Transferrin receptor binding molecules, their conjugates, and their use for the prevention or treatment of muscle diseases.

Abstract

The present invention relates to a variable domain (VHH) of a camelid heavy chain molecule that binds to TfR, and its use, for example, to transport a therapeutic molecule to muscle cells in a typically pathological state.

Description

[Technical Field]

[0001] The present invention relates to transferrin receptor (TfR) binding molecules, conjugate compounds involved in such TfR binding molecules, and their uses. More specifically, the present invention relates to variable domains (VHHs) of camelid heavy chain molecules that bind to TfR on the surface of muscle cells, and their use in preventive or therapeutic settings, for example, to transport nucleic acid molecules, particularly oligonucleotide therapeutic agents, into muscles. [Background technology]

[0002] In the muscular system, muscle tissue is classified into three distinct types: skeletal muscle tissue, cardiac muscle tissue, and smooth muscle tissue. Each type of muscle tissue in the human body has a unique structure and specific role. Muscle diseases generally involve muscle weakness or dysfunction that can lead to life-threatening complications. Many examples of such diseases, including various forms of muscular dystrophy and neuromuscular disorders, have been described for their characteristics. Muscular dystrophy is a group of genetic disorders in which muscles gradually weaken, leading to an increasing level of impairment. Various types of muscular dystrophy exist, each associated with the potential for ultimate loss of strength and deformation. These include Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), facioscapulohumeral muscular dystrophy (FSHD), Pompe disease, and familial hypertrophic cardiomyopathy. Many muscle diseases are monogenic disorders associated with gain-of-function or loss-of-function mutations, which can have dominant or recessive phenotypes. For example, activating mutations have been identified in genes encoding ion channels, structural proteins, metabolic proteins, and signaling proteins that contribute to muscle diseases. For instance, DMD is caused by a mutation in the DMD gene (Xp21.2) located on the short arm (p) of the X chromosome. Despite advances in our understanding of the genetic etiology of muscle diseases, effective treatment options remain extremely limited.

[0003] Neuromuscular disorders affect the nerves that control voluntary muscles and the nerves that transmit sensory information to the brain. These represent multifaceted abnormal conditions that can lead to death from complete muscle wasting and atrophy, and are often incurable or never curable. Neuromuscular disorders can affect the peripheral nervous system (PNS), muscle tissue, and central nervous system (CNS), and include spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), Charcot-Marie-Tooth disease (CMT), multiple sclerosis (MS), Huntington's disease (HD), and others.

[0004] Currently, there are no oligonucleotides under development for treating myopathy that act in muscle tissue after local CNS administration (e.g., intrathecal or intraventricular). While some oligonucleotides have been proposed to act on the CNS symptoms of such myopathy after local CNS administration, the lack of co-delivery to muscle tissue necessitates further systematic administration to address peripheral muscle symptoms (Ait Benichou et al., 2022; Bizot et al., 2020). RNA therapeutics are a new class of drugs designed to prevent and / or treat specific diseases using RNA-based molecules. These include a diverse group of oligonucleotide-based drugs, such as antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), double-stranded RNAs (dsRNAs), and microRNAs (miRNAs), which can be designed to selectively interact with biological targets in disease scenarios that are currently unaddressable or inadequately addressed by small molecule-based drugs or monoclonal antibodies. These represent a promising therapeutic strategy for regulating gene expression and addressing mutations in several pathological conditions, including myopathy. Furthermore, RNA-based therapies have the potential to modulate the entire disease pathway, thereby offering better treatment options that target the pathophysiological mechanisms of various disorders, potentially leading to better patient outcomes. Moreover, many RNA-based therapies have already been approved by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA), and an increasing number of treatments in various phases of clinical trials are demonstrating the efficacy of such RNA therapies for a variety of diseases. However, their anionic charge, as well as their sensitivity to ribonucleases present in both bloodstream and tissue, makes it difficult for therapeutic RNAs to effectively enter cells and function on their own. Delivery remains a central challenge in the therapeutic application of RNA therapies. RNA therapies are transported to their target cells using available delivery systems. Various strategies are employed, including direct conjugation to lipid particles (LNPs) and delivery agents (e.g., cholesterol).In recent years, significant progress has been made using N-acetylgalactosamine (GalNAc) as a target ligand for liver-specific asialoglycoprotein receptors (Nair JK et al., Tai, W, Debacker et al.). Indeed, it has been demonstrated that GalNAc-siRNA conjugates can effectively suppress liver-expressed genes. However, effective strategies for preventing or treating muscle diseases are still needed. [Overview of the project]

[0005] This invention relates to specific VHH molecules that favorably target transferrin receptors (TfRs) in muscle tissue, and their use for delivering various therapeutic agents to muscle. More specifically, the invention provides conjugate compounds comprising such VHH molecules that are optimized for muscle identification and for mediating the effective functional delivery of oligonucleotide therapeutic agents to muscle tissue. The invention demonstrates that the conjugate compounds of the invention can effectively accumulate in muscle, deliver the conjugated therapeutic agent to muscle tissue, and are therefore suitable for the effective prevention and treatment of muscle diseases.

[0006] Therefore, the object of the present invention relates to a VHH molecule that binds to TfR in muscle tissue.

[0007] A further object of the present invention relates to a conjugate compound comprising one or more VHH molecules that bind to TfR in muscle tissue.

[0008] A particular object of the present invention is a conjugate compound comprising (i) one or more VHH molecules of the formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and (ii) one or more oligonucleotides, wherein the VHH molecules bind to TfR on the surface of muscle cells.

[0009] Preferably, the muscle cells are skeletal cells, cardiomyocytes, or muscle cancer cells.

[0010] The preferred VHH molecules in the present invention bind to TfR with affinity (Kd) of 0.01 nM to 4 μM, or 0.01 nM to 2500 nM, or 0.01 nM to 1000 nM, or 0.01 nM to 500 nM, or 0.01 nM to 100 nM, or 0.1 nM to 4 μM, or 0.1 nM to 2500 nM, or 0.1 nM to 1000 nM, or 0.1 nM to 500 nM, or 0.1 nM to 100 nM.

[0011] In another embodiment, the preferred VHH molecule in the present invention binds to human, non-human primate, and / or rodent TfR1 on the surface of muscle cells.

[0012] In a particular embodiment, the VHH molecule in the present invention is CDR1 containing sequences selected from sequence numbers 1, 5, 9, 13, 17, 19, 67, 69, 125, 175, 179, 182, 184, 186, 190, 194, 198, 201, 205, 392, 410, 413, 426, 434, 437, 607, 610, 671-674, 710, 711, and / or CDR2 containing sequences selected from sequence numbers 2, 6, 10, 14, 21, 23, 71, 73, 75, 113, 115, 128, 160, 162, 164, 166, 169, 171, 176, 187, 191, 195, 199, 202, 206, 416, 419, 431, 608, 611, and 712, and / or • Includes a CDR3 containing sequences selected from sequence numbers 3, 7, 11, 15, 25, 27, 29, 31, 33, 77, 79, 81, 83, 85, 117, 119, 121, 123, 177, 180, 188, 192, 196, 200, 203, 207, 452, 455, 609, 612, 713-715, and 741-744.

[0013] In other specific embodiments, the VHH molecule in the present invention is sequence number 1, 2, and 3; or sequence number 5, 6, and 7; or sequence number 9, 10, and 11; or sequence number 13, 14, and 15; sequence number 17, 2, and 3; or sequence number 19, 2, and 3; or sequence number 1, 21, and 3; or sequence number 1, 23, and 3; or sequence number 1, 2, and 25; or sequence number 1, 2, and 27; or sequence number 1, 2, and 29; or sequence number 1, 2, and 31; or sequence number 1, 2, and 33; or sequence number Sequence numbers 67, 2, and 3; or Sequence numbers 69, 2, and 3; or Sequence numbers 1, 71, and 3; or Sequence numbers 1, 73, and 3; or Sequence numbers 1, 75, and 3; or Sequence numbers 1, 2, and 77; or Sequence numbers 1, 2, and 79; or Sequence numbers 1, 2, and 81; or Sequence numbers 1, 2, and 83; or Sequence numbers 1, 2, and 85; or Sequence numbers 392, 2, and 3; or Sequence numbers 1, 113, and 3; or Sequence numbers 1, 115, and 3; or Sequence numbers 1, 2, and 117; or Sequence numbers 1, 2, and 119; or SEQ ID NOs: 1, 2, and 121; or SEQ ID NOs: 1, 2, and 123; or SEQ ID NOs: 125, 2, and 3; or SEQ ID NOs: 17, 73, and 3; or SEQ ID NOs: 17, 128, and 3; or SEQ ID NOs: 5, 160, and 7; or SEQ ID NOs: 5, 162, and 7; or SEQ ID NOs: 5, 164, and 7; or SEQ ID NOs: 5, 166, and 7; or SEQ ID NOs: 9, 169, and 11; or SEQ ID NOs: 9, 171, and 11; or SEQ ID NOs: 175, 176, and 177; or SEQ ID NOs: 179, 176, and 180 ; or SEQ ID NOs. 182, 176, and 177; or SEQ ID NOs. 184, 176, and 177; or SEQ ID NOs. 186, 187, and 188; or SEQ ID NOs. 190, 191, and 192; or SEQ ID NOs. 194, 195, and 196; or SEQ ID NOs. 198, 199, and 200; or SEQ ID NOs. 201, 202, and 203; or SEQ ID NOs. 205, 206, and 207; or SEQ ID NOs. 410, 6, and 7; or SEQ ID NOs. 413, 6, and 7; or SEQ ID NOs. 5, 416, and 7; or SEQ ID NOs. 5, 419, and 7;or SEQ ID NOs. 426, 6, and 7; or SEQ ID NOs. 5, 431, and 7; or SEQ ID NOs. 434, 6, and 7; or SEQ ID NOs. 437, 6, and 7; or SEQ ID NOs. 5, 6, and 452; or SEQ ID NOs. 5, 6, and 455; or SEQ ID NOs. 607, 608, and 609; or SEQ ID NOs. 610, 611, and 612; or SEQ ID NOs. 671, 2, and 3; or SEQ ID NOs. 672, 2, and 3; or SEQ ID NOs. 673, 6, and 7; or SEQ ID NOs. 674, 6, and 7; or SEQ ID NOs. 1, 2, and 713; or SEQ ID NOs. 5, 6, and 714; or SEQ ID NOs. 67 Includes 4, 164, and 7; or SEQ ID NOs. 710, 6, and 7; or SEQ ID NOs. 5, 6, and 715; or SEQ ID NOs. 674, 712, and 7; or SEQ ID NOs. 711, 6, and 7; or SEQ ID NOs. 673, 6, and 741; or SEQ ID NOs. 673, 6, and 742; or SEQ ID NOs. 673, 6, and 743; or SEQ ID NOs. 673, 431, and 741; or SEQ ID NOs. 673, 431, and 742; or SEQ ID NOs. 673, 431, and 743; or SEQ ID NOs. 673, 6, and 7; or SEQ ID NOs. 674, 6, and 7.

[0014] In a further specific embodiment, the VHH molecule comprises an amino acid sequence selected from one of the following: SEQ ID NOs: 213-271, 273-299, 412, 415, 418, 421, 423, 425, 428, 430, 433, 436, 439, 441, 443, 445, 447, 449, 451, 454, 457, 613-615, 675-678, 701-709, and 766-786, wherein the aforementioned amino acid sequences optionally include tags and / or linkers.

[0015] In a further specific embodiment, the VHH molecule comprises an amino acid sequence selected from any one of SEQ ID NOs: 214, 273, 276 - 284, 412, 415, 418, 421, 423, 425, 428, 430, 433, 436, 439, 441, 443, 445, 447, 449, 451, 454, 457, 677, 678, 702 - 709, and 766 - 786. In a further specific embodiment, the VHH molecule comprises an amino acid sequence selected from any one of SEQ ID NOs: 213, 216 - 271, 274, 275, 675, 676, and 701. In a further specific embodiment, the VHH molecule comprises an amino acid sequence selected from any one of SEQ ID NOs: 215, and 285 - 299. In a further specific embodiment, the VHH molecule comprises an amino acid sequence selected from any one of SEQ ID NOs: 613 - 615. In a further specific embodiment, the VHH molecule is humanized and is preferably selected from SEQ ID NOs: 87 - 92, 130 - 149, 152 - 154, 236 - 241, 252 - 271, 273 - 275, 752 - 765, and 773 - 786.

