RAGE receptor binding molecule, its conjugate, and its use for detecting, preventing or treating lung disease

Abstract

The present invention relates to a variable domain (VHH) molecule consisting solely of a camel heavy chain that binds to RAGE, and to the use of the same molecule, for example, to transport a pharmacokinetic or diagnostically important molecule to lung cells and organs in pathological conditions including infectious, inflammatory, cancerous, or rare lung diseases.

Description

[Technical Field]

[0001] The present invention relates to an advanced glycation end product receptor (RAGE) binding molecule and its use. More specifically, the present invention relates to a variable domain (VHH) molecule consisting only of the heavy chain of a camelid animal that binds to RAGE on the surface of cells, particularly lung epithelial cells, and its use, for example, to transport a molecule of interest for pharmaceutical or diagnostic purposes to lung cells to detect or treat lung diseases such as cancer, infectious or inflammatory lung disease. [Background technology]

[0002] The treatment of lung diseases, including lung cancer, pneumonia, and lung infections such as tuberculosis, is a challenging problem in clinical practice. This is because conventional drug delivery systems cannot effectively deliver drugs to the lungs via systemic pathways. Therefore, lung-targeted drug delivery systems that can deliver therapeutic agents to the lungs to increase drug concentration in lung tissue while reducing drug distribution in other organs and tissues are an ideal strategy sought after by physicians, especially those aiming to treat lung diseases. Several strategies have been developed to date, including lung inhalation. However, drugs delivered by inhalation are subject to multiple clearance mechanisms that are major barriers to drug absorption after lung administration (He, Gui, J. et al., 2022). Furthermore, an inhaler is required to administer the drug. Therefore, there is a need to develop more efficient strategies that can enhance the transport of therapeutic molecules, particularly to the lungs.

[0003] Over the past 30 years, various cell membrane receptor targeting strategies have been developed to optimize drug delivery. However, despite the advances made in the field of drug delivery, the need for drugs that can improve drug access to the lungs remains unmet in this technology.

[0004] The inventors hereby provide such advantageous ligands. In fact, the inventors have discovered and, for the first time herein, ligands that target a receptor expressed at high levels in the lung and at low levels in other tissues, namely the Advanced Glycation End Product Receptor (RAGE). These ligands consist of variable domains (VHHs) of heavy-chain-only antibodies found in camelids. Some of the VHHs of the present invention exhibit interspecies reactivity to human and mouse RAGE. The inventors demonstrate and, as disclosed herein, that the identified RAGE-targeted VHHs can deliver a cargo, preferably human IgG1 Fc, to the lung at high levels in a time-dependent manner.

[0005] Overexpression and activation of RAGE by ligands such as advanced glycation end products (AGEs) are associated with inflammatory processes found in some neurodegenerative diseases, diabetic nephropathy, non-diabetic vascular diseases, acute liver injury, lung injury, and malignant tumors (Sims, Rowe et al. 2010). RAGE is highly expressed in the lungs under non-pathological conditions (Chavakis, Bierhaus et al. 2004, Khaket, Kang et al. 2019). Small molecules acting as RAGE antagonists have been developed and are undergoing clinical trials, and therapeutic antibodies conjugated to drugs for treating specific cancers (endometrial cancer) have been evaluated in the preclinical stage (Healey, Pan-Castillo et al. 2019). VHH molecules are also being developed as human RAGE antagonists (Mohammed, Zeng et al. 2021). However, cross-reactive VHH-type molecules that can be used as vectors to deliver molecules of therapeutic interest to the lungs have not yet been developed. In particular, conventional technologies do not provide efficient VHH-type molecules that can be used to detect (diagnose), prevent, or treat human lung diseases. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] This invention provides a novel conjugating molecule that can be used to effectively transport molecules to the lungs. More specifically, the invention discloses a VHH molecule ("VHH"), particularly a VHH that can conjugate to both non-human and human RAGE and deliver pharmaceuticals, typically drugs, and especially biological agents, to the lungs. The invention demonstrates that the VHH molecule of the invention can effectively accumulate in the lungs and deliver conjugated therapeutic or imaging agents in vivo into organs, particularly into lung cells. Thus, such a VHH would be a highly advantageous molecule for therapeutic or diagnostic use.

[0007] Therefore, the object of the present invention relates to VHH molecules that bind to RAGE in humans and non-humans (e.g., rodents such as mice or rats).

[0008] A further object of the present invention is a VHH molecule that binds to human and / or non-human RAGE on the surface of lung cells. [Means for solving the problem]

[0009] The preferred VHH of the present invention can bind to RAGE in humans and rodents, particularly mice, and target lung cells, and has an affinity (Kd) for RAGE of less than 10 μM, for example, 0.1 nM to 10 μM, preferably 1 nM to 10 μM.

[0010] The preferred substance is a VHH molecule of the formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, which binds to the advanced glycation end product receptor (RAGE) on the surface of lung cells.

[0011] In certain embodiments, the VHH molecule comprises or consists of one or more of the following sequences: -CDR1 sequence selected from sequence numbers 1, 5, 9, 13 and 17, - A CDR2 sequence selected from sequence numbers 2, 6, 10, 14 and 18, and / or - A CDR3 sequence selected from sequence numbers 3, 7, 11, 15, and 19.

[0012] In another specific embodiment, the VHH molecule of the present invention is: - A CDR1 sequence selected from SEQ ID NOs: 1, 5, 9, 13, or 17, or a variant thereof having at least 60% amino acid identity with any one of the aforementioned sequences over its entire length. - A CDR2 sequence selected from SEQ ID NOs: 2, 6, 10, 14, or 18, or a variant thereof having at least 60% amino acid identity with any one of the aforementioned sequences over its entire length, and / or - A CDR3 sequence selected from SEQ ID NOs: 3, 7, 11, 15, or 19, or a variant thereof having at least 60% amino acid identity with any one of the aforementioned sequences over its entire length. It comprises or consists of the following: The aforementioned VHH has RAGE binding ability on the surface of lung cells.

[0013] In another particular embodiment, the VHH molecule comprises or consists of SEQ ID NOs: 1, 2 and 3, or SEQ ID NOs: 5, 6 and 7, or SEQ ID NOs: 9, 10 and 11, or SEQ ID NOs: 13, 14 and 15, or SEQ ID NOs: 17, 18 and 19.

[0014] In a more specific embodiment, the VHH molecule comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 4, 8, 12, 16, and 20, wherein the amino acid sequence optionally comprises a tag and / or a linker.

[0015] In yet another specific embodiment, the VHH molecule is i) preferably arranged [ka] Q tags, myc tags, or tags that include or consist of such a feature [ka] , tags such as a poly-His tag, a poly-Arg tag, a poly-Lys tag, a HA tag, a FLAG tag, a GFP tag, a CBP tag, a Strep II tag, a sortase tag, a SNAP tag, or a combination thereof, and / or ii) a linker such as a Gly linker or an Ala linker.

[0016] In further particular embodiments, the VHH molecule comprises i)

Chemical formula

Chemical formula

[0017] The present invention also relates to a chimeric agent (also interchangeably referred to herein as a "conjugate") comprising one or more VHHs as defined above conjugated to at least one additional compound / molecule. This additional compound may be a separate VHH or a molecule that is not a VHH.

[0018] More generally, the at least one additional compound can be a stabilizing group or a scaffold, which can be selected from an antibody or a fragment thereof (such as an Fc fragment), a VHH molecule, PEG, a serum albumin protein, or a serum albumin binding site (e.g., a protein, a peptide, or a chemical molecule). In another embodiment, the at least one additional compound can be a therapeutic, diagnostic, or imaging compound, or a vehicle comprising such a therapeutic, diagnostic, or imaging compound. In a further particular embodiment, the chimeric agent / conjugate can comprise both types of additional compounds, i.e., i) a stabilizing group or a scaffold, and ii) a therapeutic, diagnostic, or imaging compound, or a vehicle comprising the same.

[0019] The molecule conjugated to VHH may be, for example, any active compound useful in medicine (also referred to herein as "substance of interest"), such as a "diagnostic agent" such as a tracer, or a "therapeutic agent" such as a peptide, polypeptide, protein, antibody or its fragments, and nucleic acid. The chimeric agent may further comprise a vehicle comprising the substance of interest. This vehicle may be selected from, for example, viruses, virus-like particles (VLPs), cell-derived vesicles (CDVs), exosomes, lipid vehicles, and polymer vehicles, and is preferably a lipid nanoparticle (LNP), micelle, or liposome.

[0020] As described herein, chimeric agents may also include, in addition to or instead of the active compound, a stabilizing group (e.g., Fc, IgG, albumin, an albumin-binding molecule also referred herein as an albumin-binding moiety, or e.g., PEG) for extending the plasma half-life of the VHH or conjugate. Thus, a particular chimeric agent of the present invention comprises i) at least one (one or more) VHH molecules, ii) a stabilizing group, iii) an active compound (typically a therapeutic, diagnostic, or imaging compound), and optionally iv) a vehicle (e.g., a conjugate VHH-Fc-therapeutic agent) in any order.

[0021] The present invention further provides a pharmaceutical or diagnostic composition comprising the chimeric agent as defined above and an optionally appropriate (pharmaceutically acceptable) support or excipient.

[0022] The present invention also provides nucleic acids, vectors, and (recombinant) host cells that encode or contain the VHH molecules or chimeric agents defined above or described herein.

[0023] The present invention further provides a method for producing a chimeric agent, comprising covalently or noncovalently conjugating one or more VHHs as defined above to a molecule, drug, or scaffold.

[0024] Another object of the present invention relates to the VHH molecules or chimeric agents defined above for preparing and using pharmaceuticals or diagnostic agents, or for use as pharmaceuticals or diagnostic agents.

[0025] Another object of the present invention relates to the use of the VHH molecule as defined above to increase the biological activity and / or pulmonary delivery of any substance of interest, which is typically a diagnostic or therapeutic substance.

[0026] Another object of the present invention relates to a method for improving the distribution of molecules to a lung site, comprising coupling the molecule to the VHH defined above.

[0027] Another object of the present invention is a method for treating a disease condition in a subject, comprising administering the conjugate defined above to the subject.

[0028] Another object of the present invention is a method for imaging a specific cell type, target tissue or organ, typically lung cells or the lung organ, in a subject, the method comprising the step of administering the conjugate defined above to the subject.

[0029] Another object of the present invention is an improved method for treating a lung lesion in a subject requiring such treatment with a substance of interest, typically a therapeutic agent or drug, the method comprising the step of administering to the subject VHH, preferably a conjugate as defined above.

[0030] This invention can be used in all mammals, and especially in all humans. [Brief explanation of the drawing]

