer micelle.

30. The conjugate for use according to any one of claims 25 to 29, wherein the particles comprise a pharmaceutically acceptable compound, such as a pharmaceutically active compound or a diagnostic compound.

31. A conjugate for use according to any one of claims 25 to 30, intended for use as a medicament.

32. 32. The conjugate for use according to claim 31, wherein said use is therapeutic, diagnostic, prophylactic and / or both therapeutic and diagnostic.

33. A conjugate for use according to any one of claims 31 to 32 for use in the treatment of kidney diseases.

34. A conjugate for use according to any one of claims 31 to 33 for use in the treatment of chronic kidney disease.

35. 31. Use of a complex as defined in any one of claims 25 to 30, comprising contacting said complex with a cell expressing ITLN-1 in vitro or ex vivo to bind to, target, purify, induce uptake of, or transfect said cell.

36. 31. An in vitro or ex vivo method for binding to, targeting to, purifying, inducing (endosomal) uptake of or transfecting cells expressing ITLN-1, comprising contacting said cells in vitro or ex vivo with a complex as defined in any one of claims 25 to 30.

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