[0016] In other specific embodiments, the oligonucleotide in the present invention is selected from any single - stranded or double - stranded oligonucleotide, such as small interfering RNA (siRNA), small activating RNA (saRNA), gapmer, antisense oligonucleotide (ASO), shRNA, miRNA, aptamer RNA, and bridged nucleic acid (BNA), etc.

[0017] Another object of the present invention relates to a conjugate compound in which the VHH molecule is conjugated to an oligonucleotide by a covalent or non - covalent bond, either directly or via at least one conjugation linker. The conjugate compound may further comprise at least one additional compound.

[0018] In certain embodiments, additional compounds include a half-life extension moiety, or a stabilizing group, or a scaffold such as an antibody or a fragment thereof (such as an Fc fragment), a VHH molecule, PEG, a serum albumin protein, and a serum albumin binding moiety, etc., preferably an Fc fragment. Preferably, the Fc fragment is an Fc heterodimer comprising a modified Fc having the sequence of SEQ ID NO: 664 in the knob arm and a modified Fc having the sequence of SEQ ID NO: 665 in the hole arm.

[0019] Another object of the present invention relates to a pharmaceutical composition comprising a conjugate compound as described herein and a pharmaceutically acceptable support, carrier, or excipient.

[0020] The VHH molecules, conjugate compounds, and pharmaceutical compositions of the present invention can be administered by any conventional route, preferably parenterally, systemically, intravenously, intramuscularly, subcutaneously, intracerebrally, intraventricularly, or intrathecally. The present invention can be used in any mammalian subject, particularly a human subject. This is suitable for the treatment of any muscle disease or neuromuscular disease, such as myopathy, cardiomyopathy, muscular dystrophy (such as DMD, BMD, FSHD, Pompe disease, or familial hypertrophic cardiomyopathy, etc.), neuromuscular disease (such as ALS, SMA, MS, HD, CMT, etc.), or muscle cancer (such as rhabdomyosarcoma or leiomyosarcoma, etc.).

Brief Description of the Drawings

[0021] [Figure 1] Figure 1 shows the concentration-dependent binding and apparent binding affinity values (Ki / app) of VHH-oligo conjugates (such as B8-siSOD1m, C5-siSOD1m, B8-MALAT1-ASO, B8-siSOD1h, or C5-siSOD1h, etc.) and free VHH (B8 or C5) under competition with a reference fluorescent TfR-binding VHH for mouse, cynomolgus monkey, or human TfR expressed in mouse Neuro-CHO cells stably expressing cynomolgus monkey TfR, or human MCF-7 cells, respectively. [Figure 2]Figure 2 shows the concentration-dependent downregulation of mRNA or lncRNA levels after free uptake in human MCF-7 cells or MIA PaCa-2 cells of either (A) a TfR-binding VHH-siRNA conjugate targeting human SOD1 mRNA (VHH-siSOD1h, where VHH is B8, C5, or C5V8) or an unbinding conjugate (C5neg-siSOD1h), or (B) a TfR-binding VHH-ASO conjugate targeting human and mouse MALAT-1 long non-coding RNA (VHH-MALAT1-ASO, where VHH is B8). [Figure 3] Figure 3 shows the potential downregulation of mRNA or lncRNA of TfR-binding VHH-oligoconjugates after free uptake in mouse Neuro-2A cell lines. (A) Figure 3A shows the concentration-dependent downregulation of SOD1 mRNA levels obtained with TfR-binding VHH-siRNA conjugates (VHH-siSOD1m, VHH is C5) or unbinding conjugates (C5neg-siSOD1m). (B) Figure 3B shows the downregulation of SOD1 mRNA levels obtained for various examples of TfR-binding VHH-siRNA conjugates targeting mouse SOD1 mRNA (i.e., VHH-siSOD1m or VHH-thiol-Mal-siSOD1m or VHH-ΔHis-siSOD1m, where VHH is C5, B8, B8h1, C5V1, C5V13, C5h18, C5h19, or C5V7 or including them), and the apparent binding affinity values ​​(Ki / app) of the tested conjugates to TfR evaluated in mouse Neuro-2A cells as shown in Figure 1. (C) Figure 3C shows the downregulation of MALAT-1 lncRNA levels obtained with the VHH-MALAT1-ASO conjugate (VHH-MALAT1-ASO, VHH is B8) targeting human and mouse MALAT-1 long non-coding RNA. [Figure 4]Figure 4 shows the muscle-specific downregulation of mouse SOD1 mRNA levels after a single systemic (intravenous IV or subcutaneous SC) administration of a VHH-siRNA conjugate (VHH is C5 or B8) in wild-type C57Bl / 6 mice. [Figure 5] Figure 5 shows the TfR-specific and muscle-specific downregulation of mouse SOD1 mRNA levels after a single subcutaneous administration of the TfR-conjugated VHH-siSOD1 conjugate (VHH is B8) in wild-type C57Bl / 6 mice. [Figure 6] Figure 6 shows the dose-dependent downregulation of mouse SOD1 mRNA levels in skeletal muscle (gastrocnemius) or cardiac muscle after a single subcutaneous administration of the TfR-conjugated VHH-siSOD1m conjugate (VHH is B8) in wild-type C57Bl / 6 mice. [Figure 7] Figure 7 shows the time course of mouse SOD1 mRNA downregulation in muscle tissue (e.g., gastrocnemius, diaphragm, cardiac tissue) after a single subcutaneous (SC) administration of the TfR-bound VHH-siSOD1m conjugate (VHH is B8) in wild-type C57Bl / 6 mice. [Figure 8] Figure 8 shows the concentration-dependent binding and apparent binding affinity (Ki / app) of the TfR-binding VHH-hFc-siSOD1 conjugate (VHH is C5 or B8) to mouse and human TfR expressed in mouse Neuro-2A cells and human MCF-7 cells, respectively, under competition with reference fluorescent TfR-binding VHH. [Figure 9] Figure 9 shows the concentration-dependent downregulation of human or mouse SOD1 mRNA levels after free uptake of a TfR-conjugated VHH-hFc-siSOD1 conjugate (VHH is C5 or B8) targeting human or mouse SOD1 mRNA in (A) human breast MCF-7 cells and (B) mouse Neuro-2A cells, respectively. [Figure 10]Figure 10 shows the muscle-specific and dose-dependent downregulation of mouse SOD1 mRNA levels, estimated ED50 values ​​in muscle tissue, and maximum downregulation effect (Max KD) in wild-type C57Bl / 6 mice after (A) a single intravenous administration of the TfR-bound VHH-hFc-siSOD1m conjugate (VHH is C5) or (B) a single subcutaneous administration of the TfR-bound VHH-hFc-siSOD1m-5'VP conjugate (VHH is C5). [Figure 11] Figure 11 shows the muscle-specific downregulation of mouse SOD1 mRNA levels after a single subcutaneous (SC) administration of the TfR-binding VHH-siSOD1m conjugate (VHH is C5 or B6) in hTfR1+ / +-KI mice. Downregulation of mouse SOD1 mRNA levels was not observed in any of the test samples analyzed after treatment with non-conjugated siSOD1m. Both hTfR-binding conjugates induced similar potent and muscle-tissue-selective effects, showing approximately 70–80% knockdown in the gastrocnemius and diaphragm, and approximately 40–50% knockdown in the myocardium, while showing no effect in the liver or lungs. [Figure 12] Figure 12 shows the time course of SOD1 mRNA downregulation in various muscle tissues of Anubis baboons (such as gastrocnemius, quadriceps, or anterior tibialis muscle tissue) after a single intravenous administration of the TfR-linked VHH-siSOD1h conjugate (VHH is C5). [Figure 13]Figure 13 shows the general structure of the VHH-oligo conjugate of the present invention, which may consist of (A)(i) an oligo portion that may be a drug based on any oligonucleotide such as single-stranded ASO or double-stranded siRNA, (ii) VHH, and (iii) a linking portion that may consist of a half-life extension component in which the oligo and VHH are linked at two different sites, as well as (B~E) detailed structures of some of the conjugates evaluated in the experimental section, such as the VHH-siRNA conjugate (B), VHH-hFc-siRNA conjugate (C), VHH-thiol-Mal-siRNA (D), or VHH-ASO conjugate (E). [Figure 14] Figure 14 shows an example of a conjugation strategy to produce a VHH-oligo(A) or heterodimeric VHH-hFc-oligo(B) conjugate with a stable linker. Such an overall conjugation strategy involves convergent synthesis by (i) site-specific introduction of an azide linker to VHH(A) or heterodimeric VHH-hFc(B), and (ii) parallel modification of oligonucleotides to introduce a constrained alkyne group complementary to the azide functional group. In the final step, both the functionalized azide-VHH(A) or VHH-hFc-azide(B) and the alkyne-oligo precursor are linked together, preferably using a copper-free click reaction, to produce a VHH-oligo or VHH-hFc-oligo conjugate with a stable linker. [Figure 15] Figure 15 shows the downregulation of mouse SOD1 mRNA levels in muscle tissue of B-hTfR mice after a single intraventricular (ICV) administration of the TfR-bound VHH-siSOD1 conjugate (VHH is B8h1 or B8V32) compared with the lipophilic C16(palmitic acid)-siSOD1 conjugate or the unbound C5neg-siSOD1 conjugate; VHH-siSOD1m-5'VP, single ICV in hTfR1+ / + mice, 7 days. [Figure 16]Figure 16 shows the downregulation of mouse SOD1 mRNA levels in muscle tissue of B-hTfR mice after a single subcutaneous (SC) administration of a TfR-conjugated VHH-hFc-siSOD1 conjugate (VHH is E8, C10a, C5, C5V30, B8V31, B8V32, C5h9, C5V5, or C5h18) at a dose of 4.5 mg / kg (siRNA molar equivalent). The binding affinity of each conjugate tested to human TfR was evaluated using surface plasmon resonance; VHH-hFc-siSOD1m-5'VP, 4.5 mg / kg equivalent siRNA, single SC in hTfR1+ / + mice, 14 days. [Figure 17] Figure 17 shows the downregulation of mouse SOD1 mRNA levels in muscle tissue of B-hTfR mice after multiple IV bolus injections (3 times every other day (Q2D × 3)) of TfR-bound VHH-hFc-siSOD1 conjugates (VHH being B8V40, B8V32, B8V31, or B8V31h5) at 1.5 mg / kg (siRNA molar equivalent). The binding affinity of each conjugate tested to human and rhesus / cynomolgus monkey TfR was evaluated using surface plasmon resonance; VHH-hFc-siSOD1m-5'VP, 1.5 mg / kg equivalent siRNA, 3 IV injections every other day for 14 days in B-hTfR mice. [Modes for carrying out the invention]

[0022] The present invention provides a novel transferrin receptor (TfR) conjugate that can be used to deliver molecules such as therapeutic agents, imaging agents, or diagnostic agents to muscle. More specifically, the present invention discloses an improved VHH molecule that binds to TfRs in muscle tissue, and its use. Thus, TfR-targeted drugs are particularly suitable for the treatment of any muscle or neuromuscular disease, such as myopathy, cardiomyopathy, muscular dystrophy (DMD, BMD, FSHD, Pompe disease, or familial hypertrophic cardiomyopathy, etc.), and neuromuscular diseases (ALS, SMA, MS, HD, or CMT, etc.).

[0023] TfRs are involved in iron transport to cells and organs via their ligand, transferrin (Tf). While this receptor is abundant in blood cells and lungs (Chan and Gerhardt), it has been shown to be highly expressed in the brain endothelium (Jefferies et al., Pardridge et al.). This receptor is used to deliver pharmacological drugs across the blood-brain barrier (Johnsen et al., International Publication 2012 / 075037, International Publication 2016 / 208695, International Publication 2020 / 144233, etc.). Furthermore, TfRs have been described as playing a crucial role in myogenesis (Ying Li et al.). In addition to its role in myogenesis, TfRs play significant functional roles in iron homeostasis and systemic metabolism in muscle tissue (Ying Li et al., Barrientos T. et al., Xu W. et al.). TfRs are involved in the uptake of iron transported by their transferrin ligands and in regulating cell proliferation (Neckers and Trepel 1986, Ponka and Lok 1999).