[0031] [Figure 1]RAGE expression in the tissues of pigs, rhesus monkeys, rats, and mice. (A) Western blots performed on the whole membrane fractions of the following organs in pigs, non-human primates (NHP, rhesus monkeys), rats, and mice: heart, muscle, kidney, cornea, retina, bladder, adrenal gland, pancreas, testis, stomach, lung, and liver. The amount of loaded protein is shown. (B) Immunohistochemical (IHC) detection of RAGE in frozen sections (18 μm) of rat (B1) and mouse (B3) lungs with anti-RAGE antibody (R&D Systems #Mab1179) followed by donkey anti-rat A488 secondary antibody (green). Cell nuclei are labeled with Hoechst #33258 (blue). When IHC is performed using only the secondary antibody, no green labeling is visible in the lungs of rats (B2) and mice (B4). [Figure 2] Validation of CHO cell lines expressing human or mouse RAGE. (A) Map of plasmid constructs used to generate various h / mRAGE-GFP expressing cell lines. (B) Validation of receptor expression in CHO cell lines overexpressing human-type RAGE by immunocytochemistry. Cell nuclei were labeled with Hoechst#33258 (blue). RAGE was detected with anti-RAGE antibody, followed by detection with Alexa594 conjugated secondary antibody (red). Co-labeling of RAGE-EGFP (green) and anti-RAGE antibody is shown in yellow in the merged image. (C) Validation of receptor expression in CHO cell lines by Western blotting of cell membrane specimens using RAGE-specific antibody, followed by HRP conjugated secondary antibody. [Figure 3]Apparent Kd determination of VHH on CHO cell lines expressing hRAGE and mRAGE. (A) CHO-hRAGE-EGFP cells and CHO-mRAGE-EGFP cells were incubated at 4°C for 1 hour while increasing the concentration of VHH and detected with mouse anti-6His (1 / 1000) and Alexa647 conjugate anti-mouse secondary antibody (1 / 400). Measurements were performed using flow cytometry. The ratio of fluorescence intensity for each point was normalized by the corresponding EGFP signal (receptor expression) to generate arbitrary units. Data are presented as mean ± SEM of at least three independent experiments. (B) Characteristics of selected VHH: molecular weight (Da); apparent Kd (nM) on human RAGE; apparent Kd (nM) on mouse RAGE. Data are presented as mean ± SEM of at least three independent experiments. [Figure 4] Determination of the apparent Kd of VHH-siRNA in mRAGE-expressing CHO cell lines. (A) CHO-mRAGE-EGFP cells were incubated at 4°C for 1 hour with increasing VHH-siRNA concentration and detected with mouse anti-6His (1 / 1000) and Alexa647 conjugate anti-mouse secondary antibody (1 / 400). Measurements were performed using flow cytometry. (B) Characteristics of selected VHH-siRNAs: Molecular weight (Da); apparent Kd against mouse RAGE (nM). [Figure 5] Cell binding / uptake of VHH-Fc in mRAGE-expressing CHO cells. These are representative confocal micrographs of CHO-mRAGE-EGFP cells (green) incubated with VHH1-Fc, VHH2-Fc, VHH3-Fc, VHH4-Fc, VHH5-Fc, and 50nM control VHHctrl-Fc at 37°C for 1 hour. After PFA fixation, the cell membrane was permeabilized with X-100 and detected using Alexa594 conjugated anti-hFc antibody (1 / 800) (red). Cell nuclei were labeled with Hoechst#33342 at 0.5 μg / ml (blue). In the merged image, co-labeling is shown in yellow / orange. [Figure 6]Cell binding / uptake of VHH-Fc in hRAGE-expressing CHO cells. These are representative confocal micrographs of CHO-hRAGE-EGFP cells (green) incubated with VHH1-Fc, VHH2-Fc, VHH3-Fc, VHH4-Fc, VHH5-Fc, and 50nM control VHHctrl-Fc at 37°C for 1 hour. After PFA fixation, the cell membrane was permeabilized with X-100 and detected using Alexa594 conjugated anti-hFc antibody (1 / 800) (red). Cell nuclei were labeled with Hoechst#33342 at 0.5 μg / ml (blue). In the merged image, co-labeling is shown in yellow / orange. [Figure 7] Apparent Kd determination of VHH-Fc and Fc-VHH on CHO cell lines expressing hRAGE and mRAGE. (A) CHO-hRAGE-EGFP and CHO-mRAGE-EGFP cells were incubated at 4°C for 1 hour with increasing concentrations of VHH-Fc or Fc-VHH and detected using Alexa647 conjugated anti-hFc antibody (1 / 400). Measurements were performed using flow cytometry. The ratio of fluorescence intensity for each point was normalized by the corresponding EGFP signal (receptor expression) to generate arbitrary units. Data are presented as mean ± SEM of at least three independent experiments. (B) Characteristics of selected VHH-Fc and Fc-VHH: molecular weight (Da); apparent Kd (nM) relative to human RAGE; apparent Kd (nM) relative to mouse RAGE. Data are presented as mean ± SEM of at least three independent experiments. [Figure 8]Competitive assay between VHH and VHH1-Fc. (A) Principle of the competitive test. In the first step, CHO-mRAGE-EGFP cells were incubated with diluted series of competitive cells at 4°C for 1 hour. Next, tracers at EC80-90 were added and incubated at 4°C for 1 hour. Subsequently, tracers were detected using an appropriate detection system. Measurements were performed using flow cytometry. (B) CHO-mRAGE-EGFP cells were incubated with competitive cells (VHH). Next, tracers at EC80 (VHH1-Fc) were added and detected with Alexa647 conjugate anti-hFc antibody (1 / 400). [Figure 9] Identification of the mRAGE domain interacting with VHH-Fc. (A) Schematic diagrams of the full-length and truncated constructs of mRAGE. In the extracellular portion of mRAGE, the domain was sequentially truncated, generating two truncated variants. An HA tag was added to confirm the proper extracellular localization of the extracellular domain. (B) Representative confocal micrographs of CHO cells transiently expressing mRAGE, HA-mRAGE, mRAGE-ΔV (=ΔV), and mRAGE-ΔV-C1 (=ΔV-C1) (all green), incubated with 250 nM VHH5-Fc at 37°C for 1 hour, fixed with PFA, and detected with Alexa594 conjugated anti-hFc antibody (1 / 800, red). Cell nuclei were labeled with Hoechst#33342 (blue) at 0.5 μg / ml. In the merged image, co-labeling is shown in yellow / orange. (C) This table summarizes the binding characteristics between anti-HA antibodies or VHH-Fc cells, along with full-length and truncated mRAGEs transiently expressed in CHO cells, as determined by immunocytochemistry experiments. (+) indicates positive binding, and (-) indicates no binding. [Figure 10]Cellular binding / colocalization of VHH-Fc on RAGE in mouse lungs. Immunocytochemical analysis was performed on the lungs of mice injected via tail vein with VHHctrl-Fc, VHH1-Fc, and VHH5-Fc at 35 nmol / kg. Mice were perfused with saline 48 hours post-injection, and lungs were incubated overnight in 4% PFA. Lungs were thoroughly washed with PBS 1X and incubated in 30% sucrose for 2 days. Double immunofluorescence staining of lung tissue was performed on lung sections with anti-RAGE (Alexa Fluor488, green) and VHH-Fc (Alexa Fluor594, red) antibodies. Cell nuclei were labeled with Hoechst#33342 (blue) at 0.5 μg / ml. In merged images, colabeling is shown in yellow / orange (right panel). Representative images were taken with a confocal microscope at 20x and 63x magnification. [Figure 11]A. Distribution and pulmonary uptake of VHH-Fc fusions in WT C57Bl / 6 mice. VHHctrl-Fc, VHH1-Fc, VHH4-Fc, and VHH5-Fc were infused into the tail vein at 35 nmol / kg, and mice were perfused with saline at 2, 6, 18, 48, 96, or 168 hours post-infusion. The amount of VHH-Fc in plasma and lung was evaluated by ELISA. Plasma VHH-Fc concentration (A, C) and percentage of infused volume per gram of tissue in plasma (B, D). Lung VHH-Fc concentration (E), percentage of infused volume per gram of lung tissue (F), and lung-to-plasma ratio (G). Data are expressed as mean ± SD. N=4-12 per group per time point; *p≦0.05, **p≦0.01, ***p≦0.001 for VHH1-Fc, 4, or 5 vs. VHHctrl-Fc. Distribution of VHH-Fc fusions and uptake into the liver and kidneys in B.WT C57Bl / 6 mice. VHHctrl-Fc, VHH1-Fc, VHH4-Fc, and VHH5-Fc were infused into the tail vein at 35 nmol / kg, and mice were perfused with saline at 6, 18, 48, 96, or 168 hours post-infusion. The amount of VHH-Fc in the liver and kidneys was evaluated by ELISA. Liver VHH-Fc concentration (H), percentage of infused dose per gram of liver tissue (I), and liver-to-plasma ratio (J). VHH-Fc concentration in the kidney (K), percentage of infusion volume per gram of renal tissue (L), and kidney-to-plasma ratio (M). Data are expressed as mean ± SD. N=4-12 per group per time point. *p≦0.05, **p≦0.01, ***p≦0.001 for VHH1-Fc, 4, or 5 vs. VHHctrl-Fc. [Figure 12] VHH conjugate strategies. Using either chemical conjugates or recombinant fusions, VHHs can be used to vectorize a number of types of molecules, including, but not exhaustively, imaging agents and radiotherapeutic agents, as well as proteins including small organic molecules, dyes, peptides, antibodies, nucleic acids including siRNA and antisense oligonucleotides (ASOs), nanoparticles (NPs), or liposomes. Furthermore, VHHs can be used to vectorize molecules in the form of monovalent (VHH) or polyvalent (VHHn) conjugates. [Figure 13] Characterization of VHH-RAGE LNPs, in vitro delivery of mLuc mRNA to h / mRAGE-GFP CHO cells, and in vivo distribution in C57 / Bl6 mice after 6 hours. (A) LNPs were functionalized with either VHHctrl or VHH5, loaded with mLuc mRNA, and characterized by DLS analysis to determine their Z-mean size and PDI (polydispersion index). Data represent the mean of N=3 measurements. In addition, LNPs were characterized for lipid concentration using a standard cholesterol assay and for mRNA concentration using a RiboGreen assay to estimate the N / P ratio. (B) CHO-hRAGE-EGFP or CHO-mRAGE-EGFP cells (20,000 cells per well in a 96-well plate) were incubated with bare LNPs, VHHctrl-LNPs, and VHH5-LNPs at concentrations equivalent to 1.25 μg mLuc mRNA / mL in OptiMEM culture medium at 37°C for 6 hours. The ability of naked or functionalized LNPs to deliver mLuc mRNA into cells was evaluated by quantifying the luminescence produced by translated luciferase proteins using the ONE-Glo® Luciferase Assay System (Promega). Luminescence was measured using the GloMax Navigator (Promega). Data represent relative light units (RLU) ± SEM mean. N=3 per group per condition;***p≦0.001 for VHH5-LNP vs VHHctrl. (C) VHHctrl-LNP and VHH5-LNP were injected into the tail vein of C57 / Bl6 mice at a mLuc mRNA equivalent of 20 μg / 200 μL per mouse, and organs were harvested 6 hours post-injection. Lungs, muscles (gastrocnemius), hearts, kidneys, and brains were pulverized, and luminescence was quantified using the ONE-Glo® Luciferase Assay System. Data represent relative light units (RLU) ± SEM mean. For each LNP preparation, N=3 / 4 mice, in the case of VHH5-LNP versus VHHctrl, p≦0.05;**p≦0.01. [Modes for carrying out the invention]

[0032] The present invention provides a novel RAGE-binding agent that can be used to deliver molecules such as therapeutic agents, imaging agents, or diagnostic agents to the lungs. More specifically, the present invention discloses an improved VHH molecule that binds to RAGE and its use.

[0033] Advanced glycation end product receptors (RAGEs) are 45 kDa transmembrane receptor members of the immunoglobulin superfamily. Structurally, full-length human RAGE consists of three major domains: - A V-type (variable) domain followed by two C-type (constant) domains (usually called C1 and C2); - A single hydrophobic transmembrane domain; and -Short charged intracellular cytoplasmic domains mainly involved in signal transduction (Neeper et al., J. Biol. Chem. 1992).

[0034] In addition to its full-length membrane-bound form, RAGE also exists in a soluble form. Soluble RAGE contains only the N-terminal and extracellular domains and is produced by alternative splicing or proteolytic degradation of RAGE by ADAM10 or matrix metalloproteinases (Raucci A et al. FASEB J.2008, Yonekura H et al. Biochem J.2003, Hudson B et al. FASEB J.2008).

[0035] In adult humans, RAGE is expressed in most tissues, but at very low levels except in the lungs (Brett J et al. Am J Pathol. 1993). RAGE recognizes a variety of ligands, including advanced glycation end products (AGEs), high-mobility box proteins (HMGB1), macrophage antigen 1 (Mac1), S-100 protein, β-amyloid peptide, and DNA. Most of these ligands bind RAGE to its V domain. S100A12, Aβ, and S100A6 have been reported to bind RAGE at their C1 / C2 domains (Lee and Park. Genomics Inform. 2013, Ostendorp et al. EMBO J. 2007, Leclerc E et al., J Biol Chem. 2007).

[0036] Ligand binding to RAGE initiates a multicellular cascade of responses leading to inflammation. Activation of transcription factors NF-κB, MAP kinase, ERK1 and ERK2, or p21ras, triggers transcription of several target genes, including cytokines (IL-1β, IL-6, TNF-α), adhesion molecules, and RAGE itself (Bongarzone S et al., J Med Chem. 2017). This positive feedback loop between RAGE and NF-κB maintains and amplifies the RAGE signal, which contributes to chronic pathological inflammation in many diseases (Bierhaus A et al. Diabetes. 2001, Sparvero J et al. J Trans Med. 2009, Dong H et al. Front Immunol. 2022). The diversity of the signaling cascade suggests that different RAGE ligands can activate different signaling pathways in different cell types.

[0037] The inventors hereby advantageously demonstrate that conjugating a drug or diagnostic agent to a RAGE-targeting VHH is advantageous for delivering such molecules of interest, such as diagnostic agents or drugs, to tissues that preferentially express RAGE, particularly the lungs, for the diagnosis or treatment of disease.

[0038] Using membrane samples purified from cells expressing high levels of hRAGE and mRAGE, the inventors generated and selected VHH molecules that bind to both human and non-human RAGE. They showed that these VHH molecules retain their RAGE-binding ability when fused to human IgG1 Fc regions or siRNA. They also showed that the VHH molecules represent efficient lung delivery. They ultimately demonstrated that the RAGE-targeted VHH molecules of the present invention can also be used for vectorization of lipid nanoparticles (LNPs), and that such conjugates can be used to effectively transport and deliver various diagnostic or therapeutically interested molecules, such as mRNA / LNPs, to the lungs.

[0039] Therefore, the present invention provides a novel RAGE-binding molecule that can serve as a drug useful for targeting the lungs.

[0040] Therefore, an object of the present invention relates to a VHH molecule, the VHH molecule which binds to both human RAGE and non-human (e.g., rodent such as rat or mouse) RAGE. Preferably, the VHH binds to RAGE-expressing lung tissue. The present invention also relates to a chimeric agent comprising such a VHH, the manufacture of the same, a composition comprising the same, and the use of the same.

[0041] VHH molecule VHH molecules correspond to the variable region of camelid antibodies that naturally lack a light chain and consist only of heavy chains. VHHs are very small in size, approximately 15 kDa. They contain single-chain molecules that can bind to their congener antigens using a single domain. The antigen-binding surface of VHHs is usually convex (or protruding) compared to the surface of conventional antibodies, which are typically flat or concave. More specifically, VHHs consist of four framework regions (i.e., FRs) whose sequence and structure are defined as conserved, and three complementarity-determining regions (i.e., CDRs) that exhibit high variability in both sequence content and structural conformation and participate in antigen binding to provide antigen specificity. Compared to conventional human antibody VHs, several amino acids are substituted in the FR2 region and complementarity-determining regions (CDRs) of VHHs. For example, highly conserved hydrophobic amino acids in the FR2 region (such as Val42, Gly49, Leu50, and / or Trp52) are often substituted with hydrophilic amino acids (Phe42, Glu49, Arg50, Gly52), which makes the overall structure more hydrophilic and contributes to high stability, solubility, and resistance to aggregation.

[0042] The VHH molecule according to the present invention is a polypeptide comprising (or consisting of, or essentially consisting of) an antigen-binding domain of a heavy-chain-only antibody (HcAb).

[0043] To generate VHH molecules with suitable properties, the inventors tested over 700 RAGE-bound VHHs from a library of VHHs produced by immunization of llamas with RAGE immunogen. After analyzing the binding and specificity of the clones, the inventors further selected approximately 70 clones with the required specificity and interspecies binding. All of the clones were sequenced, and their structures were analyzed and compared. The relevant domains and sequences of preferred VHHs are provided in the experimental section and sequence list. The properties of the VHHs and their conjugates are also described in the experimental section.

[0044] The VHH molecule of the present invention is typically, formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 It comprises or consists of, where FRn represents the framework region and CDRn represents the complementarity determination region (where n is, for example, 1, 2, 3, or 4).

[0045] In certain embodiments, the VHH molecule of the present invention comprises an amino acid sequence selected from SEQ ID NOs: 1, 5, 9, 13, or 17, or a variant thereof having at least 60%, particularly at least 65%, 70%, or 75%, for example at least 80% or 85%, preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity with any one of the sequences over its entire length (preferably the preferred identity percentage for a particular sequence is the percentage corresponding to an integer number of amino acids), or comprises a CDR1 domain consisting of such variants, the variants retaining RAGE binding ability. Preferred VHH molecules of the present invention include a CDR1 domain having an amino acid sequence selected from SEQ ID NOs: 1, 5, 9, 13, or 17, or a variant thereof having several amino acid modifications, for example, at least three amino acid modifications, preferably up to three or two amino acid modifications, and in certain embodiments up to one amino acid modification.

[0046] The "% identity" between amino acid (or nucleic acid) sequences can be determined by techniques known in the art. Typically, the % identity between two nucleic acids or amino acid sequences is determined by computer programmatic means such as GAP (Program Manual for the Wisconsin Package, Version 8, August 1996, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) provided in the GCG program package (Needleman, S.B. and Wunsch, CD, (1970), Journal of Molecular Biology, 48, 443-453). The percentage of identity between two sequences represents the identity over the entire length of the sequence. As described above, the preferred identity percentage for a particular sequence is preferably the percentage corresponding to an integer of amino acids in both the reference sequence (e.g., SEQ ID NOs: 1, 5, 9, 13, or 17, or any other reference sequence identified herein, such as SEQ ID NOs: 2, 3, 6, 7, 10, 11, 14, 15, 18, or 19) and its variants.

[0047] A specific example of the VHH molecule of the present invention is a CDR1 sequence comprising, or essentially consisting of, SEQ ID NOs: 1, 5, 9, 13, or 17.

[0048] In more specific embodiments, the VHH molecule of the present invention comprises an amino acid sequence selected from SEQ ID NOs: 2, 6, 10, 14, or 18, or a variant thereof having at least 60%, particularly at least 65%, 70%, or 75%, for example at least 80% or 85%, preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length, or comprises a CDR2 domain comprising such variants, the variants retaining RAGE binding ability. A preferred VHH molecule of the present invention comprises a CDR2 domain having an amino acid sequence selected from SEQ ID NOs: 2, 6, 10, 14, or 18, or a variant thereof having several amino acid modifications, for example at least three amino acid modifications, preferably up to three or two amino acid modifications, and in specific embodiments up to one amino acid modification.

[0049] A specific example of the VHH molecule of the present invention is a CDR2 sequence comprising, or essentially consisting of, SEQ ID NOs: 2, 6, 10, 14, or 18.

[0050] In further specific embodiments, the VHH molecule of the present invention comprises an amino acid sequence selected from SEQ ID NOs: 3, 7, 11, 15, or 19, or a variant thereof having at least 60%, particularly at least 65%, 70%, or 75%, for example at least 80% or 85%, preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length, or comprises a CDR3 domain consisting of such variants, the variants retaining RAGE binding ability. A preferred VHH molecule of the present invention comprises a CDR3 domain having an amino acid sequence selected from SEQ ID NOs: 3, 7, 11, 15, or 19, or a variant thereof having several amino acid modifications, for example at least three amino acid modifications, preferably up to three or two amino acid modifications, and in a particular embodiment up to one amino acid modification.

[0051] A specific example of the VHH molecule of the present invention is a CDR3 sequence comprising, or essentially consisting of, SEQ ID NOs: 3, 7, 11, 15, or 19.

[0052] Furthermore, in certain embodiments, the VHH molecule of the present invention is: - A CDR1 domain comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 1, 5, 9, 13, or 17, or a variant thereof having at least 60%, particularly at least 65%, 70%, or 75%, for example at least 80% or 85%, preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, more preferably at least 95% amino acid identity with any one of the sequences over its entire length; and - A CDR2 domain comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 2, 6, 10, 14, or 18, or a variant thereof having at least 60%, particularly at least 65%, 70%, or 75%, for example at least 80% or 85%, preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, more preferably at least 95% amino acid identity with any one of the sequences over its entire length; and - CDR3 domains comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 3, 7, 11, 15, or 19, or a variant thereof having at least 60%, particularly at least 65%, 70%, or 75%, for example at least 80% or 85%, preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, more preferably at least 95% amino acid identity with any one of the sequences over its entire length. Equipped with, The aforementioned VHH has RAGE binding ability.

[0053] In further specific embodiments, the VHH molecule of the present invention is: - A CDR1 sequence selected from SEQ ID NOs: 1, 5, 9, 13, or 17, or a variant thereof having at least 60% amino acid identity with any one of the aforementioned sequences over its entire length. - A CDR2 sequence selected from SEQ ID NOs: 2, 6, 10, 14, or 18, or a variant thereof having at least 60% amino acid identity with any one of the aforementioned sequences over its entire length, and / or - A CDR3 sequence selected from SEQ ID NOs: 3, 7, 11, 15, or 19, or a variant thereof having at least 60% amino acid identity with any one of the aforementioned sequences over its entire length. Equipped with, The aforementioned VHH has RAGE binding ability on the surface of lung cells.

[0054] In further specific embodiments, the VHH molecule of the present invention is: - A CDR1 sequence selected from SEQ ID NOs: 1, 5, 9, 13, or 17, or a variant thereof having at least 60% amino acid identity with any one of the aforementioned sequences over its entire length. - A CDR2 sequence selected from SEQ ID NOs: 2, 6, 10, 14, or 18, or a variant thereof having at least 60% amino acid identity with any one of the aforementioned sequences over its entire length, and / or - A CDR3 sequence selected from SEQ ID NOs: 3, 7, 11, 15, or 19, or a variant thereof having at least 60% amino acid identity with any one of the aforementioned sequences over its entire length. Equipped with, The aforementioned VHH binds to RAGE on the surface of lung cells with an affinity (Kd) of 0.1 nM to 10 μM, preferably 1 nM to 10 μM.

[0055] In a preferred embodiment, the VHH molecule of the present invention is: -CDR1 domains having amino acid sequences selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, and 17, and variants thereof having up to 3, 2, or 1 amino acid modifications; and -CDR2 domains having amino acid sequences selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, and 18, and variants thereof having up to 3, 2, or 1 amino acid modifications; and - CDR3 domains having amino acid sequences selected from the group consisting of SEQ ID NOs: 3, 7, 11, 15, and 19, and variants thereof having up to 3, 2, or 1 amino acid modifications. It is equipped with.

[0056] In a more preferred embodiment, the VHH molecule of the present invention comprises CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 domains are: - Sequence IDs 1, 2, and 3; or - Sequence IDs 5, 6, or 7; or - Sequence IDs 9, 10, and 11; or - Sequence IDs 13, 14, and 15; or - Sequence IDs 17, 18 or 19; or The variants defined above, preferably those having a maximum of 3, 2, or 1 amino acid modifications, are described above. It comprises or consists of these.

[0057] A preferred VHH molecule of the present invention comprises an FR domain as defined below.

[0058] In certain embodiments, the FR1 domain comprises or consists of the sequence number 75 shown below, or variants thereof having at least 85% amino acid identity across the entire length of this sequence, for example, at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably at least 90%, more preferably at least 95% amino acid identity: [ka]

[0059] More preferably, the bolded amino acid residues are present, and variations occur only at other positions.

[0060] In a specific embodiment, E at position 1 may be replaced with Q.

[0061] In a specific embodiment, V at position 5 may be replaced with Q.

[0062] In a specific embodiment, E at position 6 may be replaced with Q.

[0063] In a specific embodiment, G at position 10 may be replaced with K or A.

[0064] In a specific embodiment, L at position 11 can be replaced with V or E.

[0065] In a specific embodiment, P at position 14 can be replaced with A.

[0066] In a specific embodiment, A at position 23 may be replaced with V or T.

[0067] In a specific embodiment, A at position 24 can be replaced with V.

[0068] More preferably, FR1 contains up to four amino acid modifications, even more preferably up to three, and even more preferably up to two amino acid modifications, with reference to this sequence, using non-bold amino acid residues.

[0069] In a further specific embodiment, FR1 has an amino acid sequence selected from one of the following amino acid sequences: -QVQLVQSGGGLVQPGGSLRLSCAVS(Sequence ID 76); -QVQLVQSGGGLVQAGGSLRLSCAAS(Sequence ID 77); -QVQLVQSGGGLVQAGGSLRLSCVAS(Sequence ID 78); -EVQLVESGGGLVQAGGSLRLSCVAS(Sequence ID 79); -EVQLVESGGGLVQPGGSLRLSCAAS (Sequence ID 80), Or their variants as defined above.

[0070] Examples of such variants are given below for illustrative purposes only: [ka]

[0071] Any combination of these mutations may exist in other mutants of interest.

[0072] In certain embodiments, the VHH molecule of the present invention comprises SEQ ID NO: 87 shown below, or a variant thereof having at least 85%, for example, at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity with this sequence over its entire length, preferably at least 90%, or at least 95%, amino acid identity, or comprising an FR2 domain consisting of such variants: [ka]

[0073] More preferably, the bolded amino acid residues are present, and variations occur only at other positions.

[0074] In a specific embodiment, M at position 1 may be replaced with I or V.

[0075] In a specific embodiment, G at position 2 can be replaced with A.

[0076] In a specific embodiment, Y at position 4 can be replaced with F.

[0077] In a specific embodiment, Q at position 6 may be replaced with R.

[0078] In a specific embodiment, A at position 7 may be replaced with R.

[0079] In a specific embodiment, K at position 10 can be replaced with E.

[0080] In a specific embodiment, Q at position 11 may be replaced with E.

[0081] In a specific embodiment, R at position 12 may be replaced with L.

[0082] In a specific embodiment, L at position 14 may be replaced with F or W.

[0083] In a specific embodiment, V at position 15 can be replaced with A.

[0084] In a specific embodiment, A at position 16 may be replaced with T.

[0085] In a specific embodiment, R at position 17 may be replaced with T or L.

[0086] More preferably, FR2 contains up to six amino acid modifications, even more preferably up to five, up to three, and even more preferably up to two amino acid modifications, with reference to this sequence, using non-bold amino acid residues.

[0087] The VHH molecules described herein typically have at least one of the following amino acids in the FR2 domain: Phe42, Glu49, or Arg50 (in order of IMGT number).

[0088] In a further specific embodiment, FR2 has an amino acid sequence selected from one of the following amino acid sequences: -MGWYRQAPGKQRELAAR(sequence number 88); -MGWYRQAPGKQREWVTT(Sequence ID 89); -MAWFRQAPGEEREFVAR(array_90); -MGWYRQAPGKQLELVAL(Sequence ID 91) Or their variants as defined above.

[0089] Examples of such variants are given below for illustrative purposes only: [ka]

[0090] Any combination of these mutations may exist in other mutants of interest.

[0091] In certain embodiments, the VHH molecule of the present invention comprises an FR3 domain having or consisting of SEQ ID NO: 102 shown below, or a variant thereof having at least 85%, preferably at least 90%, more preferably at least 95% amino acid identity with this sequence over its entire length: [ka]

[0092] More preferably, the bolded amino acid residues are present, and variations occur only at other positions.

[0093] In specific embodiments, N at position 1 may be replaced with S or D.

[0094] In a specific embodiment, Y at position 2 can be replaced with A.

[0095] In a specific embodiment, A at position 3 may be replaced with L.

[0096] In a specific embodiment, D at position 4 can be replaced with A.

[0097] In a specific embodiment, S at position 5 can be replaced with F.

[0098] In a specific embodiment, K at position 7 may be replaced with R.

[0099] In a specific embodiment, N at position 16 may be replaced with T.

[0100] In a specific embodiment, A at position 17 may be replaced with T.

[0101] In a specific embodiment, N at position 19 can be replaced with K.

[0102] In a specific embodiment, T at position 20 can be replaced with A.

[0103] In a specific embodiment, V at position 21 may be replaced with L.

[0104] In a specific embodiment, N at position 26 can be replaced with I.

[0105] In a specific embodiment, S at position 27 can be replaced with N.

[0106] In a specific embodiment, K at position 29 may be replaced with E.

[0107] In a specific embodiment, P at position 30 may be replaced with L.

[0108] In a specific embodiment, V at position 35 may be replaced with R.

[0109] More preferably, FR3 contains up to seven amino acid modifications, even more preferably up to six, up to three, and even more preferably up to two amino acid modifications, with reference to this sequence, in the non-bold amino acid residues.

[0110] In a further specific embodiment, FR3 has an amino acid sequence selected from one of the following amino acid sequences: -NYLDSVKGRFTISRDNAKNTVYLQMNSLKLEDTAVYYC(Sequence ID 103); -DYAASVKGRFTISRDTAKNAVYLQMNNLKPEDTARYYC(Sequence ID 104); -SYADSVKGRFTISRDNAKNTVYLQMISLKPEDTAVYYC(Sequence ID 105); -NYADFVRGRFTISRDTTKKTLYLQMNSLEPEDTAVYYC(Sequence ID 106), Or their variants as defined above.

[0111] Examples of such mutants are shown below, but these are for illustrative purposes only: [ka]

[0112] Any combination of these mutations may exist in other mutants of interest.

[0113] In certain embodiments, the VHH molecule of the present invention comprises the following sequence number 123, or an FR4 domain comprising it, or a variant thereof having at least 85%, for example, at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity with this sequence over its entire length, preferably at least 90%, more preferably at least 95%: [ka]

[0114] More preferably, the bolded amino acid residues are present, and variations occur only at other positions.

[0115] More preferably, FR4 contains up to four amino acid modifications, even more preferably up to three, and even more preferably up to two amino acid modifications, with reference to this sequence, using non-bold amino acid residues.

[0116] Specific examples of FR4 sequences include WGKGTQVTVSS (sequence number 124) or WGQGTQVTVSS (sequence number 125).

[0117] Specific examples of the RAGE-bound VHH molecules of the present invention include, for example, molecules comprising or consisting of amino acid sequences selected from any one of the following: SEQ ID NOs: 4 (VHH1, x=0), 8 (VHH2, x=0), 12 (VHH3, x=0), 16 (VHH4, x=0), 20 (VHH5, x=0), 31 (VHH1, x=1), 32 (VHH2, x=1), 33 (VHH3, x=1), 34 (VHH4, x=1), and 35 (VHH5, x=1), each of which may or may not include a tag sequence (when x is 1).

[0118] In the examples corresponding to Sequence IDs 31, 32, 33, 34, and 35 shown above (where x is equal to 1), each VHH molecule has the following specific tag sequence of Sequence ID 36: AAAEQKLISEEDLNGAAHHHHHHGS. When x is equal to 0, as in the examples corresponding to Sequence IDs 4, 8, 12, 16, and 20, the VHH molecule does not have a tag.

[0119] In certain embodiments, the VHH molecule of the present invention is humanized.

[0120] For humanization, one or more FR domains may be further modified by one or more amino acid substitutions.

[0121] In this regard, in certain embodiments, VHH is humanized by selected modifications (e.g., amino acid substitutions) of the FR1 domain. The FR1 domain typically consists of a sequence of 25 amino acid residues. Typical humanization sites of FR1 are selected from 14P and 23A (by referring, for example, any one of SEQ ID NOs. 75, 76, 77, or 80, or any of its variants as defined herein, particularly any one of SEQ ID NOs. 81-86, as illustrated herein). A particular example of humanized FR1 is provided with SEQ ID NO: 83. A14 and / or V23 present in SEQ ID NO: 78 are specifically modified to 14P or 23A, respectively, in the humanized sequence of SEQ ID NO: 83.

[0122] In another specific embodiment, VHH is humanized by selected modifications of the FR2 domain. Typical humanization sites in FR2 are typically selected from 11G and 12L, referring to an FR2 domain consisting of a sequence of 17 amino acid residues, such as MGWYRQAPGKGLELVAR (SEQ ID NO: 126).