[0024] There are two types of transferrin receptors: TfR1 and its homologous receptor TfR2, which is mainly expressed in the liver. In the context of this invention, the term TfR is used to refer to the TfR1 homolog. TfR is a type II homodimeric transmembrane glycoprotein consisting of two identical 90 kDa subunits linked by two disulfide crosslinks (Jing and Trowbridge, 1987; McClelland et al., 1984). Each monomer has a short N-terminal cytoplasmic domain of 61 amino acids containing a YTRF (tyrosine-threonine-arginine-phenylalanine) internal translocation motif, a single hydrophobic transmembrane moiety of 27 amino acids, and a large C-terminal extracellular domain of 670 amino acids containing a trypsin cleavage site and a transferrin binding site (Aisen, 2004). Each subunit is capable of binding to a transferrin molecule. The extracellular domain has one O-glycanization site and three N-glycanization sites, the latter of which are particularly important for proper folding and transport of the receptor to the cell surface (Hayes et al., 1997). The intramembrane domain also contains palmitoylation sites, which likely fix the receptor and enable its endocytosis (Alvarez et al., 1990, Omary and Trowbridge, 1981). Furthermore, an intracellular phosphorylation site is present, but its function is unknown and it does not play a role in endocytosis (Rothenberger et al., 1987).

[0025] TfR receptors are expressed at high levels by hyperproliferative cells, whether healthy or neoplastic (Gatter et al., 1983). Numerous studies have shown higher levels of TfR expression in cancer cells compared to healthy cells. Thus, conditions such as breast cancer (Yang et al., 2001), glioma (Prior et al., 1990), lung adenocarcinoma (Kondo et al., 1990), chronic lymphocytic leukemia (Das Gupta and Shah, 1990), or non-Hodgkin lymphoma (Habeshaw et al., 1983) show increased TfR expression associated with tumor malignancy and disease stage or prognosis. Therefore, TfR-targeted drugs may be suitable for the treatment of cancer, particularly muscle cancers such as rhabdomyosarcoma and leiomyosarcoma.

[0026] The inventors used purified membrane preparations from cells expressing high levels of hTfR and mTfR to construct and select VHH molecules, particularly those that bind to both human and non-human TfR. The inventors also demonstrated that when these VHH molecules are fused to oligonucleotides such as siRNA or ASO, they retain their TfR-binding ability and are effectively delivered in vivo to muscle cells and other cell types. The VHH molecules exhibit appropriate levels of affinity and specificity to undergo appropriate endocytosis following TfR binding. Therefore, the present invention provides novel TfR-binding molecules equivalent to useful agents for drug targeting of muscle. More specifically, the inventors demonstrated that the conjugate of the present invention, comprising VHH molecules and oligonucleotides such as siRNA, binds to TfR in various muscle tissues, in wild-type mice expressing mouse TfR1, or in humanized hTfR1 expressing human TfR1. + / +In mice and non-human primates, we demonstrated that the TfR-binding conjugates mediated targeted mRNA downregulation in vivo. For example, the TfR-binding conjugates in this invention induced potent and muscle-tissue-selective effects, achieving over 70% mRNA knockdown in the gastrocnemius, diaphragm, and cardiac muscle. Among the strategies evaluated for the delivery of RNA therapeutics to their target organs / tissues is receptor-mediated transcellular transport (RMT), a physiological process in which ligands bind to their receptors expressed in organs / tissues. Interestingly, a single systemic dose of the TfR-binding VHH-siRNA conjugate induced potent and prolonged downregulation of target mRNA levels in all muscle tissues tested in mice and non-human primates (including gastrocnemius, quadriceps, and tibialis anterior), with 60% knockdown observed for over 3 months in non-human primates.

[0027] Overall, the inventors demonstrated the interspecies capabilities of the TfR-binding VHH-siRNA conjugate in rodents / NHPs / humans, in vivo in skeletal muscle and cardiomyocyte tissues, at low therapeutic doses, mediating TfR-dependent binding, functional uptake, and long-term target mRNA downregulation.

[0028] Furthermore, the inventors have shown that when the VHH molecule in the present invention is conjugated to an RNA therapeutic agent (such as siRNA or ASO) via an antibody or antibody fragment scaffold, such as the human IgG1 Fc region, it retains TfR binding ability in vitro and exhibits potent, muscle-selective targeting and functional uptake properties in vivo at low doses via systemic administration (such as intravenous (IV) or subcutaneous (SC) administration).

[0029] Furthermore, the inventors demonstrated that the conjugate of the present invention exhibits the potential for potent functional delivery at low doses in the muscle tissue of hTfR-expressing mice. Interestingly, the inventors observed that several VHH mutants and conjugates showed remarkably similar binding affinity (less than a twofold difference) between human TfR and non-human primate (rhesus / cynomolgus macaque) TfR, thus facilitating a smooth transition from preclinical settings in non-human primates to clinical studies in humans.

[0030] Surprisingly, the inventors have also demonstrated for the first time that the conjugate in this invention can also be functionally delivered to muscle tissue after local CNS administration (such as intracerebral, intraventricular (ICV), or intrathecal (IT) administration), at doses similar to those used for systemic muscular delivery. Therefore, the TfR-conjugated VHH-oligonucleotide conjugate in this invention has the potential to address myofascial and neuromuscular disorders not only by systemic administration but also by local CNS administration. Furthermore, the inventors have demonstrated for the first time that the TfR-binding VHH-oligonucleotide conjugate in the present invention can be used to simultaneously target the muscular components (particularly muscle cells) and neuronal components (particularly nervous system cells) of various disorders, such as muscle diseases preferably selected from myopathy, cardiomyopathy, muscular dystrophy (DMD, BMD, FSHD, Pompe disease, or familial hypertrophic cardiomyopathy, etc.), or neuromuscular disorders such as spinal muscular atrophy, amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, multiple sclerosis, or Huntington's disease, because the conjugate in the present invention is functionally delivered to both muscle and nervous system tissues after topical CNS (IT or ICV) administration and can simultaneously bind to TfR on the surface of both muscle cells and nervous system cells after topical CNS administration. Therefore, the conjugate in the present invention can be used to address and treat muscle and neuromuscular disorders not only by systemic administration but also by topical CNS (IT or ICV) administration.

[0031] Therefore, the object of the present invention relates to a VHH molecule, wherein the VHH molecule binds to TfRs in both human and non-human organisms (e.g., non-human primates (NHPs) or rodents such as rats or mice). Preferably, the VHH binds to TfR-expressing muscle tissue. The present invention also relates to conjugates containing such VHH, the manufacture of the same, compositions containing the same, and the use of the same.

[0032] definition Unless otherwise defined herein, all scientific and technical terms used in connection with the present invention shall have the same meaning as those commonly understood by those skilled in the art.

[0033] As used herein, the terms “treatment,” “to treat,” or “to treat” mean any action intended to improve a patient’s health condition, such as treating, suppressing, preventing, and delaying a disease, such as a muscular disease, neuromuscular disease, or cancer of the muscle, or at least one symptom of a muscular disease, neuromuscular disease, or cancer. This includes both curative and / or preventive treatments for muscular diseases, neuromuscular diseases, or cancer. In the context of cancer, this includes, among other things, symptom relief, reduction of inflammation, suppression of cancer cell growth, and / or reduction of tumor size. For example, in the case of cancer, the response to treatment is measured by a set of criteria, respectively, established by the National Cancer Institute and the Food and Drug Administration for the approval of new drugs, and includes reduced cachexia, increased survival, extended time to tumor growth, decreased tumor burden, reduced tumor load, and / or extended time to tumor metastasis, extended time to tumor recurrence, tumor response, complete remission, partial remission, stable disease, progressive disease, progression-free survival, and overall survival (Johnson et al., J.Clin.Oncol., 2009;21(7):1404-1411).

[0034] In this specification, “therapeutic dose” refers to the dose that produces a therapeutic effect under given conditions and administration schedule. This is typically the average dose of an active substance administered to significantly improve some of the symptoms associated with a disease or pathological condition. For example, when treating cancer of muscle (such as rhabdomyosarcoma or leiomyosarcoma) or other tissue, a condition, lesion, or disorder affecting muscle (such as a muscular disease or neuromuscular disease), a dose of an active substance that reduces, prevents, delays, eliminates, or stops one of the causes or symptoms of the disease or disorder may be therapeutically effective. A “therapeutic dose” of an active substance may not necessarily cure a muscular disease or disorder, but it may provide treatment for the disease or disorder by delaying, preventing, or preventing its onset, suppressing its symptoms, altering its duration or making it less severe, or accelerating the patient's recovery.

[0035] As used herein, the “VHH molecule” corresponds to the variable domain of a camelid antibody that naturally lacks a light chain and consists only of a heavy chain. VHHs have an extremely small molecular weight of approximately 12–15 kDa. They contain a single-chain molecule capable of binding to its congener antigen using a single domain. The antigen-binding surface of a VHH is usually convex (or protruding) compared to that of a normal antibody, which is usually flat or concave. More specifically, a VHH consists of four framework regions (or FRs) whose sequence and structure are defined as conserved, as well as three complementarity-determining regions (or CDRs) that exhibit high variability in both sequence content and structural conformation, which are involved in antigen binding and give antigen specificity. Compared to the VH of a normal human antibody, there are slight amino acid substitutions in the FR2 region and the complementarity-determining region (CDR) of a VHH. 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) to make the overall structure more hydrophilic, contributing to high stability, solubility, and agglutination resistance. The VHH molecules in this invention are polypeptides containing (or consisting of, or substantially consisting of) the antigen-binding domain of a heavy-chain-only antibody (HcAb). As used herein, the term "and / or" is interpreted as a specific disclosure of each of the two identified features or components, with or without the other. For example, "A and / or B" is interpreted as the specific disclosure of each, as (i) A, (ii) B, and (iii) A and B are each presented separately.

[0036] The terms "a" or "an" can refer to one or more of the elements they modify unless the context makes it clear that one or more of the elements being described are being referred to (for example, "a VHH molecule" can mean one or more VHH molecules).

[0037] VHH molecule To produce VHH molecules with suitable properties, the inventors tested over 2000 TfR-binding VHHs from a library of VHHs produced by llama immunization with TfR immunogens. Following the above-mentioned analysis of clones for binding affinity and specificity, the inventors further selected approximately 450 clones, all of which were sequenced and compared. Furthermore, VHHs with controlled / enhanced binding properties were produced by mutagenesis or humanization. The sequences of the relevant domains and preferred VHHs are listed in the Experiments section and the sequence listings. The properties of the VHHs and their conjugates are also shown in the Experiments section.

[0038] The VHH molecule of the present invention is typically, The formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 is included or consists of the formula In the formula, FRn refers to the framework domain, and CDRn refers to the complementarity determination domain.

[0039] In certain embodiments, the VHH molecule of the present invention comprises at least 60%, particularly at least 65%, 70%, or 75%, for example at least 80%, of the total length of any one of the sequences described above: SEQ ID NOs: 1, 5, 9, 13, 17, 19, 67, 69, 125, 175, 179, 182, 184, 186, 190, 194, 198, 201, 205, 392, 410, 413, 426, 434, 437, 607, 610, 671-674, 710, or 711, or any one of the sequences described above. Alternatively, it comprises a CDR1 domain comprising an amino acid sequence selected from a variant thereof that has 85% amino acid identity, preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity (the preferred percentage of identity in a particular sequence is preferably the percentage corresponding to an integer number of amino acids) and retains TfR 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, 17, 19, 67, 69, 125, 175, 179, 182, 184, 186, 190, 194, 198, 201, 205, 392, 410, 413, 426, 434, 437, 607, 610, 671-674, 710, 711, or variants thereof having several amino acid modifications, for example, at least three amino acid modifications, preferably as many as three or two amino acid modifications, and in a particular embodiment as many as one amino acid modification. 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 a computer program such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1996, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman, S.B. and Wunsch, CD, (1970), Journal of Molecular Biology, 48, 443-453). The % identity between two sequences refers to the identity over the entire length of the sequences mentioned above. As shown above, the preferred percentage of identity in a particular sequence is preferably the reference sequence (e.g., sequence numbers 1, 5, 9, 13, 17, 19, 67, 69, 125, 175, 179, 182, 184, 186, 190, 194, 198, 201, 205, 392, 410, 413, 426, 434, or 437, 607, 610, 671-674, 710, 711), or any other reference sequence specified herein, e.g., sequence numbers 2, 6, 10, 14, 21, 23, 71, 73, These are percentages corresponding to the integer number of amino acids in both 75, 113, 115, 128, 160, 162, 164, 166, 169, 171, 176, 187, 191, 195, 199, 202, 206, 608, 611, 712, 3, 7, 11, 15, 25, 27, 29, 31, 33, 77, 79, 81, 83, 85, 117, 119, 121, 123, 177, 180, 188, 192, 196, 200, 203, 207, 609, 612, or 713-715, as well as their variants.