[0123] In another specific embodiment, VHH is humanized by selected modifications of the FR3 domain. Typical humanization sites in FR3 can be selected from 17S, 21L, 27S, 29R, 30A, 35V, and any combination thereof, with reference to the FR3 domain, which typically consists of a sequence of 38, up to 39, amino acid residues.

[0124] The humanized FR3 domain therefore comprises an FR3 sequence of 38, up to 39, amino acid residues, one or more, or all of A17, V21, N27, K29, P30, and R35 (e.g., appearing in SEQ ID NO: 104), modified to 17S, 21L, 27S, 29R, 30A, and 35V, respectively.

[0125] In further specific embodiments, the FR1 and / or FR2 and / or FR3 domains are humanized.

[0126] In further specific embodiments, the VHH molecule may further comprise one or more tags suitable for, for example, purification, coupling, detection, etc.

[0127] In the context of this invention, the term “tag” includes any peptide sequence attached to the polypeptide VHH molecule of the present invention for the purpose of facilitating the easy detection or purification of expressed proteins, identifying their binding to RAGE, or for site-specific enzymatic / enzymatic conjugate purposes. The tag may be an affinity tag, an epitope tag, a site-specific conjugate tag, or a fluorescent tag.

[0128] Examples of such tags include tags comprising a glutamine residue inserted into the tag sequence, which are specifically recognized by TGase and preferably comprise the sequence LQR, myc tag (EQKLISEEDL, SEQ ID NO: 38), polyHis tag (comprising 2 to 8 histidine residues, preferably 6 to 8 His residues, e.g., His6 (SEQ ID NO: 37)), polyArg tag (comprising 2 to 8 arginine residues), polyLys tag (comprising 2 to 8 lysine residues), HA tag, FLAG tag or GFP tag, CBP tag, Strep II tag, saltase tag, SNAP tag, or a combination thereof, or a Q tag consisting of these.

[0129] Typically, one or more tags are located at the C-terminus of the VHH.

[0130] In further specific embodiments, the VHH molecule may further comprise one or more linkers.

[0131] In the context of this invention, the terms “linker” or “spacer” include one or more amino acid residues (typically 1 to 10 amino acid residues) used to link between the VHH molecule of the present invention and a tag, or between the various tags described herein, provided that the linker does not specifically bind to the target protein which is RAGE. The linker may be any amino acid residue, such as glycine (Gly), alanine (Ala), serine (Ser), cysteine ​​(Cys), leucine (Leu), asparagine (Asn), lysine (Lys), or a combination thereof. Such peptide linkers differ from conjugate linkers that can be introduced between the VHH and the compound of interest, where the compound of interest is a difunctional or polyfunctional agent containing alkyl, aryl, or peptide groups, such as esters, aldehydes, alkyl or aryl acids, anhydrides, sulfidyl or carboxyl groups, cyanogen bromide or cyanogen chloride, carbonyl diimidazole, succinimide esters, or groups derived from sulfonic acid halides.

[0132] As a further example, the VHH of the present invention may preferably comprise a Gly linker located at the C-terminus of the VHH. The Gly linker may comprise, for example, 2 to 7 Gly residues, such as 3, 4 (SEQ ID NO: 39), 5 (SEQ ID NO: 40), or 6 (SEQ ID NO: 41) Gly repeats. Specific examples of Gly linkers include Gly3, Gly4 (SEQ ID NO: 39), Gly5 (SEQ ID NO: 40), or SerGlySerGly5 (SEQ ID NO: 41).

[0133] In certain embodiments, the VHH of the present invention may comprise a Gly linker and a Q tag, preferably located at the C-terminus. A more specific example of such a VHH is the following structure: VHH-Gly linker-Q tag, where the Gly linker comprises or consists of 2 to 6 Gly residues, and the Q tag preferably comprises or consists of an LQR.

[0134] For example, VHH has the following additional sequence at its C-terminus. [ka] It may have the following characteristics, with underlined being the myc tag and double underlined being the His6 tag (the remaining residues are from linkers such as the Ala linker AAA, or are produced by cloning).

[0135] As another example, VHH has the following additional sequence at its C-terminus. [ka] It may be equipped with a spacer, a bold C is a free cysteine ​​available for site-specific chemical conjugates, and a double underline is a His tag (the remaining residues are either linkers or generated by cloning).

[0136] As already taught above in this specification, specific examples of such tagged RAGE-bound VHH molecules of the present invention are molecules comprising or consisting of amino acid sequences selected from any one of SEQ ID NOs: 31 (VHH1), 32 (VHH2), 33 (VHH3), 34 (VHH4), and 35 (VHH5), where x is 1.

[0137] As another example, the VHH of the present invention may comprise an LQR sequence located at the C-terminus of the VHH, or a Q tag consisting of the LQR sequence.

[0138] As a further example, VHH has the following additional sequence at its C-terminus ("C-ter") [ka] It may have the following characteristics: underlined is a Q tag, and bold is a Gly linker. Other examples include: [ka] There is.

[0139] In a preferred embodiment of the present invention, VHH comprises the additional sequence of sequence number 42.

[0140] In further specific embodiments, the VHH of the present invention may comprise an Ala linker, a His tag, a Gly linker, and a Q tag. Preferably, the linkers and tags are located at the C-terminus of the VHH. In other embodiments, the Q tag may be located at least at the N-terminus ("N-ter") of the VHH. A more specific example of such a VHH is the following structure: VHH-Ala linker-His tag-Gly linker-Q tag, where the Ala linker preferably comprises 3 residues, the His tag comprises 2 to 7 His residues, preferably 6 His residues, the Gly linker comprises 2 to 6 Gly residues, preferably 3 residues, and the Q tag preferably comprises or consists of an LQR.

[0141] As another example, VHH has the following additional sequence at its C-terminus. [ka] The tags may include: underlined are Q tags, bold are Ala and Gly linkers, and double underlined are His tags. Other examples are: [ka] That is the case.

[0142] In a preferred embodiment of the present invention, VHH is [ka] It includes an additional array.

[0143] Further specific examples of the RAGE-binding VHH molecule of the present invention include VHH molecules that competitively inhibit the binding of the VHH defined above to human RAGE and non-human RAGE. The term "competitively inhibit" means that the VHH can reduce, inhibit, or substitute the binding of the reference VHH to RAGE in vitro or in vivo. Competitive assays can be performed using standard methods such as competitive ELISA or other binding assays. Typically, a competitive binding assay includes recombinant lung cells or membrane specimens expressing RAGE, optionally bound to a solid substrate, unlabeled test VHH (or a phage expressing it), and labeled reference VHH (or a phage expressing it). Competitive inhibition is measured by determining the amount of labeled VHH bound in the presence of test VHH. Typically, test VHH is present in excess, about 5 to 500 times the amount of reference VHH. Typically, in the case of ELISA, test VHH is 100 times in excess. If an excess test VHH inhibits or substitutes at least 70% of the binding of a reference VHH to RAGE, it is considered to competitively inhibit the reference VHH. A preferred competitive VHH binds to an epitope that shares common amino acid residues.

[0144] As shown in the experimental section, VHH molecules can bind to RAGE in vitro and in vivo. They exhibit sufficient affinity, with apparent Kd values ​​ranging from 0.1 nM to 10 μM, particularly in the range of 1 nM to 1 μM. Furthermore, preferred molecules (such as those containing VHH1, VHH2, and VHH5) bind to both human and mouse RAGE. Moreover, the binding of the VHHs of the present invention to the human RAGE receptor does not compete with the binding of endogenous RAGE (native) ligands(s), and therefore does not affect the normal function of the ligands. Conjugates generated with such VHH molecules have further been shown to bind to RAGE in vitro and accumulate in the lungs and / or lung cells. Thus, such VHHs would be potent agents for lung targeting and drug delivery.

[0145] The VHHs of the present invention can be synthesized by any technique known to those skilled in the art (e.g., chemical synthesis, biosynthesis, gene synthesis). They can be stored as is or formulated in the presence of the substance of interest or acceptable excipients.

[0146] For chemical synthesis, commercially available equipment is used that can incorporate VHH sequences containing one or more peptide-mimicking bonds, which may particularly include the insertion of a methylene (-CH2-) or phosphate (-PO2-) group, a secondary amine (-NH-), or an oxygen (-O-) or N-alkyl peptide, as well as natural and unnatural amino acids such as D enantiomas and residues with hydrophobic and sterically hindered side chains different from those of their natural homologs (so-called exotic, i.e., non-coding amino acids).

[0147] During synthesis, various chemical modifications can be introduced, for example, by positioning lipid (or phospholipid) derivatives or components of liposomes or nanoparticles at the N-terminus or C-terminus, or in the side chains, to incorporate the VHH of the present invention into a lipid membrane such as a liposome consisting of one or more lipid layers or bilayers, or a nanoparticle. Liposomes and nanoparticles are examples of “vehicles” that can be conjugated with one or more VHH molecules of the present invention.

[0148] The VHH of the present invention can also be obtained from the nucleic acid sequence encoding it, as further described in the following sequence numbers 21-30 (see Table 3).

[0149] Conjugate A further object of the present invention relates to at least one additional compound, in particular to a conjugate comprising one or more VHH molecules as defined above, conjugated to at least one additional molecule, drug, or compound of interest, e.g., a scaffold of interest (also interchangeably referred to herein as a “chimeric agent”).

[0150] This additional compound may be a separate VHH or a molecule that is not a VHH.

[0151] The at least one additional molecule, drug, or compound of interest may be any molecule, drug, or compound, such as a stabilizing group (also interchangeably referred herein as a “half-life extension portion” or “scaffold”), a therapeutic (i.e., active) compound, a drug, or a medical agent, diagnostic agent, imaging compound, tracer, or a vehicle comprising such a therapeutic, diagnostic, or imaging compound.

[0152] In certain embodiments, the chimeric agent / conjugate may comprise both types of additional compounds, namely i) a stabilizing group, a half-life extension moiety or scaffold, and ii) a therapeutic, diagnostic or imaging compound, or a vehicle comprising them.

[0153] The stabilizing group or half-life extending portion extends the plasma half-life of VHH or the conjugate.

[0154] Therapeutic compounds are selected from, for example, peptides, polypeptides, proteins, antibodies, nucleic acids, and any fragments thereof.

[0155] Examples of conjugate molecules, drugs, or compounds of interest include, but are not limited to, small chemical molecules (e.g., chelating agents, antibiotics, antivirals, immunomodulators, antitumor agents, anti-inflammatory agents, or adjuvants); peptides, polypeptides, or proteins (e.g., enzymes, hormones, cytokines, apolipoproteins, growth factors, antigens, antibodies or parts of antibodies, adjuvants, etc.); nucleic acids (e.g., RNA or DNA of human, viral, animal, eukaryotic, prokaryotic, plant, or synthetic origin, including, for example, coding genes, inhibitory nucleic acids such as ribozymes, antisense oligonucleotides (ASOs), interfering nucleic acids (siRNA), small activated RNA (saRNA), mRNA, whole genomes or parts thereof, plasmids, etc.); and any chemical substances such as lipid (nano) particles, exosomes, viruses, cell-derived vesicles (CDVs) such as markers or tracers. In general, “molecule, drug, or compound of interest” can be any drug (active) component, whether chemical, biochemical, natural, or synthetic. Generally, the expression "small chemical molecules, drugs, or compounds" refers to pharmaceutically relevant molecules with a maximum molecular weight of 1,000 daltons, typically between 300 and 700 daltons.

[0156] The vehicle can be selected from, for example, viruses, virus-like particles (VLPs), cell-derived vesicles (CDVs), exosomes, lipid vehicles, and polymer vehicles, and is preferably a lipid nanoparticle (LNP), micelle, or liposome.

[0157] Conjugate compounds are typically drugs (e.g., small drugs, nucleic acids or polypeptides, e.g., antibodies or fragments thereof) or imaging agents suitable for treating or detecting lung diseases, such as infectious, immune, or cancerous lung lesions.

[0158] The chimeric agent may, in addition to or instead of the compound of interest, include a stabilizing group for extending the plasma half-life of the VHH or conjugate. Thus, a particular chimeric agent of the present invention comprises i) at least one VHH, e.g., multiple VHH molecules, ii) a stabilizing group, iii) a compound of interest, typically a compound for therapeutic, diagnostic, or imaging purposes, and optionally iv) a vehicle, in any order.

[0159] In certain embodiments described herein, the compound of interest is also a group that enables the stabilization of the VHH molecule(s) of the present invention.

[0160] The stabilizing group can be any group known to have a substantial plasma half-life (e.g., at least 1 hour, at least 1 day, or at least 1 week) and to have essentially no harmful biological activity. Examples of such stabilizing groups include, for example, Fc fragments of immunoglobulins, VHH molecules or their variants, albumin, particularly large human serum proteins such as human serum albumin (HSA), or serum albumin-binding molecules, or antibodies or fragments thereof such as IgG or PEG molecules.

[0161] In certain embodiments, the stabilizing group is an Fc fragment. More preferably, the stabilizing group is an Fc fragment of IgG1, for example, an Fc fragment of human IgG1.

[0162] In another specific embodiment, the conjugate according to the present invention comprises a stabilizing group which is an albumin-binding moiety that binds to albumin with affinity preferably about 1 nM to about 10 μM, thereby improving the pharmacokinetic profile of the compound of interest by the gradual release of the conjugate from albumin. Such albumin-binding moieties include, for example, fragments of Evans blue (EB) dye, fatty acids and their derivatives such as the C16 group and the 4-(p-iodophenyl)butytril (PIB) group, the 89D03 peptide, and the ABD035 protein (a 46-residue 3-helix bundle albumin-binding domain).

[0163] VHH can be conjugated to the N-terminus, C-terminus, or both of the stabilizing group. When the stabilizing group is an Fc fragment, conjugation typically occurs via gene fusion. The resulting protein can remain as a monomer or a polymer, depending on the nature of the stabilizing group. In the case of an Fc fragment, the fusion protein Fc-VHH or VHH-Fc usually forms a homodimer.

[0164] In the conjugate compounds of the present invention, coupling can be performed by any acceptable coupling means, taking into account the chemical properties, interference, and number of conjugates. Therefore, coupling can be performed by one or more covalent, ionic, hydrogen, hydrophobic, or van der Waals bonds that are cleavable or non-cleavable in a physiological medium or intracellularly, preferably cleavable when the present invention is used in the context of delivering at least one active agent to a lung site. Furthermore, coupling can be performed at various reactive groups, particularly one or more terminal and / or one or more internal or lateral reactive groups. Coupling can also be performed using genetic engineering.