[0040] Specific examples of the VHH molecules of the present invention include CDR1 sequences containing or substantially consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 19, 67, 69, 125, 175, 179, 182, 184, 186, 190, 194, 198, 201, 205, 392, 410, 413, 426, 434, 437, 607, 610, 671-674, 710, or 711.

[0041] In further specific embodiments, the VHH molecule of the present invention is at least 60% of its total length, particularly less than any one of the sequences described above: SEQ ID NOs: 2, 6, 10, 14, 21, 23, 71, 73, 75, 113, 115, 128, 160, 162, 164, 166, 169, 171, 176, 187, 191, 195, 199, 202, 206, 416, 419, 431, 608, 611, or 712, or any one of the sequences described above. The CDR2 domain comprises an amino acid sequence selected from a variant having at least 65%, 70%, or 75%, for example, at least 80% or 85% amino acid identity, preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity, and retaining TfR binding ability. The preferred VHH molecules of the present invention include a CDR2 domain having an amino acid sequence selected from SEQ ID NOs: 2, 6, 10, 14, 21, 23, 71, 73, 75, 113, 115, 128, 160, 162, 164, 166, 169, 171, 176, 187, 191, 195, 199, 202, 206, 416, 419, 431, 608, 611, or 712, or from variants thereof having several amino acid modifications, for example, at least three amino acid modifications, preferably as many as three or two amino acid modifications, and as many as one amino acid modification in a particular embodiment.

[0042] Specific examples of the VHH molecules of the present invention include CDR2 sequences containing or substantially consisting of SEQ ID NOs: 2, 6, 10, 14, 21, 23, 71, 73, 75, 113, 115, 128, 160, 162, 164, 166, 169, 171, 176, 187, 191, 195, 199, 202, 206, 416, 419, 431, 608, 611, or 712.

[0043] In further specific embodiments, the VHH molecule of the present invention is at least 60% of its total length, particularly at least, of the sequence numbers 3, 7, 11, 15, 25, 27, 29, 31, 33, 77, 79, 81, 83, 85, 117, 119, 121, 123, 177, 180, 188, 192, 196, 200, 203, 207, 452, 455, 609, 612, or 713-715, or 741-744, or any one of the sequences described above. The CDR3 domain comprises an amino acid sequence selected from a variant thereof that has 65%, 70%, or 75%, for example, at least 80% or 85% amino acid identity, preferably at least 80%, more preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity, and that retains TfR binding ability. The preferred VHH molecules of the present invention include a CDR3 domain having an amino acid sequence selected from the following variants: SEQ ID NOs: 3, 7, 11, 15, 25, 27, 29, 31, 33, 77, 79, 81, 83, 85, 117, 119, 121, 123, 177, 180, 188, 192, 196, 200, 203, 207, 452, 455, 609, 612, 713-715, or 741-744, or several amino acid modifications, for example, at least three amino acid modifications, preferably as many as three or two amino acid modifications, and in a particular embodiment as many as one amino acid modification.

[0044] Specific examples of the VHH molecules of the present invention include CDR3 sequences containing or substantially consisting of SEQ ID NOs: 3, 7, 11, 15, 25, 27, 29, 31, 33, 77, 79, 81, 83, 85, 117, 119, 121, 123, 177, 180, 188, 192, 196, 200, 203, 207, 452, 455, 609, 612, 713-715, or 741-744.

[0045] In further specific embodiments, the VHH molecule of the present invention is • Sequence numbers 1, 5, 9, 13, 17, 19, 67, 69, 125, 175, 179, 182, 184, 186, 190, 194, 198, 201, 205, 392, 410, 413, 426, 434, 437, 607, 610, 671-674, 710, or 711, or any one of the sequences listed above, at least 60% of its total length, especially at least 65%, 70%, or 7 A CDR1 domain comprising or consisting of an amino acid sequence selected from a variant having 5%, for example, at least 80% or 85% amino acid identity, preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity, more preferably at least 95% amino acid identity, and • Sequence numbers 2, 6, 10, 14, 21, 23, 71, 73, 75, 113, 115, 128, 160, 162, 164, 166, 169, 171, 176, 187, 191, 195, 199, 202, 416, 419, 431, 206, 608, 611, or 712, or for any one of the sequences mentioned above, at least 60% of its total length, especially at least 65%, 70%, or 7 A CDR2 domain comprising or consisting of an amino acid sequence selected from a variant having 5%, for example, at least 80% or 85% amino acid identity, preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity, more preferably at least 95% amino acid identity, and • Sequence numbers 3, 7, 11, 15, 25, 27, 29, 31, 33, 77, 79, 81, 83, 85, 117, 119, 121, 123, 177, 180, 188, 192, 196, 200, 203, 207, 452, 455, 609, 612, 713-715, or 741-744, or any one of the sequences mentioned above, at least 60% of its total length, especially at least 65%, 70%, or A CDR3 domain comprising an amino acid sequence selected from variants having 75%, for example, at least 80% or 85% amino acid identity, preferably at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity, more preferably at least 95% amino acid identity, The aforementioned VHH has TfR binding ability.

[0046] 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, 17, 19, 67, 69, 125, 175, 179, 182, 184, 186, 190, 194, 198, 201, 205, 392, 410, 413, 426, 434, 437, 607, 610, 671-674, 710, 711, and their variants having at most three, two, or one amino acid modification, • CDR2 domains having amino acid sequences selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 21, 23, 71, 73, 75, 113, 115, 128, 160, 162, 164, 166, 169, 171, 176, 187, 191, 195, 199, 202, 206, 416, 419, 431, 608, 611, 712, and their variants having at most three, two, or one amino acid modification, and The CDR3 domain has an amino acid sequence selected from the group consisting of sequence numbers 3, 7, 11, 15, 25, 27, 29, 31, 33, 77, 79, 81, 83, 85, 117, 119, 121, 123, 177, 180, 188, 192, 196, 200, 203, 207, 452, 455, 609, 612, 713-715, 741-744, and their variants having at most three, two, or one amino acid modification.

[0047] In a more preferred embodiment, the VHH molecule of the present invention comprises CDR1, CDR2, and CDR3, wherein the domains of CDR1, CDR2, and CDR3 described above are, • Sequence IDs 1, 2, and 3, or, • Sequence IDs 17, 2, and 3, or, • Sequence IDs 19, 2, and 3, or • Sequence IDs 67, 2, and 3, or, • Sequence IDs 69, 2, and 3, or • Sequence IDs 1, 21, and 3, or, • Sequence IDs 1, 23, and 3, or, • Sequence numbers 1, 71, and 3, or, • Sequence IDs 1, 73, and 3, or, • Sequence IDs 1, 75, and 3, or, • Sequence IDs 1, 2, and 25, or, • Sequence IDs 1, 2, and 27, or, • Sequence IDs 1, 2, and 29, or, • Sequence IDs 1, 2, and 31, or, • Sequence IDs 1, 2, and 33, or, • Sequence IDs 1, 2, and 77, or, • Sequence IDs 1, 2, and 79, or, • Sequence IDs 1, 2, and 81, or, • Sequence IDs 1, 2, and 83, or, • Sequence IDs 1, 2, and 85, or, • Sequence IDs 5, 6, and 7, or, • Sequence IDs 9, 10, and 11, or, • Sequence IDs 13, 14, and 15, or, • Sequence IDs 392, 2, and 3, or • Sequence numbers 1, 113, and 3, or, • Sequence IDs 1, 115, and 3, or, • Sequence IDs 1, 2, and 117, or, • Sequence IDs 1, 2, and 119, or, • Sequence IDs 1, 2, and 121, or, • Sequence IDs 1, 2, and 123, or, • Sequence IDs 125, 2, and 3, or • Sequence IDs 17, 73, and 3, or • Sequence IDs 17, 128, and 3, or • Sequence IDs 5, 160, and 7, or, • Sequence IDs 5, 162, and 7, or, • Sequence IDs 5, 164, and 7, or, • Sequence IDs 5, 166, and 7, or, • Sequence IDs 9, 169, and 11, or, • Sequence numbers 9, 171, and 11, or, • Sequence IDs 175, 176, and 177, or, • Sequence IDs 179, 176, and 180, or, • Sequence IDs 182, 176, and 177, or, • Sequence IDs 184, 176, and 177, or, • Sequence IDs 186, 187, and 188, or, • Sequence IDs 190, 191, and 192, or, • Sequence IDs 194, 195, and 196, or, • Sequence IDs 198, 199, and 200, or, • Sequence IDs 201, 202, and 203, or, • Sequence IDs 205, 206, and 207, or, • Sequence IDs 410, 6, and 7, or, • Sequence IDs 413, 6, and 7, or, • Sequence IDs 5, 416, and 7, or, • Sequence IDs 5, 419, and 7, or, • Sequence IDs 426, 6, and 7, or, • Sequence IDs 5, 431, and 7, or, • Sequence IDs 434, 6, and 7, or, • Sequence IDs 437, 6, and 7, or • Sequence IDs 5, 6, and 452, or, • Sequence IDs 5, 6, and 455, or, • Sequence IDs 607, 608, and 609, or, • Sequence IDs 610, 611, and 612, or • Sequence IDs 671, 672, and 673, or, • Sequence IDs 672, 672, and 673, or, • Sequence IDs 673, 6, and 7, or, • Sequence IDs 674, 6, and 7, or, • Sequence IDs 1, 2, and 713, or, • Sequence IDs 5, 6, and 714, or, • Sequence IDs 674, 164, and 7, or, • Sequence IDs 710, 6, and 7, or, • Sequence IDs 5, 6, and 715, or, • Sequence IDs 674, 712, and 7, or, • Sequence IDs 711, 6, and 7, or, • Sequence IDs 673, 6, and 741, or, • Sequence IDs 673, 6, and 742, or, • Sequence IDs 673, 6, and 743, or, • Sequence IDs 673, 6, and 744, or, • Sequence IDs 673, 431, and 741, or, • Sequence IDs 673, 431, and 742, or, • Sequence IDs 673, 431, and 743, or, • Sequence IDs 673, 6, and 7, or, • Sequence IDs 674, 6, and 7, or, The variants include or consist of the variants defined above, preferably having at most three, two, or one amino acid modification.

[0048] In other preferred embodiments, the VHH molecule of the present invention comprises CDR1, CDR2, and CDR3, wherein the domains of CDR1, CDR2, and CDR3 are C5 or a variant of CDR3 (as shown in Table 1), and these are respectively • Sequence IDs 1, 2, and 3, or, • Sequence IDs 13, 14, and 15, or, • Sequence IDs 17, 2, and 3, or, • Sequence IDs 19, 2, and 3, or • Sequence IDs 67, 2, and 3, or, • Sequence IDs 69, 2, and 3, or • Sequence IDs 1, 21, and 3, or, • Sequence IDs 1, 23, and 3, or, • Sequence numbers 1, 71, and 3, or, • Sequence IDs 1, 73, and 3, or, • Sequence IDs 1, 75, and 3, or, • Sequence IDs 1, 2, and 25, or, • Sequence IDs 1, 2, and 27, or, • Sequence IDs 1, 2, and 29, or, • Sequence IDs 1, 2, and 31, or, • Sequence IDs 1, 2, and 33, or, • Sequence IDs 1, 2, and 77, or, • Sequence IDs 1, 2, and 79, or, • Sequence IDs 1, 2, and 81, or, • Sequence IDs 1, 2, and 83, or, • Sequence IDs 1, 2, and 85, or, • Sequence IDs 392, 2, and 3, or • Sequence numbers 1, 113, and 3, or, • Sequence IDs 1, 115, and 3, or, • Sequence IDs 1, 2, and 117, or, • Sequence IDs 1, 2, and 119, or, • Sequence IDs 1, 2, and 121, or, • Sequence IDs 1, 2, and 123, or, • Sequence IDs 125, 2, and 3, or • Sequence IDs 17, 73, and 3, or • Sequence IDs 17, 128, and 3, or • Sequence IDs 671, 672, and 673, or, • Sequence IDs 672, 672, and 673, or, • Sequence IDs 1, 2, and 713, or, The variants include or consist of the variants defined above, preferably having at most three, two, or one amino acid modification.

[0049] In other preferred embodiments, the VHH molecule of the present invention comprises CDR1, CDR2, and CDR3, wherein the domains of CDR1, CDR2, and CDR3 are B6 domains (as shown in Table 1), and these are respectively • Sequence IDs 9, 10, and 11, or, • Sequence IDs 9, 169, and 11, or, • Sequence numbers 9, 171, and 11, or, The variants include or consist of the variants defined above, preferably having at most three, two, or one amino acid modification.

[0050] In certain embodiments, the VHH molecule of the present invention is B6 or a variant thereof, which targets the apical domain of hTfR and comprises CDR1, CDR2, and CDR3, which are respectively • Sequence IDs 9, 10, and 11, or, • Sequence IDs 9, 169, and 11, or, • Sequence numbers 9, 171, and 11, or, • Sequence IDs 175, 176, and 177, or, • Sequence IDs 179, 176, and 180, or, • Sequence IDs 182, 176, and 177, or, • Sequence IDs 184, 176, and 177, or, The variants include or consist of the variants defined above, preferably having at most three, two, or one amino acid modification.

[0051] In other specific embodiments, the VHH molecule of the present invention targets the apical domain of hTfR and is a VHH molecule comprising CDR1, CDR2, and CDR3 (listed in Table 1), wherein CDR1, CDR2, and CDR3 are, • Sequence IDs 9, 10, and 11, or, • Sequence IDs 9, 169, and 11, or, • Sequence numbers 9, 171, and 11, or, • Sequence IDs 175, 176, and 177, or, • Sequence IDs 179, 176, and 180, or, • Sequence IDs 182, 176, and 177, or, • Sequence IDs 184, 176, and 177, or, • Sequence IDs 186, 187, and 188, or, • Sequence IDs 190, 191, and 192, or, • Sequence IDs 194, 195, and 196, or, • Sequence IDs 198, 199, and 200, or, • Sequence IDs 201, 202, and 203, or, • Sequence IDs 205, 206, and 207, or, The variants include or consist of the variants defined above, preferably having at most three, two, or one amino acid modification.

[0052] In other preferred embodiments, the VHH molecule of the present invention comprises CDR1, CDR2, and CDR3, wherein the domains of CDR1, CDR2, and CDR3 are domains of B8 or its variants (as shown in Table 1), and these are respectively • Sequence IDs 5, 6, and 7, or, • Sequence IDs 5, 160, and 7, or, • Sequence IDs 5, 162, and 7, or, • Sequence IDs 5, 164, and 7, or, • Sequence IDs 5, 166, and 7, or, • Sequence IDs 410, 6, and 7, or, • Sequence IDs 413, 6, and 7, or, • Sequence IDs 5, 416, and 7, or, • Sequence IDs 5, 419, and 7, or, • Sequence IDs 426, 6, and 7, or, • Sequence IDs 5, 431, and 7, or, • Sequence IDs 434, 6, and 7, or, • Sequence IDs 437, 6, and 7, or • Sequence IDs 5, 6, and 452, or, • Sequence IDs 5, 6, and 455, or, • Sequence IDs 673, 6, and 7, or, • Sequence IDs 674, 6, and 7, or, • Sequence IDs 5, 6, and 714, or, • Sequence IDs 674, 164, and 7, or, • Sequence IDs 710, 6, and 7, or, • Sequence IDs 5, 6, and 715, or, • Sequence IDs 674, 712, and 7, or, • Sequence IDs 711, 6, and 7, or, • Sequence IDs 673, 6, and 741, or, • Sequence IDs 673, 6, and 742, or, • Sequence IDs 673, 6, and 743, or, • Sequence IDs 673, 6, and 744, or, • Sequence IDs 673, 431, and 741, or, • Sequence IDs 673, 431, and 742, or, • Sequence IDs 673, 431, and 743, or, • Sequence IDs 673, 6, and 7, or, • Sequence IDs 674, 6, and 7, or, The variants include or consist of the variants defined above, preferably having at most three, two, or one amino acid modification.

[0053] In other specific embodiments, the VHH molecules of the present invention are cross-reactive with human, mouse, and / or non-human primate species, as detailed in Tables 5 and 6 below. Preferred VHH molecules of the present invention include an FR domain as defined below.

[0054] In certain embodiments, the FR1 domain includes or comprises a variant thereof having at least 58% of the amino acid identity over its entire length, for example, at least 60%, 62%, 64%, 66%, 68%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably having at least 80% amino acid identity: E[VQL]VE[SGG]GL[V]QP[G]G[SL]K[L]S[C]AAS(Sequence ID 35). More preferably, the amino acid residues in square brackets are present, and mutations occur only at other positions.

[0055] In a specific embodiment, the first E may be replaced with Q.

[0056] In a specific embodiment, the fifth V may be replaced with Q.

[0057] In a specific embodiment, the sixth E may be replaced with Q.

[0058] In a specific embodiment, the tenth G may be replaced with K or A.

[0059] In specific embodiments, the 11th L may be replaced with V or E.

[0060] In a specific embodiment, the 14th P may be replaced with A.

[0061] In a specific embodiment, the 16th G may be replaced with D.

[0062] In other specific embodiments, the 19th K may be replaced with R.

[0063] In other specific embodiments, the 23rd A may be replaced with V or T.

[0064] In other specific embodiments, the 25th S may be replaced with D.

[0065] More preferably, FR1 includes at least four amino acid modifications, even more preferably three, and even more preferably two, in amino acid residues that are not bold with respect to this sequence. In a preferred embodiment, the amino acid modification is at the 19th R.

[0066] In a further specific embodiment, FR1 has an amino acid sequence selected from one of the following amino acid sequences: EVQLVESGGGVVQPGGSLKLSCVAS (Sequence ID 36), EVQLVESGGGVVQPGGSLRLSCAAS (Sequence ID 37), EVQLVESGGGLVQPGGSLRLSCTAS (Sequence ID 38), or, EVQLVESGGGEVQPGGSLKLSCVAS (SEQ ID NO: 39), or their variants.

[0067] Further specific examples of FR1 of the VHH molecule in this invention are described below (see also Table 3): EVQLVESGGGVVQPGGSLKLSCAAS (Sequence ID 331), EVQLVESGGGLVQPGGSLRLSCAAS (Sequence ID 332), EVQLVESGGGVVQPGGSLRLSCAAD (Sequence ID 333), EVQLVESGGGVVQPGGSLRLSCVAS (Sequence ID 400), QVQLVQSGGGLVQAGGSLTLSCTAS (Sequence ID 334), EVQLVESGGGLVQAGGSLRLSCTAS (Sequence ID 335), QVQLVQSGGGLVQPGGSLRLSCAAS (Sequence ID 336), EVQLVESGGGLVQAGDSLRLSCTAS (Sequence ID 337), QVQLVQSGGGLVQAGGSLRLSCAAS (Sequence ID 338), EVQLVQSGGGLVQAGGSLRLSCAAS (Sequence ID 339), EVQLVESGGGLVQPGESLRLSCTAS (Sequence ID 340), EVQLVESGGGLVQPGGSLRLSCVSS (Sequence ID 341), EVQLVESGGGLVQAGDSLRLSCAAS (Sequence ID 619), or, VQLVESGGRLVQAGGSLRLSCTAS (Sequence ID 620).

[0068] In certain embodiments, the VHH molecule of the present invention comprises an FR2 domain comprising or consisting of a variant thereof having at least 58%, for example, at least 60%, 62%, 64%, 66%, 68%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length: MR[W]YRQA[P]G[K]QRELVAT(Sequence ID 40). More preferably, the amino acid residues in square brackets are present and mutations occur only at other positions.

[0069] In specific embodiments, the first M may be replaced with I or V.

[0070] In specific embodiments, the second R may be replaced with G, H, or S.

[0071] In a specific embodiment, the fourth Y may be replaced with F or V.

[0072] In a specific embodiment, the fifth R may be replaced with G.

[0073] In specific embodiments, the sixth Q may be replaced with R or E.

[0074] In a specific embodiment, the seventh A may be replaced with R.

[0075] In specific embodiments, the 9th G may be replaced with I or E.

[0076] In a specific embodiment, the 11th Q may be replaced with E, G, I, or D.

[0077] In a specific embodiment, the 12th R may be replaced with L.

[0078] In specific embodiments, the 13th E may be replaced with N or H.

[0079] In a specific embodiment, the 14th L may be replaced with F, W, S, or Q.

[0080] In specific embodiments, the 15th V may be replaced with Q or I.

[0081] In specific embodiments, the 16th A may be replaced with M or S.

[0082] In a specific embodiment, the 17th T may be replaced with G or S.

[0083] More preferably, FR2 includes at most six amino acid modifications in amino acid residues that are not bold with respect to this sequence, even more preferably five, at most three, and even more preferably two. In a preferred embodiment, the amino acid modifications are the fourth V and / or the eleventh G and the twelfth L and / or the fourteenth W and / or the sixteenth S and / or the seventeenth G.

[0084] In certain embodiments, the VHH molecule of the present invention comprises at least one of the following amino acids in the FR2 domain: Phe42, Glu49, Arg50, or Gly52 (according to IMGT numbering).

[0085] In a further specific embodiment, FR2 has an amino acid sequence selected from one of the following amino acid sequences: IRWYRQAPGKQREFVAG (Sequence ID 41), MRWYRQAPGKQREWVAG (Sequence ID 42), MGWFRRAPGKERELVAS (Sequence ID 43), VRWYRQRPGKQREWVAG (SEQ ID NO: 44), or their variants.

[0086] Further specific examples of FR2 of the VHH molecule in this invention are described below (see also Table 3): IRWVRQAPGKGLEWVAG (Sequence ID 342), IRWYRQAPGKGLEFVAG (Sequence ID 343), IRWVRQAPGKGLEFVAG (Sequence ID 344), IRWYRQAPGKGREFVAG (Sequence ID 345), IRWVRQAPGKQREFVAG (Sequence ID 346), IRWYRQAPGKGLEWVAG (Sequence ID 347), MRWYRQAPGKGLEWVAG (Sequence ID 348) MRWYGQAPGKQREWVAG (Sequence ID 349), MRWYREAPGKQREWVAG (Sequence ID 350), MRWYRQAPIKQREWVAG (Sequence ID 351), MRWYRQAPGKIREWVAG (Sequence ID 352), MGWFRRAPGKERNLVAS (Sequence ID 353), MGWFRRAPGKERESVAS (Sequence ID 354), MGWFRRAPGKERELQAS (Sequence ID 355), MGWFRRAPEKERELVAS (Sequence ID 356), MGWFRRAPGKDRELVAS (Sequence ID 357), MSWVRQAPGKGRELVAS (Sequence ID 358), MGWFRRAPGKERELIAS (Sequence ID 359), LAWHRQIPGKEREWVAG (Sequence ID 360), MAWHRQAPGKERLWVAG (Sequence ID 361), VGWYRQAPGEQRVLVAH (Sequence ID 362), MGWFRQAPGKEREFVAA (Sequence ID 363), MGWYRQAPGKQRELVAV (Sequence ID 364), MGWFRQTPGKEREFVAA (Sequence ID 365), MRWYRQAPGKQREQVAG (Sequence ID 458), MRWYRQAPGKQREFVAG (Sequence ID 459), MRWYRQAPGKQRHWVAG (Sequence ID 460), MIWYRQAPGKQREWVAG (Sequence ID 461), MEWYRQAPGKQREWVAG (Sequence ID 462), MRWYRQAPGKQREWVAA (Sequence ID 463), MRWYRQAPGKQREWVAK (Sequence ID 464), IGWFRQAPGKEREKVSC (Sequence ID 621), MHWFRQAPGKEREFVGA (Sequence ID 622), IRWYSQAPGKQREFVAG (Sequence ID 716), MRWYRQAPGKQREWVSG (Sequence ID 720) MRWYRQAPGKQ[L]EWVAG (Sequence ID 787) MRWYRQAPGK[G]REWVAG (Sequence ID 788) MRWYRQAPGK[GL]EWV[S]G (Sequence ID 789), or, MRWYRQAPGK[G]REWV[S]G (Sequence ID 790).