[0165] The interaction is desirable to be strong enough to prevent the VHH from dissociating from the active substance before it reaches its site of action (i.e., the lung site). For this reason, the preferred coupling in the present invention is covalent, but non-covalent coupling may also be employed. The compound of interest can be coupled to the VHH either at its terminal (N-terminus or C-terminus) or at one of the side chains of the constituent amino acids of the sequence (Majumdar S. and Siahaan TJ., “Peptide-mediated targeted drug delivery”, Med Res Rev., May 2012;32(3):637-58). The compound of interest can be coupled directly to the VHH or indirectly via a conjugate linker or spacer. Regardless of whether a spacer is required, means of covalent chemical coupling include, for example, difunctional or polyfunctional agents selected from esters, aldehydes or alkyl or aryl acids, alkyl, aryl, thiol or peptide groups, anhydrides, sulfidyl or carboxyl groups, groups derived from cyanogen bromide or cyanogen chloride, carbonyl diimidazole, succinimide esters or sulfonic acid halides.

[0166] Exemplary strategies for conjugating the VHH of the present invention into molecules or scaffolds are disclosed in Figure 11.

[0167] In certain embodiments, coupling (or conjugate) is performed by gene fusion. Such a strategy can be used when the coupled molecule is a peptide or polypeptide. In such cases, a nucleic acid molecule encoding VHH fused to the molecule is prepared and expressed in any suitable expression system to generate the conjugate.

[0168] In another specific embodiment, coupling (or conjugate) is performed using thiol / maleimide chemistry techniques. To carry out this reaction, an additional peptide tag is typically added to its C-terminus. [ka] A fused VHH is used.

[0169] Since VHH contains only the cysteine ​​involved in disulfide crosslinking, only the additional cysteine ​​introduced into the tag chemically reacts with maleimide. This allows for specific conjugation between VHH and the maleimide derivative molecule of interest. The reaction proceeds in two steps. First, a mild reducing agent such as 2-MEA (2-mercaptoethanol), TCEP (tris(2-carboxyethyl)phosphine), or DTT (dl-1,4-dithiothreitol) is used. [ka] Reduce it. [ka] During the generation process, additional cysteine ​​present in the tag is added to another [ka] It can react with the cysteine ​​supported on it, and free [ka] and [ka] The structure becomes a mixture of the dimer and the other. In the second stage, [ka] By reacting this with a maleimide-functionalized molecule of interest in the pH range of 6.5 to 7.5, a stable VHH molecular conjugate can be formed via covalent bonding.

[0170] In another specific embodiment, coupling (or conjugate) is carried out by an enzymatic reaction. In particular, site-specific conjugate to VHH can be performed using the transglutaminase enzyme (TGase). TGase catalyzes the formation of a stable isopeptide bond between (i) the side chain of a glutamine residue inserted into a tag sequence (i.e., Q tag) specifically recognized by TGase and (ii) an amino-functionalized donor substrate. In this regard, we have developed a specific tag sequence (named "Q tag") that is recognized by TGase and can be used to couple the VHH of the present invention to any molecule of interest, in particular a chemical or drug. For this purpose, VHH is prepared by gene fusion and the following tags are added in tandem (typically at its C-terminus): first an optional trialanine linker, then an optional His tag, then an optional small triglycine linker, and finally a Q tag. The triglycerin linker widens the spacing between Q tags, making it easier for TGase to access glutamine, while the His tag is intended to facilitate the purification of VHH and its more functionalized versions.

[0171] The common conjugate strategy developed is a convergent synthesis based on a process that includes the following: 1) To introduce a reactive moiety to the Q-tagged glutamine of VHH for further conjugation to the molecule of interest. For this purpose, a heterobifunctional conjugate linker having two different reactive ends can be treated with TGase, one being a suitable primary amine group for TGase and the other being an orthogonal reactive moiety. Typical examples of such orthogonal reactive groups include azides, restricted alkynes such as DBCO (dibenzocyclooctin) or BCN (bicyclo[6.1.0]nonine), tetrazines, TCO (transcyclooctene), and free or protected thiols. 2) Introducing a reactive group complementary to the one incorporated into the VHH Q tag into the molecule of interest. Typical examples of such orthogonal reactive groups include azides, restricted alkynes such as DBCO or BCN, tetrazines, TCOs, and free or protected thiols. 3) Conjugate both the functionalized VHH and the molecule through its complementary reactive group.

[0172] This specification also describes a method for coupling two molecules by a TGase coupling reaction using the Q tag defined above. A further object of the present invention is the VHH of the present invention comprising the Q tag. A further object of the present invention is the VHH molecule of the present invention comprising a linker such as a Gly linker and the Q tag.

[0173] The preferred VHH of the present invention has the following structure: VHH-Linka-Myc-Linka-His m It has, Here, VHH is any VHH molecule, The linker is any molecular linker such as Ala or Gly linker (preferably the two linkers are different), m is an integer from 0 to 8, preferably 6 or 8.

[0174] In certain embodiments, the present invention relates to a conjugate comprising a VHH covalently bonded to a chemical substance. Preferred variants of such a conjugate include one VHH and one chemical substance.

[0175] In another specific embodiment, the present invention relates to a conjugate comprising a VHH covalently bound to a nucleic acid. The nucleic acid may be an antisense oligonucleotide ("ASO"), a ribozyme, an aptama, mRNA, or siRNA. Preferred variants of such a conjugate include one VHH and one nucleic acid molecule.

[0176] In another specific embodiment, the present invention relates to a conjugate comprising a VHH covalently bonded to a peptide. The peptide may be an active molecule, bait, tag, ligand, etc. Preferred variants of such a conjugate include one VHH and one peptide.

[0177] In another embodiment, the present invention relates to a conjugate comprising a VHH covalently bonded to a dye.

[0178] In another embodiment, the present invention relates to a conjugate comprising VHH covalently bonded to nanoparticles and / or liposomes, such as lipid particles or nanoparticles ("LNPs"). The nanoparticles and / or liposomes may be filled with or functionalized with an activator. Preferred variants of such a conjugate include multiple VHH molecules coupled to each nanoparticle or liposome.

[0179] In a further embodiment, the conjugate comprises an antibody or fragment thereof coupled with one or more VHH molecules.

[0180] Typically, the VHH molecule couples to the C-terminus or N-terminus of a heavy chain or light chain, or both, or to the C-terminus or N-terminus of an Fc fragment. In a preferred embodiment, the VHH molecule couples to the N-terminus of a heavy chain. In a more preferred embodiment, the VHH molecule couples to the C-terminus of a heavy chain.

[0181] In another specific embodiment, the conjugate comprises or consists of a single VHH molecule coupled to an antibody fragment that may be a heavy chain or a light chain. In this aspect, the VHH molecule is indiscriminately coupled to the C-terminus or N-terminus of the chain. Preferably, it is coupled to the C-terminus.

[0182] The present inventors also describe herein a method for preparing a conjugate compound as defined above, characterized in that it comprises the step of coupling VHH with a molecule or scaffold, preferably by a chemical, biochemical or enzymatic pathway, or by genetic engineering.

[0183] In the chimeric agent of the present invention, if multiple VHHs are present, they may bind to similar or different binding domains.

[0184] nucleic acids, vectors, and host cells Further aspects of the present invention relate to nucleic acids encoding the VHH as defined above, or their conjugates (where the conjugate portion is an amino acid sequence). The nucleic acid may be single-stranded or double-stranded. The nucleic acid may be DNA (e.g., cDNA or gDNA), RNA (e.g., mRNA or gRNA), or a mixture thereof. It may be in single-stranded or double-stranded form, or it may be a mixture of the two. It may comprise modified nucleotides comprising, for example, modified conjugates, modified purine or pyrimidine bases, or modified sugars. It may be prepared by any method known to those skilled in the art, including chemosynthesis, recombination, and / or mutagenesis. The nucleic acid according to the present invention may be inferred from the amino acid sequence of the VHH molecule according to the present invention, and the use of codons may be adapted depending on the host cell to which the nucleic acid is to be transcribed. These steps may be carried out according to methods well known to those skilled in the art, some of which are described in the reference manual by Sambrook et al. (Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual, Third Edition, Cold Spring Harbor).

[0185] Specific examples of such nucleic acid sequences include sequences comprising any one of sequence numbers 21, 23, 25, 27, or 29, their complementary sequences, and fragments thereof that lack or contain the optional tag code portion appearing in sequence numbers 22, 24, 26, 28, or 30. Domains encoding CDR1 (sequence numbers 52-56), CDR2 (sequence numbers 57-61), and CDR3 (sequence numbers 62-66) are underlined. The tag code portion is shown in bold in Table 3 (sequence number 67: GCGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGGGCCGCACATCACCACCATCACCATGGGAGCTAG).

[0186] The present invention also relates to vectors containing such nucleic acids under the control of regulatory sequences (e.g., promoters, terminators, etc.) in an optional manner. The vector may be a plasmid, virus, cosmid, phagemid, artificial chromosome, etc. In particular, the vector may comprise nucleic acids of the present invention operably linked to regulatory regions, i.e., regions comprising one or more regulatory sequences. Optionally, the vector may comprise several nucleic acids of the present invention operably linked to several regulatory regions.

[0187] The term "control sequence" refers to a nucleic acid sequence necessary for the representation of a coding region. Control sequences can be endogenous or heterologous. Well-known control sequences and those currently used by those skilled in the art are preferred. Such control sequences include, but are not limited to, promoters, signal peptide sequences, and transcription terminators.

[0188] The term "operably linked" means a configuration in which the control array is positioned appropriately relative to the code array, thereby directing the expression of the code region.

[0189] The present invention further relates to the use of nucleic acids or vectors according to the present invention for transforming, transfecting, or transducing host cells, or for producing compositions comprising pharmaceutical compositions for transforming, transfecting, or transducing host cells.

[0190] The present invention also provides host cells comprising one or more nucleic acids and / or one or more vectors of the present invention.

[0191] The term "host cell" also includes any offspring of a parent host cell that are no longer identical to the parent host cell due to mutations that occur during replication. Suitable host cells can be prokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast, plant, insect, or mammalian cells). Specific examples of such cells include E. coli strains, CHO cells, Saccharomyces strains, plant cells, and sf9 insect cells.

[0192] use The VHH molecule of the present invention binds to RAGE, thereby enabling the molecule to be targeted and delivered to RAGE-expressing lung cells.

[0193] In the context of this invention, binding is preferably specific, thereby resulting in binding to RAGE with higher affinity than binding to any other antigen in the same species. The preferred VHH molecules of the present invention bind to human RAGE and mouse RAGE.

[0194] Therefore, the present invention relates to a method for targeting / delivering a compound to / through RAGE-expressing lung cells, comprising coupling the compound to at least one VHH of the present invention.

[0195] The present invention further relates to the use of VHH as defined above as a vector for the transport of compounds to / through RAGE-expressing lung cells.

[0196] The present invention also relates to the use of VHH as defined above for preparing drugs ( / pharmaceuticals) that can target lung sites.

[0197] The present invention also relates to a method for enabling or improving the delivery of a compound of interest to a lung site, comprising coupling the compound of interest to the VHH molecule of the present invention.

[0198] As described above in this specification, the VHH of the present invention can be used to transport or deliver any compound to the lungs, such as chelating agents, small drugs, amino acids, peptides, polypeptides, proteins, lipids, nucleic acids, viruses, liposomes, and exosomes.

[0199] Vehicles such as viruses, virus-like particles (VLPs), cell-derived vesicles (CDVs), exosomes, lipid vehicles, or polymer vehicles may be used to transport or deliver the conjugate (including VHH), preferably lipid nanoparticles (LNPs), micelles, or liposomes.

[0200] The present invention also relates to pharmaceutical compositions, particularly diagnostic or therapeutic compositions, comprising at least one VHH or chimeric ( / conjugate) compound that is associated with or present in a vehicle, or not associated with a vehicle, for example, in the context of a therapeutic composition, comprising a VHH-drug conjugate as defined above, and one or more pharmaceutically acceptable supports, carriers, or excipients. The present invention also relates to diagnostic compositions, particularly, comprising a VHH or chimeric ( / conjugate) compound that is associated with or present in a vehicle, or not associated with a vehicle, for example, comprising a VHH-diagnostic or medical imaging agent conjugate compound as defined above.

[0201] The conjugate can be used in any pharmaceutically acceptable salt form. The expression “pharmaceutically acceptable salt” means, for example, non-limitingly, the vector or conjugate loaded with at least one pharmaceutically acceptable base or acid addition salt, hydrate, ester, solvate, precursor, metabolite or stereoisomer of the substance of interest.

[0202] The term "pharmaceutically acceptable salt" generally refers to a non-toxic salt that can be prepared by reacting a free base with a suitable organic or inorganic acid. These salts preserve the biological effects and properties of the free base. Representative examples of such salts include acetate, N-methylglucamine ammonium, amsonate (4,4-diaminostilbene-2,2'-disulfonate), benzenesulfonate, benzoate, bicarbonate, bisulfate, tartrate, borate, hydrobromide, bromide, butyrate, cansylate, carbonate, hydrochloride, chloride, citrate, clavulanate, dichlorhydrate, diphosphate, edetate, calcium edetate, edisylate, estrate, esylate, fumarate, gluceptate, gluconate, glutamate, glycolyl arsanylate, hexafluorophosphate, hexylresorcinate, hydravamin, hydroxynaphthoate, iodide, isothionate, and lactate. Examples of water-soluble and water-insoluble salts include lactobionates, laurates, malates, maleates, mandelates, mesylates, methyl bromide, methyl nitrate, methyl sulfate, mucinates, napsylates, nitrates, 3-hydroxy-2-naphthoates, oleates, oxalates, palmitates, pamoates (1,1-methylene-bis-2-hydroxy-3-naphthoates, or emboates), pantothenates, phosphates, picrates, polygalacturonates, propionates, p-toluenesulfonates, salicylates, stearates, acetates, succinates, sulfates, sulfosalicylates, sulfamates, tannates, tartrates, theocrates, tosylates, triethiodies, trifluoroacetates, and valerians.