[0087] In certain embodiments, the VHH molecule of the present invention is sequence number 45 as shown below, or at least 58% of its total length relative to this sequence, for example, at least 60%, 62%, 64%, 66%, 68%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 8 The FR3 domain includes or comprises a variant having 9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity, preferably at least 70% amino acid identity: YYAD[S]VKG[RF]T[I]S[RDN]AK[N]TVY[LQ]MNS[L]KPE[DTA]V[Y]Y[C] (SEQ ID NO: 45). More preferably, the amino acid residues in square brackets are present and mutations occur only at other positions.

[0088] In a specific embodiment, the first Y may be replaced with N.

[0089] In a specific embodiment, the second Y may be replaced with A.

[0090] In a specific embodiment, the third A may be replaced with P or I.

[0091] In specific embodiments, the fourth D may be replaced with S or N.

[0092] In a specific embodiment, the 17th A may be replaced with S.

[0093] In a specific embodiment, the 29th K may be replaced with R.

[0094] In a specific embodiment, the 30th P may be replaced with A.

[0095] More preferably, FR3 includes at most seven amino acid modifications in amino acid residues that are not bold with respect to this sequence, more preferably six, at most three, and even more preferably two.

[0096] In a further specific embodiment, FR3 has an amino acid sequence selected from one of the following amino acid sequences: NYADSMKGRFTISRDNTKNAVYLQIDSLKPEDTAVYYC (Sequence ID 46), NYPDSAKGRFTISRDNAKNTVYLQIDSLKPEDTAVYYC (Sequence ID 47), YAISSVKGRFTISRDNAENTVFLQMNSLKPDDTAVYYC (Sequence ID 48), or, NYPDSMKGRFTISRDNAKNTVYLQINSLKSEDTAVYYC (SEQ ID NO: 49), or their variants.

[0097] Further specific examples of FR3 of the VHH molecule in this invention are described below (see also Table 3): NYADSMKGRFTISRDNTKNALYLQIDSLRPEDTAVYYC (Sequence ID 366), NYADSVKGRFTISRDNTKNTLYLQIDSLRPEDTAVYYC (Sequence ID 367), NYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYC (Sequence ID 368), NYADSVKGRFTISRDNTKNTLYLQINSLRPEDTAVYYC (Sequence ID 369), NYADSMKGRFTISRDNTKNTLYLQMNSLRPEDTAVYYC (Sequence ID 370), NYADSVKGRFTISRDNAKNTLYLQIDSLRPEDTAVYYC (Sequence ID 371), NYADSVKGRFTISRDNTKNTLYLQMNSLRPEDTAVYYC (Sequence ID 372), NYADSVKGRFTISRDNTKNALYLQMNSLRPEDTAVYYC (Sequence ID 373), NYADSVKGRFTISRDNTKNTLYLQMDSLRPEDTAVYYC (Sequence ID 374), NYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC (Sequence ID 375), NYADSVKGRFTISRDNAKNAVYLQMNSLRPEDTAVYYC (Sequence ID 376), NYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYC (Sequence ID 377), NYADSMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYC (Sequence ID 378), NYPDSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYC (Sequence ID 379), YYADGMRGRFTISRDNSENTVSLQMNNLKPEDTAVYYC (Sequence ID 380), YYANSMKERFTISRDNAQNTVSLQISSLKPEDTAVYYC (Sequence ID 381), YYADGMKGRFTISRDNAENTVSLQINSLKPEDTAIYYC (Sequence ID 382), YYADSSVKGRFTISRDNAENTVSLQMNSLKPEDTAVYYC (Sequence ID 383), SYRDSVKGRFTISRDNAKNTVFLQMNSLEPEDTGVYYC (Sequence ID 384), SYADSVKGRFTISRDDAKNTVYLQMDNLTPEDTAVYFC (Sequence ID 385), EYKDSVKGRFTISRDNARNTIYLEMKNLKPEDTAIYYC (Sequence ID 386), DYADGVMGRFTISRNSALNTVYLQMDSLKSTDTGVYVC (Sequence ID 387), KYGDSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYC (Sequence ID 388), TYADSVKGRFTISRDNAKNTVYLQMNSLEPTDTAVYYC (Sequence ID 389), YYADSVKGRFTISRDTVKDMVYLQMNSLKPEDTAVYYC (Sequence ID 623), EYADSVKGRFTISRDNAKSTVYLQMNNLKPEDTAVYYC (Sequence ID 624), VYPDSAKGRFTISRDNAKNTVYLQIDSLKPEDTAVYYC (Sequence ID 625), FYPDSAKGRFTISRDNAKNTVYLQIDSLKPEDTAVYYC (Sequence ID 626), NYADSMKGRLTISRDNTKNAVYLQIDSLKPEDTAVYYC (Sequence ID 717), NYPDIAKGRFTISRDNAKNTVYLQIDSLKPEDTAVYYC (Sequence ID 718), NYPDSAKGRFTISEDNAKNTVYLQIDSLKPEDTAVYYC (Sequence ID 719), NYPDSVKGRFTISRDNAKNTAYLQMNSLRAEDTAVYYC (Sequence ID 791), NYPDSAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYC (Sequence ID 792), NYPDSAKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYC (Sequence ID 793), NYPDSAKGRFTISRDNSKNTVYLQMDSLRPEDTAVYYC (sequence number 794), or, NYPDSAKGRFTISRDNAKNTVYLQMDSLRPEDTAVYYC (Sequence ID 795).

[0098] In certain embodiments, the VHH molecule of the present invention includes or comprises an FR4 domain having at least 58% of the amino acid identity over its entire length, for example, at least 60%, 62%, 64%, 66%, 68%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably at least 90% amino acid identity, of the sequence such as SEQ ID NO: W[G]Q[GTQVT]V[S]S(SEQ ID NO: 50). More preferably, the amino acid residues in square brackets are present and mutations occur only at other positions.

[0099] More preferably, FR4 includes at most four amino acid modifications in amino acid residues that are not bolded with respect to this sequence, more preferably at most three amino acid modifications, and even more preferably at most two amino acid modifications. An example for a specific description of the FR4 sequence is Sequence ID No. 50.

[0100] Other specific examples of FR4 in the present invention are as follows: WGQGTLVTVSS (Sequence ID 390), WGKGTQVTVSS (Sequence ID 391), or, WGRGTQVTVSS (Sequence ID 670).

[0101] Specific examples of the TfR-binding VHH molecules of the present invention are molecules containing or consisting of an amino acid sequence selected from any one of the following: SEQ ID NOs: 213-271, 273-299, 412, 415, 418, 421, 423, 425, 428, 430, 433, 436, 439, 441, 443, 445, 447, 449, 451, 454, 457, 613-615, 675-678, and 701-709 (see Table 1). In the examples corresponding to sequence numbers 213-271, 273-299, 412, 415, 418, 421, 423, 425, 428, 430, 433, 436, 439, 441, 443, 445, 447, 449, 451, 454, 457, 613-615, 675-678, 701-709, and 766-786, the VHH molecule does not contain any tag sequences (when x is 0, as detailed in Table 1 below).

[0102] Other examples of the TfR-bound VHH molecules of the present invention are SEQ ID NOs: 4, 8, 12, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86-92, 114, 116, 118, 120, 122, 124, 126, 127, 129-149, 152-159, 161, 163, 165, 167, 168, 170, 172-174, 178, 181, 183, 185, 189, 193, 197, 204, 208, 393, 411, 414, 41 A molecule containing or consisting of an amino acid sequence selected from one of the following: 7, 420, 422, 424, 427, 429, 432, 435, 438, 440, 442, 444, 446, 448, 450, 453, 456, 616-618, 679-682, and 691-700, and 745-765 (listed in Table 1 below. In these examples, when x is equal to 1, each VHH molecule contains the following specific SEQ ID NO: 51 tag sequence: AAAEQKLISEEDLNGAAHHHHHHGS).

[0103] In certain embodiments, the VHH of the present invention is humanized. For humanization, one or more FR and / or CDR domains may be (further) modified by one or more amino acid substitutions.

[0104] In this regard, in certain embodiments, VHH is humanized by selective modification (e.g., amino acid substitution) of the FR1 domain. The FR1 domain typically consists of a sequence of 25 amino acid residues. Typical humanization sites in FR1 are 19R or 23A, or both (e.g., any one of SEQ ID NOs. 35-39, 331, 332, 400, or any of their variants as defined herein). Thus, specific examples of such humanized FR1 include SEQ ID NOs. 37, 331, and 400, in which K19 and / or V23 are modified to 19R and 23A, respectively.

[0105] Another humanization site in FR1 is 11L, and therefore, specific examples of such humanized FR1 include sequence number 332, in which V11 is modified to 11L and K19 and V23 are modified to 19R and 23A, respectively.

[0106] In other specific embodiments, VHH is humanized by selective modification of the FR2 domain. Typical humanization sites in FR2 are selected from 1M, 2S or 2H, 4V, 11G, 12L, 14W, or combinations thereof (for example, with respect to any one of SEQ ID NOs. 40-44, 342-348, or any variant thereof as defined herein).

[0107] Therefore, a specific example of such humanized FR2 is sequence number 41, in which one or more or all of I1, R2, Y4, Q11, R12, and F14 are modified to 1M, 2S or 2H, 4V, 11G, 12L, and 14W, respectively.

[0108] Other specific examples of humanized FR2 include Sequence ID No. 342, in which Y4, Q11, R12, and F14 are modified to 4V, 11G, 12L, and 14W, respectively.

[0109] Other specific examples of humanized FR2 include Sequence ID No. 343, in which Q11 and R12 are modified to 11G and 12L, respectively.

[0110] Other specific examples of humanized FR2 include sequence number 344, in which Y4, Q11, and R12 are modified to 4V, 11G, and 12L, respectively.

[0111] Other specific examples of humanized FR2 include sequence number 345, which modifies Q11 to 11G.

[0112] Other specific examples of humanized FR2 include sequence number 346, in which Y4 is modified to 4V.

[0113] Other specific examples of humanized FR2 include Sequence ID No. 347, in which Q11, R12, and F14 are modified to 11G, 12L, and 14W, respectively.

[0114] Other specific examples of humanized FR2 include Sequence ID No. 348, in which Q11 and R12 are modified to 11G and 12L, respectively.

[0115] Other specific examples of humanized FR2 include sequence number 787, in which R12, L14, and T17 are modified to 12L, 14W, and 17G, respectively.

[0116] Other specific examples of humanized FR2 include sequence number 788, in which Q11, L14, and T17 are modified to 11G, 14W, and 17G, respectively.

[0117] Other specific examples of humanized FR2 include sequence number 789, which modifies Q11, R12, A16, and T17 to 11G, 12L, 16S, and 17G, respectively.

[0118] Other specific examples of humanized FR2 include sequence number 790, which modifies Q11, A16, and T17 to 11G, 16S, and 17G, respectively.

[0119] In other specific embodiments, VHH is humanized by selective modification of the FR3 domain. Typical humanization sites in FR3 are selected from 6V, 17A or S, 20T, 21L, 25M, 26N, 29R, 30A, and any combination thereof (for example, with respect to any one of sequence numbers 45-49, 366-379, or any variant thereof as defined herein). A specific example of such humanized FR3 is sequence number 46, in which one or more or all of M6, T17, A20, V21, I25, D26, and K29 ​​are modified to 6V, 17A, 20T, 21L, 25M, 26N, and 29R, respectively.

[0120] Other specific examples of humanized FR3 include sequence number 366, which modifies V21 and K29 ​​to 21L and 29R, respectively.

[0121] Other specific examples of such humanized FR3s include sequence number 367, which modifies M6, A20, V21, and K29 ​​to 6V, 20T, 21L, and 29R, respectively.

[0122] Other specific examples of humanized FR3 include Sequence ID 368, in which M6, T17, A20, V21, I25, D26, and K29 ​​are modified to 6V, 17A, 20T, 21L, 25M, 26N, and 29R, respectively.