[0203] The compositions of the present invention advantageously comprise a pharmaceutically acceptable support, carrier, or excipient. The pharmaceutically acceptable support, carrier, or excipient can be selected from conventionally used carriers depending on each mode of administration. Depending on the intended mode of administration, the compound may be in the form of a solid, semi-solid, or liquid. In the case of solid compositions such as tablets, pills, powders, or granules, either free or contained in gelatin capsules, the active ingredient may be combined with: a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and / or glycine; b) lubricants, e.g., silica, talc, stearic acid, its magnesium or calcium salt, and / or polyethylene glycol; c) binders, e.g., magnesium silicate and aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone; d) disintegrants, e.g., starch, agar, alginic acid or its sodium salt, or effervescent mixtures; and / or d) absorbents, dyes, flavorings, and sweeteners. Excipients may include, for example, mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and pharmaceutical-grade analogues. In the case of semi-solid compositions such as suppositories, the excipients can be, for example, emulsions or oily suspensions, or polyalkylene glycol-based materials such as polypropylene glycol. Liquid compositions, especially those encapsulated in injections or soft capsules, can be prepared by dissolving or dispersing the active substance in a pharmaceutically pure solvent such as water, saline solution, aqueous dextrose, glycerol, ethanol, oil, or analogs thereof.

[0204] The compositions or conjugates of the present invention can be administered by any suitable route, and in a non-limiting manner, by parenteral routes such as in the form of formulations that can be injected subcutaneously, intravenously or intramuscularly; by oral routes (i.e., peros) such as in the form of coated or uncoated tablets, gelatin capsules, powders, pellets, suspensions or oral solutions (one such form for oral administration may be either immediate-release, sustained-release, or delayed-release); by rectal routes, such as in the form of suppositories; by topical routes, particularly transdermal routes (e.g., in the form of patches, pomades or gels); by nasal routes, such as in the form of aerosols and sprays; by sublingual routes; or by intraocular routes.

[0205] Preferably, the VHH or conjugate of the present invention, or a composition comprising such VHH or conjugate, is administered intravenously or subcutaneously.

[0206] The pharmaceutical compositions of the present invention typically comprise an effective dose of the VHH or conjugate of the present invention. “Therapeutic effective dose” as used herein means a dose that produces a therapeutic effect in a given symptom and administration schedule. It is typically the average dose of the active substance administered to significantly improve some of the symptoms associated with a disease or condition. For example, in treating lung cancer, a lesion, injury, or disorder affecting the lungs, a dose of the active substance that reduces, prevents, delays, eliminates, or stops one of the causes or symptoms of the disease or disorder would be therapeutically effective. A “therapeutic effective dose” of the active substance does not necessarily cure the lung disease or disorder, but provides treatment for the disease or disorder, thereby delaying, inhibiting, or preventing its onset, or reducing its symptoms, or altering its duration, for example, reducing its severity or accelerating the patient's recovery.

[0207] The "therapeutic effective dose" of the VHH or conjugate of the present invention is, for example, about 1 mg to about 100 mg per kilogram of body weight of the subject to whom the VHH or conjugate is administered.

[0208] It is understood that the "therapeutic dose" for a particular person depends on a variety of factors, including the activity / effect of the active substance, the timing of its administration, the route of administration, its toxicity, its elimination rate and metabolism, drug combinations / interactions, and the severity of the disease (or disorder) being treated for preventive or therapeutic purposes, as well as the patient's age, weight, overall health, sex, and / or diet.

[0209] In the context of imaging or diagnosis, exposure levels are proportional to the activity of the selected radiotrace at the time of injection and depend on the time it remains in the body (until it is physically or biologically removed). They clearly depend on the selected tracer. Activity at injection is assessed in millions of becquerels (MBq or megabecquerels). However, many physicians still rely on the widely used conventional millicury units (1 mCi is equivalent to 37 MBq).

[0210] The amount of radioactivity injected varies greatly depending on the test; for example, 1 mCi for a kidney scintigraphy using iodine-123 and approximately 27 mCi (1000 MBq) for a cardiac scintigraphy using technetium. Naturally, the amount of exposure is proportional to the injected radioactivity, but also to the radioactivity of the radioactive material itself. These radiation exposure characteristics vary significantly depending on the diagnostic or therapeutic application, the nature of the radiation, the specific radioactivity, the duration of its presence in the body, and how radioisotopes are distributed within the patient's body.

[0211] Therefore, the pharmacologically effective dose when using the VHH or conjugate of the present invention for imaging or diagnosis is, for example, about 1 mCi to about 40 mCi.

[0212] Depending on the coupling substance, the conjugates and compositions of the present invention can be used for imaging, diagnosis, prevention and / or treatment of conditions or disorders affecting the lungs, such as infections, asthma, bronchial asthma, and inflammatory conditions such as chronic obstructive pulmonary disease (COPD), also known as emphysema, and / or cancer. The VHH of the present invention has the ability to target RAGE-expressing cells, particularly lung cells, and / or to pass through the lung cell membrane. RAGE is found in greater quantities in the lungs compared to other organs. RAGE is also expressed in lung endothelial cells.

[0213] In this regard, the present invention relates to the use of the pharmaceutical conjugates or pharmaceutical compositions (in particular therapeutic compositions) described herein in a non-limiting manner to prevent or treat lung conditions or disorders such as lung tumors (where the tumor is benign or malignant, i.e., carcinogenic), particularly metastatic lung cancer, or bacterial, viral, parasitic or fungal infections of the lung, or any other known lung disease.

[0214] In the context of the present invention, lung cancer is also identified as lung carcinoma or malignant lung tumor. Lung tumors include, for example, non-small cell lung cancer or carcinoma (NSCLC) or small cell lung cancer or carcinoma (SCLC).

[0215] In the context of this invention, infectious lung disease refers to bacterial infections such as pneumonia or tuberculosis, or viral infections such as infection caused by SARS-CoV-2. Infectious lung disease may also be caused by parasitic or fungal infections.

[0216] In certain aspects, an infectious disease is a zoonotic disease caused by a virus, bacteria, parasite, or fungus.

[0217] In the context of the present invention, infectious pathology refers to parasitic infections such as pneumocystis, echinococcosis, porocephaliasis, aspergillosis, paragonimiasis, infections caused by penicillum marneffei, schistosomiasis, ascariasis, hookworm infection, filariasis, canine filariasis, tropical pulmonary eosinophilia, toxocariasis, amebiasis, and malignant tertian malaria.

[0218] In the context of the present invention, infectious pathology is a fungal infection caused by endemic or opportunistic fungi including, for example, Aspergillus (which may cause invasive aspergillosis), Cryptococcus (which may cause cryptococcosis), and Pneumocystis (which may cause pneumonia).

[0219] In the context of the present invention, genetic and / or rare diseases that affect the lungs include, for example, cystic fibrosis, pulmonary hypertension, berylliosis, and interstitial lung diseases including hypersensitivity pneumonitis, lymphangioleiomyomatosis (LAM), pulmonary alveolar proteinosis (PAP) syndrome, Hermansky-Pudlak syndrome (HPS), Birt-Hogg-Dubé syndrome (BHD), pulmonary Langerhans cell histiocytosis (PLCH), diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH), pulmonary alveolar microlithiasis (PAM), alpha1-antitrypsin deficiency (alpha1), or generalized lymphatic anomaly (GLA) (also known as lymphangiomatosis), and other rare lung diseases.

[0220] The present invention also relates to VHHs, conjugates, or pharmaceutical compositions (particularly diagnostic compositions) as described hereinabove for use in imaging and / or diagnosing lung lesions or disorders such as lung tumors (where the tumor is a benign or malignant lung tumor), particularly lung metastases, infectious lung lesions caused by bacteria, viruses, parasites, or fungi, or genetic and / or rare diseases of the lung.

[0221] The present invention particularly relates to VHHs, conjugates, or pharmaceutical compositions as described hereinabove for use in imaging and / or diagnosing the presence of lung tumors or lung metastasis cancer cells.

[0222] The present invention also relates to VHHs, conjugates or pharmaceutical compositions as described above for imaging, diagnosing, preventing and / or treating hereditary and / or rare diseases such as, but not limited to, cystic fibrosis, pulmonary hypertension, berylliosis or hypersensitivity pneumonitis, interstitial lung diseases, lymphangioleiomyomatosis (LAM), pulmonary alveolar proteinosis (PAP) syndrome, Hermansky-Pudlak syndrome (HPS), Birt-Hogg-Dubé syndrome (BHD), pulmonary Langerhans cell histiocytosis (PLCH), diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH), pulmonary alveolar microlithiasis (PAM), alpha1-antitrypsin deficiency (alpha1), or GLA / lymphangiomatosis.

[0223] The present invention also relates to VHHs, conjugates or pharmaceutical compositions as defined above herein, wherein the conjugate agent is or comprises a virus or virus-like particle such as a recombinant virus. The present invention can actually be used to increase the RAGE-enriched lung tissue delivery of recombinant (e.g., replication-defective or attenuated) viruses (adenovirus, adeno-associated virus, lentivirus, retrovirus, etc.) or virus-like particles used in gene therapy. Coupling to the virus or VLP can be performed, for example, by coupling to the capsid protein of the virus.

[0224] The present invention also relates to a method for preventing or treating any of the above symptoms or diseases by administering the VHH, conjugate or composition of the present invention to a subject in need thereof.

[0225] The present invention also relates to the use of the VHH, conjugate or composition of the present invention for the manufacture of a medicament for treating any of the above symptoms or diseases.

[0226] Other aspects and advantages of the present invention will become apparent from the following embodiments, but these embodiments are essentially illustrative and do not limit the scope of this application. [Examples]

[0227] Example I Evaluation of RAGE expression in various tissues The inventors analyzed the cell membrane expression profiles of RAGE in various tissues (Figure 1-A) of rats, mice, pigs, and non-human primates (NHPs, rhesus macaques) by Western blotting. The ProteoExtract intracellular proteome extraction kit (Calbiochem, La Jolla, California, USA) was used to prepare membrane extracts. The membrane extracts were quantified using the BioRad DC Protein Assay (Bio-Rad, Hercules, California, USA) according to the manufacturer's instructions. Membrane proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 4-12% polyacrylamide gels and transferred to nitrocellulose membranes (Thermo Fisher Scientific). The protein was probed with a primary rabbit polyclonal antibody against RAGE (1 / 1000, abcam#ab37647), followed by a 1 / 30,000 diluted HRP-conjugated donkey anti-rabbit IgG secondary antibody (Jackson ImmunoResearch, 711-035-152). Finally, the protein was detected using chemiluminescence (ECL; Cytiva).

[0228] As shown in Figure 1-A, full-length RAGE (approximately 55 kDa) is expressed at high levels primarily in the lungs of rats, mice, pigs, and non-human primates. RAGE is also expressed in rat corneas and at low or undetectable levels in membrane extracts of other tissues, including the heart, muscle, kidney, retina, bladder, adrenal gland, pancreas, testes, and stomach.

[0229] High levels of RAGE expression and tissue distribution in rat and mouse lungs were confirmed by immunocytochemistry (Figure 1-B). Immunohistochemistry involved transcardiac perfusion with 0.9% NaCl followed by perfusion with 50 mL of PBS 1X-4% paraformaldehyde (PFA). Mouse or rat lungs were then excised and rapidly frozen on dry ice in mounting medium (OCT). Cryostat (Leica CM-3050-S) sections (10 μm thick) were stored at -80°C. Organ sections were first permeabilized with a solution of PBS 1X, 0.3% Triton X-100, and 10% normal donkey serum (NDS), and blocked at room temperature for 1 hour. Next, sections were incubated overnight at 4°C with anti-RAGE (rat, 1 / 500, R&D Systems #MAB1179), followed by incubation at room temperature for 2.30 hours with anti-rat Alexa488 (donkey, 1 / 800, Jackson ImmunoResearch #712-545-153). Nuclei were stained with Hoechst (0.5 μg / mL, Life Technologies). A control without primary antibody was used, but no immunostaining was observed. Sections were mounted on superfrosted glass slides using Prolong Gold Antifading reagent (Life Technologies). Images were taken and processed using an Apotome microscope (Zeiss, Jena, Germany) and Zen software (Zeiss).

[0230] These data indicate that RAGE is highly expressed in the lungs and therefore a viable target for the delivery of therapeutic molecules.

[0231] Example II Construction of CHO cell lines that stably express human and mouse RAGE. A prerequisite for the identification and characterization of RAGE-binding VHH was the establishment of a stable eukaryotic cell line (Chinese hamster ovary cell, CHO) that constitutively and at high rates expressed human RAGE (hRAGE) and mouse RAGE (mRAGE). These cell lines were then used to i) identify and characterize drugs that bind to receptors expressed on the cell surface in their intrinsic configuration, and ii) test whether the receptors could internalize such drugs by endocytosis.

[0232] To construct these cell lines, cDNAs encoding hRAGE and mRAGE were cloned using sequence information available in the database (access numbers NM_001136.4 and NM_007425.3, respectively). A primer necessary for cDNA amplification by RT-PCR was selected (see Table 1 below), and its ends (bold) contained the restriction sites (SacI and SacII) necessary for cloning in the pEGFP-N1 expression vector (Clontech) (Figure 2-A). [Table 1]

[0233] Using total RNA prepared from human or mouse brain, cDNA fragments encoding h / mRAGE were amplified by RT-PCR. After amplification, the PCR product corresponding to hRAGE was digested with SacI-SacII restriction enzymes, ligated with a pEGFP-N1 expression vector (Clontech), and digested with the same restriction enzymes. mRAGE was introduced into the pEGFP-N1 vector by mutagenesis. Upon introduction into eukaryotic cells, this vector enables the expression of h / mRAGE fused to EGFP at its C-terminus, i.e., the end of its intracellular domain, under the control of a CMV promoter. Competent E. coli DH5α bacteria were transformed, isolated colonies were obtained, plasmid DNA was prepared, and both strands of the construct were fully sequenced for validation.

[0234] Transient transfection was performed in CHO-K1 cells, and stable transfectants were selected by limiting dilution and antibiotic (G418) resistance. These cell lines were amplified while maintaining the selection pressure.

[0235] For fixed (PFA) cell lines, immunocytochemistry was performed using a rat anti-RAGE primary antibody (R&S Systems #MAB1179) diluted 1 / 200 and then an anti-rat A594 conjugate secondary antibody diluted 1 / 800, and confocal microscopy images were taken after that. In Figure 2-B, it was confirmed that EGFP (green) and the anti-RAGE antibody (red) co-localized, and thus the receptor was well-expressed, especially on the cell membrane.

[0236] Membrane expression of the receptor of the expected size was confirmed by Western blot on the cell membrane of the h / mRAGE-GFP CHO cell line extracted with the ProteoExtract Subcellular Proteome Extraction Kit. A protein corresponding to the total size (95 kDa) of EGFP and h / mRAGE was detected with the anti-RAGE antibody (Figure 2-C). The CHO K1 wild-type (WT) cell line was used as a negative control, and no protein was detected with the anti-RAGE antibody.

[0237] These data confirm the expression of the h / mRAGE receptor on the cell surface of the generated CHO cell lines. These cells can be used to immunize llamas to generate VHHs that bind to RAGE.