[0123] Other specific examples of humanized FR3 include sequence number 369, which modifies M6, A20, V21, D26, and K29 ​​to 6V, 20T, 21L, 26N, and 29R, respectively.

[0124] Other specific examples of humanized FR3 include Sequence ID 370, which modifies A20, V21, I25, D26, and K29 ​​to 20T, 21L, 25M, 26N, and 29R, respectively.

[0125] Other specific examples of humanized FR3 include Sequence ID No. 371, in which M6, T17, A20, V21, and K29 ​​are modified to 6V, 17A, 20T, 21L, and 29R, respectively.

[0126] Other specific examples of humanized FR3 include Sequence ID 372, in which M6, A20, V21, I25, D26, and K29 ​​are modified to 6V, 20T, 21L, 25M, 26N, and 29R, respectively.

[0127] Other specific examples of humanized FR3 include Sequence ID 373, in which M6, V21, I25, D26, and K29 ​​are modified to 6V, 21L, 25M, 26N, and 29R, respectively.

[0128] Other specific examples of humanized FR3 include Sequence ID 374, in which M6, A20, V21, I25, and K29 ​​are modified to 6V, 20T, 21L, 25M, and 29R, respectively.

[0129] Other specific examples of humanized FR3 include Sequence ID No. 375, in which M6, T17, A20, V21, I25, D26, K29, and P30 are modified to 6V, 17S, 20T, 21L, 25M, 26N, 29R, and 30A, respectively.

[0130] Other specific examples of humanized FR3 include sequence number 376, in which M6, T17, I25, D26, and K29 ​​are modified to 6V, 17A, 25M, 26N, and 29R, respectively.

[0131] Other specific examples of humanized FR3 include Sequence ID No. 377, in which M6, T17, A20, V21, I25, and D26 are modified to 6V, 17A, 20T, 21L, 25M, and 26N, respectively.

[0132] Other specific examples of humanized FR3 include Sequence ID 378, in which T17, A20, V21, I25, D26, and K29 ​​are modified to 17A, 20T, 21L, 25M, 26N, and 29R, respectively.

[0133] Other specific examples of humanized FR3 include sequence number 379, in which M6, I25, D26, and K29 ​​are modified to 6V, 25M, 26N, and 29R, respectively.

[0134] Other specific examples of humanized FR3 include sequence number 791, in which Y1, A3, and K29 ​​are modified to 1N, 3P, and 29R, respectively.

[0135] Other specific examples of humanized FR3 include sequence number 792, in which Y1, A3, V6, A17, K29, and P30 are modified to 1N, 3P, 6A, 17S, 29R, and 30A, respectively.

[0136] Other specific examples of humanized FR3 include Sequence ID 793, in which Y1, A3, V6, K29, and P30 are modified to 1N, 3P, 6A, 29R, and 30A, respectively.

[0137] Other specific examples of humanized FR3 include Sequence ID 794, in which Y1, A3, A17, N26, K29, N, and P30 are modified to 1N, 3P, 17S, 26D, 29R, and 30A, respectively.

[0138] Other specific examples of humanized FR3 include Sequence ID 795, in which Y1, A3, N26, K29, N, and P30 are modified to 1N, 3P, 26D, 29R, and 30A, respectively.

[0139] Specific examples of the humanized TfR-binding VHH molecules of the present invention are molecules containing or consisting of an amino acid sequence selected from any one of SEQ ID NOs: 236-241, 252-271, 273-275, 752-765, or 773-786 (see Table 1).

[0140] In further specific embodiments, the VHH molecule may further include one or more tags suitable for, for example, purification, coupling, detection, etc. In the context of this invention, the term “tag” includes any peptide sequence that is conjugated to the polypeptide VHH molecule of the present invention for the purpose of facilitating the easy detection or purification of the expressed protein, or for identifying binding to TfR, or for site-specific enzymatic / enzymatic conjugation. The tag may be an affinity tag, an epitope tag, a site-specific conjugation tag, or a fluorescent tag.

[0141] Examples of such tags include tags containing glutamine residues inserted into the tag sequence, specifically recognized by transglutaminase (TGase), and preferably containing or consisting of the LQR sequence, such as Q tags, myc tags (EQKLISEEDL, SEQ ID NO: 394), polyHis tags (containing 2-8 histidine residues, preferably 6-8 His residues, e.g., His6 (SEQ ID NO: 395) or His8 (SEQ ID NO: 396)), polyArg tags (containing 2-8 arginine residues), polyLys tags (containing 2-8 lysine residues), HA tags (e.g., YPYDVPDYA, SEQ ID NO: 397), FLAG tags (e.g., DYKDDDDK, SEQ ID NO: 398), or GFP tags, CBP tags, Strep II tags, saltase tags, SNAP tags, or combinations thereof (shown in Table 4 below).

[0142] Typically, one or more tags are located at the C-terminus of the VHH.

[0143] In a further specific embodiment, the VHH molecule may further comprise one or more linkers.

[0144] In the context of this invention, the terms “linker” and “spacer” are used interchangeably. The linker may be a peptide linker or a conjugation linker. The peptide linker comprises one or more amino acid residues, typically 1 to 10 amino acid residues, used to link the VHH molecule of the present invention to a tag, or to link various tags as described herein, provided that the linker does not specifically bind to a target protein which is a TfR. The linker may be any amino acid residue, such as glycine (Gly or G), alanine (Ala or A), phenylalanine (Phe or F), serine (Ser or S), cysteine ​​(Cys or C), leucine (Leu or L), asparagine (Asn or N), lysine (Lys or K), glutamic acid (Glu or E), glutamine (Gln or Q), proline (Pro or P), valine (Val or V), arginine (Arg or R), aspartic acid (Asp or D), or a combination thereof. The peptide linker may be mobile (e.g., any mobile hydrophilic linker) or rigid (e.g., any α-helix rigid linker).

[0145] Such peptide linkers are different from conjugation linkers that can be introduced between VHH and the compound of interest, such as difunctional or polyfunctional substances (as described herein in the “Conjugate” section) containing alkyl, aryl, thiol, azide, alkyne, nucleotide, or peptide groups, which may be derived from esters, aldehydes, or alkyl or aryl acids, anhydrides, sulfhydryl or carboxyl groups, or groups derived from cyanogen bromide or cyanogen chloride, carbonyl diimidazole, succinimide esters, or sulfonic acid halides.

[0146] As a further example, the VHH of the present invention may preferably include a linker located at the C-terminus of the VHH. The linker may include a Gly residue, or a Gly repeat of, for example, 2 to 7 Gly residues (i.e., Gly2, Gly3, Gly4, Gly5, Gly6, or Gly7, respectively). The VHH of the present invention may also include a combination of a Gly residue and a Ser residue. Specific examples of such Gly-Ser combination linkers include GlySerGlySer(GSGS, SEQ ID NO: 627), SerGlySerGly5(SGSGGGGG, SEQ ID NO: 628), (Gly4Ser)n, where n is 1 to 6, for example, any of SEQ ID NOs. 629 to 634 (shown in Table 4 below), preferably SEQ ID NOs. 629 to 631, or any linker containing such a (Gly4Ser)n sequence, for example, GlyGly(Gly4Ser)3(GGGGGGSGGGGSGGGGS, SEQ ID NO: 635).

[0147] Other specific examples of peptide linkers in the present invention include, as shown in Table 4, EAAAK (SEQ ID NO: 636), or EAAAK repeats combined with other amino acid residues, such as any of SEQ ID NOs: 637 to 641.

[0148] Other specific examples of peptide linkers in the present invention are as follows, as shown in Table 4: GG(AP)17 (SEQ ID NO: 642), ASTKGPSVFPLAP (SEQ ID NO: 643), GSAGSAAGSGEF (SEQ ID NO: 644), or KESGSVSSEQLAQFRSLD (SEQ ID NO: 645).

[0149] In certain embodiments, the VHH of the present invention may include a Gly linker and a Q tag, preferably located at the C-terminus. A more specific example of such a VHH includes the following structure: VHH-Gly linker-Q tag, where the Gly linker includes or consists of 2 to 6 Gly residues, and the Q tag includes or consists of an LQR. For example, the VHH may include the following tag sequence AAA(EQKLISEEDL)NGAA"HHHHHH"GS" (SEQ ID NO: 51) at the C-terminus, where the parentheses represent the myc tag and the quotation marks represent the His6 tag (the remaining residues are linkers such as Ala linker AAA, or residues resulting from cloning).

[0150] Specific examples of TfR-bound VHH molecules with such tags in the present invention, which include the tag sequence of sequence number 51 at the C-terminus, are 4, 8, 12, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86~92, 114, 116, 118, 120, 122, 124, 126, 127, 129~149, 152~159, 161, 163, 165, 167, 168, 170, A molecule containing or consisting of an amino acid sequence selected from one of the following: 172-174, 178, 181, 183, 185, 189, 193, 197, 204, 208, 393, 411, 414, 417, 420, 422, 424, 427, 429, 432, 435, 438, 440, 442, 444, 446, 448, 450, 453, 456, 616-618, 679-682, 691-700, and 745-765.

[0151] Specific examples of the humanized TfR-binding VHH molecules of the present invention, accompanied by a tag sequence, are molecules containing or consisting of an amino acid sequence selected from any one of SEQ ID NOs: 87-92 or 130-149, 152-154 or 752-765 (see Table 1).

[0152] As another example, VHH may contain the following tag sequence (GGGG)S[C]"HHHHHH" (SEQ ID NO: 399) at its C-terminus, where the parentheses are spacers, the square brackets C is a free cysteine ​​available for site-directed chemical conjugation, and the quotation marks are the His tag (the remaining residues are linkers or result of cloning).

[0153] As another example, the VHH of the present invention may include or consist of the sequence LQR, and may include a Q tag preferably located at the C-terminus of the VHH.

[0154] As a further example, VHH may contain the following tag sequence [GGG](LQR)(sequence number 111) at its C-terminus ("C-ter" or "C-terminus"), where the parentheses are Q tags and the square brackets are Gly linkers. Other examples include GGGGLQR(sequence number 401), GGGGGLQR(sequence number 402), GGGGGGLQR(sequence number 403), and GGGGGGGLQR(sequence number 404). In a preferred embodiment of the present invention, VHH contains the tag sequence of sequence number 111.

[0155] In further specific embodiments, the VHH of the present invention may include an Ala linker, a His tag, a Gly linker, and a Q tag. Preferably, the linker and tag are located at the C-terminus of the VHH. In other embodiments, the Q tag may be located at least at the N-terminus of the VHH. A more specific example of such a VHH includes the following structure: VHH-Ala linker-His tag-Gly linker-Q tag, where the Ala linker 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.

[0156] For example, VHH may contain the following tag sequence [AAA]"HHHHHH"[GGG](LQR)(sequence code 112) at its C-terminus, where the parentheses are Q tags, the square brackets are Ala and Gly linkers, and the quotation marks are His tags. Other examples are AAAHHHHHHGGGGLQR(sequence code 406), AAAHHHHHHGGGGGLQR(sequence code 407), AAAHHHHHHGGGGGGLQR(sequence code 408), and AAAHHHHHHGGGGGGGLQR(sequence code 409). In a preferred embodiment of the present invention, VHH includes a further sequence of sequence code 112.

[0157] As already stated herein, specific examples of such tagged TfR-bound VHH molecules in the present invention are 4, 8, 12, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86~92, 114, 116, 118, 120, 122, 124, 126, 127, 129~149, 152~159, 161, 163, 165, 167, 168, 170, A molecule containing or consisting of an amino acid sequence selected from one of the following: 172-174, 178, 181, 183, 185, 189, 193, 197, 204, 208, 393, 411, 414, 417, 420, 422, 424, 427, 429, 432, 435, 438, 440, 442, 444, 446, 448, 450, 453, 456, 616-618, 679-682, 691-700, and 745-765.