[0238] Example III Generation of VHHs that Bind to RAGE Lama (Lama glama) was subcutaneously immunized four times with membrane preparations from CHO stable cell lines expressing human and mouse receptors of interest. VHH library construction was performed as previously described (Alvarez-Rueda et al., 2007, Behar et al., 2009). Briefly, mRNA encoding VHH was amplified by RT-PCR from total RNA of peripheral blood mononuclear cells isolated by Ficoll gradient and cloned into pHEN1 phagemids. Repeated selection allowed for the isolation of phages presenting VHH with strong affinity to RAGE expressed on the cell surface.

[0239] In total, over 700 clones were screened for their binding ability to RAGE, and the sequences of approximately 70 clones were determined.

[0240] VHH cells with improved binding affinity (to both mouse and human cell lines) and cell permeability were obtained. Representative VHH cells include VHH1, VHH2, VHH3, VHH4, and VHH5 (see also sequence list). These VHH cells do not bind to control CHO cells.

[0241] The amino acid sequences of each of these VHHs are listed in the sequence list. Based on these studies, various VHHs that bind to RAGE were generated and are included in the claimed invention.

[0242] Example IV Determination of VHH-RAGE binding affinity The binding properties of VHH with affinity for RAGE were tested using flow cytometry, and the apparent affinity (K d app ) was determined. All experiments were conducted in 96-well plates with 2 × 10⁶ 5The procedure was performed using individual cells / well with shaking at 4°C. CHO cell lines or CHO WT cells expressing EGFP-fused RAGE were saturated with 2% PBS / BSA solution for 30 minutes to avoid nonspecific binding, followed by incubation with purified VHH at concentrations of 50 μM to 0.5 nM for 1 hour. After washing once with 2% PBS / BSA, cells were incubated with anti-6His tag antibody (mouse) for 1 hour, washed twice with 2% PBS / BSA, and incubated with Alexa647 conjugate anti-mouse secondary antibody for 1 hour. After washing twice with 2% PBS / BSA, cells were fixed by incubation in 2% PBS / PFA for 15 minutes, washed once with PBS, and finally resuspended in PBS. Fluorescence levels were assessed using an Attune NxT flow cytometer (Thermo Fisher Scientific).

[0243] Three of the five VHHs (VHH1, 2, and 5) bound to both human and mouse RAGE cells, causing a concentration-dependent shift in signal (Figure 3-A). Two VHHs (VHH3 and 4) bound only to mouse RAGE cells. Under control conditions, no nonspecific labeling was observed when cells were incubated with the control VHH (VHHctrl). Furthermore, no labeling was detected for any of the VHHs tested in CHO WT control cells (not shown). VHH K d app These values ​​were calculated using GraphPad Prism software. They ranged from 32 nM (VHH4) to 1486 nM (VHH5) on mRAGE and from 204 nM (VHH5) to 350 nM (VHH2) on hRAGE (Figure 3-B).

[0244] Based on these results, all VHHs exhibit the required binding affinity (0.1 nM to 10 μM).

[0245] Example V Generation of VHH-siRNA conjugates and evaluation of their binding affinity. The conjugate strategy included convergent synthesis via parallel modification of i) amine-functionalized siRNA and ii) VHH fused to myc and 6His tags. Amino-functionalized siRNA (in this example, siTTRm (transthyretin) and siSOD1m (superoxide dismutase 1)) was chemically modified by conjugation with a heterobifunctional linker (DBCO-NHS or BCN-NHS in the context of this particular example) to introduce a cycloalkyne moiety (DBCO: dibenzocyclooctin or BCN: bicyclooctin). VHH was site-specifically modified using the BTG (bacterial transglutaminase) enzyme, which catalyzes the formation of an isopeptide bond between the glutamine residue (Q residue) of the myc tag sequence and the amino-functionalized substrate, generating an azide VHH intermediate.

[0246] In the final stage, both the alkyne siRNA and the azide VHH were conjugated to each other by a copper-free click chemistry reaction.

[0247] The binding properties of VHH-siRNA conjugates with affinity for RAGE were tested using flow cytometry, and the apparent affinity (K) was determined. d app The following was determined. The same protocol as described in Example IV was used.

[0248] All VHH-siRNAs bound to mRAGE and induced a concentration-dependent shift in signaling (Figure 4-A), confirming that VHH conjugated to both siTTRm and siSOD1m retained binding to the receptor of interest. VHH-siRNA K d app These were calculated using GraphPad Prism software. Their range was from 6.8 nM (VHH4-siSOD1m) to 238 nM (VHH2-siSOD1m) (Figure 4-B).

[0249] These data demonstrate that the VHH of the present invention can be conjugated to therapeutic molecules such as siRNA and maintain its affinity for RAGE.

[0250] Example VI Generation of VHH-RAGE-Fc fusions and evaluation of their binding affinity. The anti-RAGE VHH molecule of the present invention was fused to an IgG Fc fragment. To construct the fusion protein, the DNA fragment encoding VHH (untagged) was amplified by PCR and cloned into a pINFUSE-IgG1-Fc2 vector (InvivoGen) to encode a human IgG1-Fc fragment containing VHH at its N-terminus or C-terminus. The fusion protein was prepared using the Expi293 Expression System according to the manufacturer's instructions (Life Technologies). 72 hours after transfection, the supernatant was collected and purified using a Protein A GraviTrap column (GE Healthcare). The purified fusion protein was quantified at 280 nm using a Nanodrop instrument (Thermo Fisher).

[0251] To confirm the binding ability of the fusion protein to RAGE, immunocytochemistry experiments were performed on mouse or human RAGE-expressing CHO cell lines fused to EGFP. In these experiments, the VHH-Fc fusion protein was incubated at 50 nM on live cells, the cells were fixed with PBS-PFA 4%, the cell membrane was permeabilized with PBS-0.1% Triton X100, and then detected using Alexa594 conjugated anti-hFc antibody, followed by imaging with a confocal microscope.

[0252] The results demonstrate that the VHHRAGE-Fc fusion of the present invention binds to / is taken up by cells expressing mRAGE or hRAGE (Figures 5 and 6). No binding of the control VHH-Fc conjugate (VHHctrl-Fc) on cells was observed, demonstrating the specificity of the interaction.

[0253] The binding properties of VHH-Fc and Fc-VHH fusion proteins with affinity for RAGE were tested using flow cytometry experiments, and the apparent affinity (K) was determined. d app) was determined. All experiments were performed at 4 °C with shaking in 96-well plates using 2 × 10 5 cells / well. CHO cell lines or CHO WT cells expressing the receptor of interest fused to EGFP were saturated with PBS / 2% BSA and subsequently incubated for 1 h with purified VHH-Fc or Fc-VHH at concentrations ranging from 12.5 μM to 6 pM. After washing twice with PBS / 2% BSA, the cells were incubated for 1 h with Alexa647-conjugated anti-hFc antibody. After final washing twice with PBS / 2% BSA, the cells were fixed by incubation with PBS / 2% PFA for 15 min, washed once with PBS, and finally resuspended in PBS. Fluorescence levels were evaluated using an Attune NxT flow cytometer (Thermo Fisher Scientific).

[0254] All VHH-Fc and Fc-VHH fusion proteins induced a concentration-dependent shift in the signal, confirming binding to the receptor of interest (Figure 7-A). Notably, VHH3-Fc and VHH4-Fc showed binding to the human receptor. VHH-Fc and Fc-VHH K d app was calculated using GraphPad Prism software (Figure 7-B). The K d app of all VHHs was significantly improved by conjugation with the Fc fragment, and the K d app of RAGE-binding VHH-Fc and Fc-VHH ranged from 1 nM to 154 nM.

[0255] This is another example demonstrating that the VHHs of the present invention can be conjugated to other therapeutic molecules (in this case the Fc fragment) and maintain their affinity for RAGE.

[0256] Example VII Competitive assay between purified VHH with affinity for RAGE and VHH1-Fc To evaluate the ability of selected VHHs to compete with each other for receptor binding, a competition assay using flow cytometry was performed. In the first step, a diluted series of competitors were incubated at 4°C for 1 hour on CHO cells expressing the target receptor of interest fused to EGFP. Next, an EC80 tracer was added, and the cells were incubated for another hour before detection using an appropriate detection system (Figure 8-A).

[0257] Exemplary experiments showed that when used as a tracer, VHH1-Fc can substitute for the binding of VHH3 and VHH4, suggesting that these three VHHs bind to the same or nearby epitopes on mRAGE (Figure 8-B).

[0258] Example VIII Determination of the VHH-RAGE-Fc binding domain on RAGE The full-length isoform of the RAGE extracellular domain contains three conserved domains: a type V immunoglobulin (Ig) domain and two type C Ig domains designated C1 and C2.

[0259] To determine the binding domain of VHH-RAGE-Fc, the inventors sequentially repressed the RAGE domain and expressed the resulting shortened constructs mRAGE-ΔV and mRAGE-ΔV-C1 (Figure 9-A). VHH-RAGE-Fc binding on the full-length constructs (mRAGE and HA-mRAGE) in transfected CHO cells was then compared. HA tags were introduced to the N-terminus of each receptor to verify proper presentation of the extracellular domain using anti-HA, impermeable immunocytochemistry. EGFP was fused at the C-terminus, allowing for easy visualization of the shortened and full-length receptors using fluorescence microscopy. Using gene synthesis (GeneCust), DNA fragments encoding amino acids 1–380 of mature full-length mRAGE, amino acids 95–380 of mRAGE-ΔV, and amino acids 204–380 of mRAGE-ΔV-C1 were generated. The synthesized DNA fragments were cloned into the pEGFP-N1 plasmid after restriction and ligation with XhoI EcoRI. CHO WT cells were transfected with various plasmid constructs using jetPEI®, according to the manufacturer's instructions.

[0260] For immunocytochemistry, transfected viable cells were incubated with 250 nM VHH-RAGE-hFc or anti-HA antibody at 37°C for 1 hour. The cells were then fixed with PBS-PFA 4%. VHHRAGE-hFc or anti-HA antibody was detected as mouse anti-human Fc Alexa594 or donkey anti-rat Fc Alexa594, respectively, and the cells were imaged using a confocal microscope.

[0261] Anti-HA immunocytochemistry (without cell permeabilization treatment) demonstrated that mRAGE-ΔV, mRAGE-ΔV-C1, and HA-mRAGE were correctly expressed on the plasma membrane. VHH5-Fc (Figure 9-B), shown as an example, bound to full-length RAGE and all its shortened versions, meaning this VHH binds to the C2 domain. A similar binding profile was observed with VHH2-Fc. On the other hand, VHH1-Fc, VHH3-Fc, and VHH4-Fc bound to mRAGE and HA-mRAGE, but not to mRAGE-ΔV and mRAGE-ΔV-C1, meaning these VHHs bind to the V1 or VC1 domain. VHHctrl-Fc, unrelated to any of the constructs, did not bind. The results of the immunocytochemistry experiments are summarized in Figure 9-C.

[0262] These results indicate various VHH binding domains. VHH1-Fc, VHH3-Fc, and VHH4-Fc bind to the V1 or VC1 domain. VHH2-Fc and VHH5-Fc bind to the C2 domain.

[0263] Example IX Cellular binding / colocalization of VHH-Fc in mouse lung RAGE To confirm the ability of the fusion proteins to bind to RAGE in vivo, immunocytochemistry experiments were performed using the lungs of mice injected via the tail vein with VHHctrl-Fc, VHH1-Fc, and VHH5-Fc at a concentration of 35 nmol / kg. Forty-eight hours after injection, the mice were perfused with saline, and the lungs were incubated overnight in 4% PFA. The lungs were thoroughly washed with PBS 1X, incubated in 30% sucrose for two days, and then rapidly frozen. The fixed lungs were embedded in an OCT scanner and sectioned into 14 μm thick sections. Double immunofluorescence (IF) staining of lung tissue was performed on lung sections using anti-RAGE (primary antibody 1 / 200: R&D Mab1179, and secondary antibody 1 / 500 donkey anti-rat 488JIR712-545-153) and VHH-Fc (1 / 100 goat anti-human Fc594, JIR109-605-098) antibodies. Cell nuclei were labeled with Hoechst#33342. Representative images were taken with a confocal microscope at 20x and 63x magnification.

[0264] VHH-Fc administration did not alter the alveolar structure of the lungs, and as expected, strong RAGE expression was observed on the plasma membrane of lung epithelial cells. In the merged image (rightmost panel), double IF staining of RAGE and VHH-Fc shows co-localization of VHH1-Fc or VHH5-Fc with RAGE in the alveolar epithelium. No binding to RAGE was observed with VHHctrl-Fc (Figure 10).

[0265] These results indicate that VHH1-Fc and VHH5-Fc target and bind to RAGE in the lungs in vivo, while VHHctrl-Fc is unable to do so. No visible pulmonary toxicity was observed 48 hours after administration.

[0266] These results indicate significant accumulation of RAGE target conjugates (VHH1-Fc and VHH5-Fc) in the lungs.

[0267] Example X In vivo pharmacokinetics and organ uptake of VHH-Fc conjugates To evaluate the potential of VHH-Fc conjugates to target organs expressing the RAGE receptor in vivo, conjugates VHH1-Fc, VHH4-Fc, VHH5-Fc, and VHHctrl-Fc were intravenously injected into the tail vein of mice at a dose of 35 nmol / kg (n=4-12 / time point). At different time points after injection (2 hours, 6 hours, 18 hours, 48 ​​hours, 168 hours), mice were deeply anesthetized with a mixture of ketamine (100 mg / kg) and xylazine (10 mg / kg) administered intraperitoneally. Blood was collected directly from the right ventricle by cardiac puncture into a heparin sodium tube. After centrifugation at 1500 g for 10 minutes, the plasma was collected and stored at -80°C until analysis. Next, the left ventricle of the mice was extensively perfused with a heparinized 0.9% NaCl solution to remove all traces of blood from the organ. After perfusion, samples were taken from various organs (lungs, kidneys, liver), weighed, homogenized with lysis buffer (0.1% PBS Triton with antiprotease), transferred to Safe-Lock Eppendorf tubes, rapidly frozen, and stored at -80°C until in vivo analysis.

[0268] The amount of VHH-Fc in plasma and lysed organs was measured using an in-house developed anti-FcELISA. The results are shown as concentration (nM), percentage of injected dose per gram of tissue, or organ-to-plasma ratio (Figure 11).

[0269] The RAGE-binding fusion proteins VHH1-Fc, VHH4-Fc, VHH5-Fc, and VHHctrl-Fc exhibit distinct pharmacokinetic profiles (Figure 11-A, C). VHH1-Fc distributes rapidly, with 19% ID measured in plasma 2 hours post-injection (pi). The distribution phase continues for approximately 6 hours for VHH1-Fc, VHH5-Fc, and VHHctrl-Fc, at which point 19%, 41%, and 34% ID are still present in plasma, respectively (Figure 11-B, D). At 48 hours post-injection, the efflux phase for all molecules becomes similar. Plasma pharmacokinetic parameters for all VHH-Fc analyzed using Kinetica software are shown below in A, 2B, 2C, and 2D. [Table 2] Table 2: Pharmacokinetic parameters of VHH-Fc fusions injected into WT C57Bl / 6 mice. VHHctrl-Fc, VHH1-Fc, VHH4-Fc, and VHH5-Fc were injected into the tail vein at 35 nmol / kg, and mice were perfused with saline at 2, 6, 18, 48, 96, or 168 hours post-injection. The amounts of VHH-Fc in plasma (A), lung (B), liver (C), and kidney (D) were evaluated by ELISA, and pharmacokinetic parameters were calculated using Kinetica software.

[0270] Strong lung targeting was observed with all VHH-Fc agents from 18 hours post-injection (pi) to 168 hours post-injection. Peak concentrations were reached at 48 hours post-injection, with VHH1-Fc at 59.5 nM (6.9% ID), VHH4-Fc at 77.5 nM (9.5% ID), and VHH5-Fc at 192.5 nM (22.9% ID) (Figure 11-E, F). No significant accumulation was observed with the control VHHctrl-Fc (10 nM; 1% ID) (Figure 11-E, F). Similar distribution advantages were observed when evaluating the lung-to-plasma ratio (Figure 11-G). Lung targeting was confirmed for VHH1-Fc, VHH4-Fc, and VHH5-Fc, with strong accumulation sustained for up to 168 hours.

[0271] In the liver, all VHH-Fc cells showed no significant accumulation compared to VHHctrl-Fc cells and exhibited the same profile (Figure 11-H, I). In the kidneys, VHH1-Fc cells accumulated 1.6 times more than VHHctrl-Fc cells at 2 hours post-infection. VHH4-Fc and VHH5-Fc cells were identical to VHHctrl-Fc cells (Figure 11-K, L). At all time points except 168 hours, VHH1-Fc cells showed a significant advantage compared to controls when evaluating liver-to-plasma ratios and kidney-to-plasma ratios (Figure 11-J, M).

[0272] Pharmacokinetic parameters in the lungs, liver, and kidneys were estimated by non-compartmental analysis. Significant uptake into the lungs was observed for all VHH-Fc compared to VHHctrl-Fc, with VHH5-Fc showing Cmax and AUC. 0-168h The %IDmax value was the highest (Table 1B). In contrast, VHH1-Fc, VHH4-Fc, VHH5-Fc, and VHHctrl-Fc showed comparable Cmax and AUC in the liver and kidney. 0-168h , and %IDmax are also present (Table 1C, D).

[0273] These results demonstrate that the RAGE-targeted VHHs of the present invention can be used for efficient delivery of therapeutic agents to the lungs or to improve their in vivo distribution in the lungs. VHH1-Fc, VHH2-Fc, VHH3-Fc, VHH4-Fc, and VHH5-Fc, in particular VHH1-Fc, VHH4-Fc, and VHH5-Fc, exhibit the advantage of preferentially targeting the lungs compared to other organs with low RAGE expression levels (e.g., liver and kidneys).

[0274] Example XI Characterization of VHH-RAGE LNPs, in vitro mLuc mRNA delivery to h / mRAGE-GFP CHO cells, and in vivo distribution to C57 / Bl6 mice within 6 hours. To confirm the ability of VHH-RAGE to deliver purpose-specific cargo, its surface was functionalized with lipid nanoparticles (LNPs). These LNPs were loaded with Fluc luciferase mRNA, and their efficiency was measured. The prepared vectorized LNPs were compared with unvectorized LNPs (naked LNPs) and LNPs functionalized with unrelated VHH (VHHctrl). Conjugation of the vector to the LNPs was achieved by click chemistry, specifically by a SPAAC reaction between a reactive moiety BCN (bicyclo[6.1.0]nonine) selectively conjugated to VHH-RAGE and the corresponding functional group introduced via an additional lipid component (phospholipid azide; DOPE-PEG2OOO-N3) used during LNP preparation. The obtained LNPs were purified by multiple filtration steps using a 100kDa MWCO (Amicon, Sigma-Aldrich) membrane, and characterized using dynamic light scattering (DLS), a cholesterol standard assay kit, and the Quant-iT® RiboGreen RNA assay to determine mRNA concentration and encapsulation efficiency.

[0275] The hydrodynamic diameter and polydispersity index (PDI) of LNPs were determined by dynamic light scattering (DLS). Measurements were performed on samples diluted 10-fold with Dulbecco's phosphate-buffered saline (DPBS) using a Zetasizer Nano series Advanced Blue instrument (Malvern Instruments, Malvern Panalytical, Malvern, UK). Each sample was run three times for 5 minutes at 25°C with a 173° backscatter setting, and the results were averaged.

[0276] mRNA concentrations were determined using the Quant-iT RiboGreen mRNA Broad Range Assay Kit according to a standard protocol. LNPs were incubated in 0.5% (v / v) TritonX-100 (for total mRNA concentration) or DPBS (for free mRNA) at room temperature for 5 minutes. Encapsulation efficiency was calculated using the formula [(total mRNA concentration - free mRNA) / total mRNA concentration] × 100.

[0277] Cholesterol content was measured using an enzymatic spectroscopy assay (MAK043, Sigma-Aldrich) following a standard procedure, averaging two samples (diluted 10-fold with Dulbecco's phosphate-buffered saline (DPBS)).

[0278] The ability of VHH-LNPs to deliver FLuc mRNA into cells was investigated in CHO-hRAGE-EGFP and CHO-mRAGE-EGFP cell lines. Naked LNPs, VHHctrl-LNPs, and VHH5-LNPs were diluted to 1.25 μg mRNA luc / ml in OptiMEM medium (Thermo Fisher Scientific) and incubated on cells at 37°C for 6 hours (2 × 10⁵ cells / well). At the end of the incubation period, the ONE-Glo® luciferase assay system (Promega) containing a 5'-fluoroluciferin substrate was added to the cells (in a 1:1 ratio with culture medium) for 15 minutes to completely lyse the cells. The mixture was transferred to a 96-well white plate, and the luminescence generated by oxygenation of 5'-fluoroluciferin by the luciferase protein was read using a Glomax navigator (integration time 0.3 seconds).

[0279] The emission values ​​of CHO-hRAGE-EGFP and CHO-mRAGE-EGFP treated with VHH5-LNP were significantly higher than those treated with VHHctrl-LNP, while the emission values ​​measured with the two control LNPs (bare LNP and VHHctrl-LNP) were lower and similar.

[0280] These results indicate that LNP functionalization by VHH5 enhances intracellular delivery of FLuc mRNA by 3–5 times via a RAGE-dependent mechanism.

[0281] To evaluate in vivo the potential of VHH-LNP to target the lung (which has high basal RAGE expression levels), VHH5-LNP and VHHctrl-LNP were intravenously injected into the tail vein of mice at a dose of 20 μg / 200 μL of mLuc mRNA per mouse (n=3 / 4 mice per VHH-LNP construct). Six hours post-injection, mice were deeply anesthetized by intraperitoneal administration of a mixture of ketamine (100 mg / kg) and xylazine (10 mg / kg). Various organs (lungs, calf muscles, heart, kidneys, brain) were sampled, weighed, and pulverized in Precellys tubes (Bertin Technologies) with 1X passive lysis buffer from Promega (2 μl / mg tissue). The lysed tissue was centrifuged at 13,500 rpm for 10 minutes at 4°C, and the supernatant was transferred to a Safe-Lock Eppendorf tube, rapidly frozen, and stored at -80°C until bioanalysis.

[0282] The delivery and tissue distribution of FLuc mRNA by VHH-LNP was evaluated by quantifying the level of translated luciferase luminescence in each organ. 20 μl of tissue lysate was mixed with 100 μl of ONE-Glo® luciferase assay system in a 96-well white plate. After incubation for 3 minutes, luminescence was read using a Glomax navigator (integration time 0.3 seconds).

[0283] A strong luminescence signal (RLU) was measured in the lungs of mice injected with VHH5-LNP. Interestingly, this luminescence signal was nine times higher than that measured in mice injected with VHHctrl-LNP. Furthermore, the luminescence values ​​measured with VHH5-LNP in kidney, muscle, heart, and brain tissue were 6 to 22 times lower than those measured in the lungs, indicating that VHH5-LNP is superior in selectively targeting the lungs compared to other organs with low RAGE expression levels.

[0284] Overall, these results indicate that the RAGE-targeted VHH of the present invention can be used to effectively deliver therapeutic agents, such as mRNA / LNP particles, to the lungs or to improve their distribution in the lungs. [Table 3] JPEG2026512773000032.jpg247156 [Table 4] JPEG2026512773000034.jpg255156JPEG2026512773000035.jpg255153

[0285] References Bierhaus, A., S. Schiekofer, M. Schwaninger, M. Andrassy, ​​PM Humpert, J. Chen, M. Hong, et al. << Diabetes-Associated Sustained Activation of the Transcription Factor Nuclear Factor-KappaB >>. Diabetes 50, no 12 (decembre 2001): 2792-2808. https: / / doi.org / 10.2337 / diabetes.50.12.2792. Bongarzone, S., Savickas, V., Luzi, F., and Gee, AD (2017) Targeting the Receptor for Advanced Glycation Endproducts (RAGE): A Medicinal Chemistry Perspective. Journal of medicinal chemistry 60, 7213-7232. Brett, J., A. M. Schmidt, S. D. Yan, Y. S. Zou, E. Weidman, D. Pinsky, R. Nowygrod, M. Neeper, C. Przysiecki, A. Shaw and et al. (1993). "Survey of the distribution of a newly characterized receptor for advanced glycation end products in tissues." Am J Pathol 143(6): 1699-1712. Chavakis, T., Bierhaus, A., and Nawroth, P. P. (2004) RAGE (receptor for advanced glycation end products): a central player in the inflammatory response. Microbes and infection 6, 1219-1225 Dong, Hanbing, Yue Zhang, Yu Huang, et Hui Deng. << Pathophysiology of RAGE in Inflammatory Diseases >>. Frontiers in Immunology 13 (2022): 931473. https: / / doi.org / 10.3389 / fimmu.2022.931473. He, S., Gui, J., Xiong, K. et al. A roadmap to pulmonary delivery strategies for the treatment of infectious lung diseases. J Nanobiotechnol 20, 101, 2022 Healey, G. D., Pan-Castillo, B., Garcia-Parra, J., Davies, J., Roberts, S., Jones, E., Dhar, K., Nandanan, S., Tofazzal, N., Piggott, L., Clarkson, R., Seaton, G., Frostell, A., Fagge, T., McKee, C., Margarit, L., Conlan, R. S., and Gonzalez, D. (2019) Antibody drug conjugates against the receptor for advanced glycation end products (RAGE), a novel therapeutic target in endometrial cancer. J Immunother Cancer 7, 280 Hudson, Barry I., Angela M. Carter, Evis Harja, Anastasia Z. Kalea, Maria Arriero, Hojin Yang, Peter J. 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Claims

1. A VHH molecule of formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein the VHH molecule binds to the advanced glycation end product receptor (RAGE) on the surface of lung cells, and the VHH molecule: - A CDR1 sequence selected from Sequence ID No. 1, 5, 9, 13, or 17, or a variant thereof having at least 60% amino acid identity with any one of the aforementioned sequences over its entire length. - A CDR2 sequence selected from SEQ ID NOs: 2, 6, 10, 14, or 18, or a variant thereof having at least 60% amino acid identity with any one of the aforementioned sequences over its entire length, and / or - A CDR3 sequence selected from SEQ ID NOs: 3, 7, 11, 15, or 19, or a variant thereof having at least 60% amino acid identity with any one of the aforementioned sequences over its entire length. A VHH molecule possessing the following characteristics.

2. The VHH molecule according to claim 1, wherein the VHH molecule comprises SEQ ID NOs: 1, 2 and 3, or SEQ ID NOs: 5, 6 and 7, or SEQ ID NOs: 9, 10 and 11, or SEQ ID NOs: 13, 14 and 15, or SEQ ID NOs: 17, 18 and 19.

3. The VHH molecule according to any one of claims 1 and 2, wherein the VHH molecule comprises an amino acid sequence selected from any one of sequence numbers 4, 8, 12, 16, and 20.

4. The VHH molecule according to any one of claims 1 to 3, further comprising a tag and / or a linker.

5. The VHH molecule according to any one of claims 1 to 4, wherein the VHH molecule is humanized.

6. The VHH molecule according to any one of claims 1 to 5, wherein the VHH molecule binds to RAGE with an affinity (Kd) of 0.1 nM to 10 μM, preferably 1 nM to 10 μM.

7. A nucleic acid encoding a VHH molecule according to any one of claims 1 to 6.

8. A vector comprising the nucleic acid described in claim 7, preferably operably linked to a promoter.

9. Recombinant host cells comprising the nucleic acid described in claim 7, or the vector described in claim 8.

10. A conjugated compound comprising one or more VHH molecules according to any one of claims 1 to 6, conjugated to at least one additional compound.

11. The conjugate compound according to claim 10, wherein the at least one additional compound is a stabilizing group selected from an antibody or a fragment thereof, such as an Fc fragment, a VHH molecule, PEG, serum albumin protein, and a serum albumin-binding moiety.

12. The conjugate compound according to claim 10, wherein the at least one additional compound is a therapeutic, diagnostic, or imaging compound, or a vehicle comprising such a therapeutic, diagnostic, or imaging compound.

13. The conjugate compound according to claim 12, wherein the therapeutic compound is selected from peptides, polypeptides, proteins, antibodies, and nucleic acids.

14. The conjugate compound according to claim 12, wherein the vehicle is selected from a virus, a virus-like particle (VLP), a cell-derived vesicle (CDV), an exosome, a lipid vehicle, and a polymer vehicle, and is preferably a lipid nanoparticle (LNP), a micelle, or a liposome.

15. The conjugate compound according to claim 10, wherein the conjugate comprises i) one or more VHH molecules according to any one of claims 1 to 6, ii) a stabilizing group, iii) a therapeutic, diagnostic or imaging compound, and optionally iv) a vehicle, in any order.

16. A pharmaceutical composition comprising a conjugate compound according to any one of claims 10 to 15 and a pharmaceutically acceptable support.