[0158] Further specific examples of the TfR-binding VHH molecules of the present invention are VHH molecules that competitively inhibit the binding of VHH to human and non-human TfRs as defined above. The term "competitively inhibiting" indicates that the VHH can reduce, inhibit, or substitute the binding of the above-described reference VHH to TfRs in vitro or in vivo. Competitive assays can be performed using standard techniques such as competitive ELISA or other binding assays. Typically, competitive binding assays involve recombinant muscle cells or membrane preparations expressing TfRs, unlabeled test VHH (or a phage expressing it), and labeled reference VHH (or a phage expressing it), optionally bound to a solid substrate. Competitive inhibition is measured by determining the amount of labeled VHH that binds in the presence of test VHH. Typically, test VHH is present in excess, such as about 5 to 500 times the amount of reference VHH. Typically, in ELISA, test VHH is present in 100-fold excess. When an excess of test VHH inhibits or substitutes at least 70% of the binding of reference VHH to TfR, it is thought to competitively inhibit the reference VHH described above. Preferred competitive VHHs bind to epitopes that share common amino acid residues.

[0159] As shown in the Experiments section, VHH molecules bind to TfR in vitro and in vivo. They exhibit appropriate affinity for Kd values ​​of approximately 0.01 nM to 4 μM, or approximately 0.01 nM to 2500 nM, or approximately 0.01 nM to 1000 nM, or approximately 0.01 nM to 500 nM, or approximately 0.01 nM to 100 nM, or approximately 0.1 nM to 4 μM, or approximately 0.1 nM to 2500 nM, or approximately 0.1 nM to 1000 nM, as well as approximately 0.1 nM to 500 nM, or approximately 0.1 nM to 100 nM.

[0160] In the context of the present invention, K d The term is K d app , K D , and / or K Deq This refers to one of the affinity constants named K. More specifically, K d appThe term refers to the apparent binding affinity constant measured in a cell-based assay (where indirect ligand detection is performed by flow cytometry) or an enzyme-linked immunosorbent assay (ELISA) performed at 4°C, and corresponds to the ligand concentration that enables TfR binding at half of the maximum capacity in this system. K D The term refers to kinetic parameters measured from multiple ligand concentrations by surface plasmon resonance (SPR) or biolayer interferometry (BLI), namely, the association and dissociation rates (k on and k off ), respectively, and is calculated from these values. It refers to the equilibrium dissociation constant, which is calculated as the ratio of k on to k off . K Deq The term refers to the equilibrium dissociation constant estimated by SPR and corresponding to the ligand concentration required to induce half of the maximum response in SPR and BLI systems.

[0161] In certain embodiments, the VHH molecules of the present invention bind to human and non-human primate TfR with a Kd of from about 0.01 nM to about 550 nM. Preferably, the VHH molecules of the present invention bind to human and non-human primate TfR with a Kd lower than 500 nM, such as from about 0.01 nM to about 500 nM, or from about 0.1 nM to about 500 nM.

[0162] In certain embodiments, the VHH molecules of the present invention bind to human TfR with a Kd of from about 0.01 nM to about 100 nM.

[0163] In other certain embodiments, the VHH molecules of the present invention bind to cynomolgus monkey TfR with a Kd of from about 0.01 nM to about 550 nM, preferably from about 0.1 nM to about 500 nM.

[0164] In other specific embodiments, the VHH molecule of the present invention binds to mouse TfRs with a Kd of about 0.1 nM to about 4000 nM, preferably about 0.1 nM to about 2500 nM. In other specific embodiments, the conjugate of the present invention, for example comprising a VHH-hFc-siRNA variant, binds to human and non-human primate TfRs in the range of about 0.1 nM to about 1000 nM, or about 1 nM to about 1000 nM, preferably about 150 nM to about 1000 nM.

[0165] In other specific embodiments, the conjugates of the present invention, for example, comprising a VHH-hFc-siRNA variant, bind to human TfR in a range of about 1 nM to about 500 nM, preferably about 150 nM to about 400 nM.

[0166] In certain embodiments of the present invention, the VHH molecule is a humanized or non-humanized C5 or variant thereof, comprising or consisting of any of SEQ ID NOs: 213, 216-271, 274, 275, 675, 676, and 701 (listed in Table 1).

[0167] In other specific embodiments, the VHH molecule is C5 or a variant thereof, which is an interspecies VHH molecule that binds to human, non-human primate (NHP), and mouse TfR. In other specific embodiments, C5 or a variant thereof contains a tag sequence at the N-terminus and / or C-terminus. Specific examples of such variants include or consist of any of SEQ ID NOs: 4, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86-92, 114, 116, 118, 120, 122, 124, 126, 127, 129, or 130-149, 153-154, 393, 679, 680, 692.

[0168] In other specific embodiments, the VHH molecule is a humanized variant of C5 containing or consisting of any of SEQ ID NOs: 236-241, 252-271, or 274-275. In specific embodiments, the humanized variant of C5 contains a tag sequence at its N-terminus and / or C-terminus. Specific examples of such variants include or consisting of SEQ ID NOs: 87-92, or 130-149, or 153-154. Examples of humanized variants of C5 include the C5h20 variant containing or consisting of SEQ ID NOs: 265 or 143, or the C5V13h20 variant containing or consisting of SEQ ID NOs: 274 or 153.

[0169] In other specific embodiments, the VHH molecule of the present invention is B8 or a variant thereof, comprising or consisting of any of SEQ ID NOs: 214, 276-284, 412, 415, 418, 421, 423, 425, 428, 430, 433, 436, 439, 441, 443, 445, 447, 449, 451, 454, 457, 677, 678, 702-709, and 766-786 (listed in Table 1). In specific embodiments, B8 or a variant thereof contains a tag sequence at the N-terminus and / or C-terminus. Specific examples of such variants include or consist of any of the following: SEQ ID NOs: 8, 152, 155-159, 161, 163, 165, 167, 411, 414, 417, 420, 422, 424, 427, 429, 432, 435, 438, 440, 442, 444, 446, 448, 450, 453, 456, 681, 682, 693-700, or 745-765.

[0170] In other specific embodiments, the VHH molecule of the present invention is B8 or a variant thereof that is cross-reactive with human and primate species.

[0171] In other specific embodiments, the VHH molecule is a humanized or non-humanized B8 or a variant thereof, comprising an amino acid sequence selected from any one of SEQ ID NOs: 214, 273, 276-284, 412, 415, 418, 421, 423, 425, 428, 430, 433, 436, 439, 441, 443, 445, 447, 449, 451, 454, 457, 677, 678, 702-709, and 766-786.

[0172] In other specific embodiments, the VHH molecule of the present invention is B8 or a variant thereof, comprising or consisting of any of SEQ ID NOs: 677, 678, 681, 682, 702-709, or 693-700, 745-786, preferably any of SEQ ID NOs: 677, 678, 681, 682, 752-765, or 773-786.

[0173] In other specific embodiments, the VHH molecule is a humanized variant of B8 containing or consisting of SEQ ID NOs: 273, 152 (e.g., B8h1 variant), 752-765, or 773-786 (e.g., B8V31h1-5, h9, or B8V32h1, h6, h9-14 variants). In specific embodiments, preferred B8 variants of the present invention are B8h1, B8V32, B8V31, B8V40, B8V35, B8V32h14, and B8V32h6.

[0174] In other specific embodiments, the VHH molecule of the present invention is B6 or a variant thereof, comprising or consisting of any of SEQ ID NOs: 12, 168, 170, 172, 173, 174, 178, 181, 183, 185, 215, 285-293, which binds TfR (listed in Table 1). B6 or its variants in the present invention target the apical domain of TfR.

[0175] In other specific embodiments, the VHH molecule of the present invention binds to the apical domain of TfR, preferably the apical domain of TfR1, and is a VHH molecule comprising or consisting of SEQ ID NO: 215, 285-299. In the most preferred embodiment, the VHH molecule of SEQ ID NO: 215, 285-299 binds to human TfR1. In certain embodiments, such VHH molecules comprise a tag sequence at the N-terminus and / or C-terminus. Specific examples of such variants comprise or consist of any one of SEQ ID NO: 12, 168, 170, 172-174, 178, 181, 183, 185, 189, 193, 197, 204, 208, or 691.

[0176] In other specific embodiments, the VHH molecule is B6 or a variant thereof comprising an amino acid sequence selected from any one of SEQ ID NO: 215 and 285-293.

[0177] In other specific embodiments, the VHH molecule comprises or consists of an amino acid sequence selected from any one of SEQ ID NO: 613-618. Interestingly, such VHH molecules comprising or consisting of SEQ ID NO: 613-618 bind to the TfR of humans and non-human primates, but do not bind to mouse TfR.

[0178] Furthermore, the binding of the above-described VHH of the present invention to the human TfR receptor does not compete with the binding of transferrin, an endogenous TfR ligand, and thus does not affect the normal function of the above-described ligand. Conjugates made with such VHH molecules have been further shown to bind to TfR in vitro and to accumulate in muscle and / or muscle cells in vivo, indicating endocytosis. Thus, such VHH corresponds to a powerful agent for drug delivery or targeting to muscle.

[0179] The VHHs of the present invention can be synthesized by any technique known to those skilled in the art (biological or genetic synthesis, chemical, etc.). It can be stored as such or formulated in the presence of the substance of interest or any acceptable excipient. In chemical synthesis, natural and non-natural amino acids, such as D enantiomers and residues with side chains having different hydrophobicity and steric hindrance from natural homologs (so-called foreign, i.e., non-coding amino acids), or one or more peptide mimetic bonds that may particularly include the insertion of a methylene (-CH2-) group or a phosphate (-PO2-) group, a secondary amine (-NH-), an oxygen (-O-), or an N-alkyl peptide. Commercially available constructs that can introduce a VHH sequence, etc., are used. During synthesis, various chemical modifications can be introduced, such as inserting, linking, or conjugating a lipid (or phospholipid) derivative or a liposome or nanoparticle construct to the N-terminal and / or C-terminal positions, or side chains, in order to introduce the VHH of the present invention into a lipid membrane composed of one or more lipid layers or lipid bilayers, such as the lipid membrane of a liposome or nanoparticle. Liposomes and nanoparticles are examples of "vehicles" that can be conjugated to one or more VHH molecules of the present invention. Also, the VHH of the present invention can also be obtained from the nucleic acid sequence encoding it, as further described below (see Table 2 and the Sequence Listing).

[0180] Conjugate A further object of the present invention relates to a conjugate (also referred to herein as a "chimeric drug" without distinction) comprising one or more VHH molecules as defined above conjugated to at least one further compound, particularly at least one further molecule of interest, a drug, or a compound, such as at least one oligonucleotide or a scaffold of interest.

[0181] In an embodiment, the present invention is (i) one or more VHH molecules of the formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and (ii) a conjugate compound comprising one or more oligonucleotidesThe VHH molecules described above bind to TfR on the surface of muscle cells, and the VHH molecules described above are: 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; SEQ ID NOs: 17, 2, and 3; or SEQ ID NOs: 19, 2, and 3; or SEQ ID NOs: 1, 21, and 3; or SEQ ID NOs: 1, 23, and 3; or SEQ ID NOs: 1, 2, and 25; or SEQ ID NOs: 1, 2, and 27; or SEQ ID NOs: 1, 2, and 29; or SEQ ID NOs: 1, 2, and 31; or SEQ ID NOs: 1, 2 , and 33; or SEQ ID NOs. 67, 2, and 3; or SEQ ID NOs. 69, 2, and 3; or SEQ ID NOs. 1, 71, and 3; or SEQ ID NOs. 1, 73, and 3; or SEQ ID NOs. 1, 75, and 3; or SEQ ID NOs. 1, 2, and 77; or SEQ ID NOs. 1, 2, and 79; or SEQ ID NOs. 1, 2, and 81; or SEQ ID NOs. 1, 2, and 83; or SEQ ID NOs. 1, 2, and 85; or SEQ ID NOs. 392, 2, and 3; or SEQ ID NOs. 1, 113, and 3; or SEQ ID NOs. 1, 115, and 3; or SEQ ID NOs. 1, 2, and 11 7; or SEQ ID NOs: 1, 2, and 119; or SEQ ID NOs: 1, 2, and 121; or SEQ ID NOs: 1, 2, and 123; or SEQ ID NOs: 125, 2, and 3; or SEQ ID NOs: 17, 73, and 3; or SEQ ID NOs: 17, 128, and 3; or SEQ ID NOs: 5, 160, and 7; or SEQ ID NOs: 5, 162, and 7; or SEQ ID NOs: 5, 164, and 7; or SEQ ID NOs: 5, 166, and 7; or SEQ ID NOs: 9, 169, and 11; or SEQ ID NOs: 9, 171, and 11; or SEQ ID NOs: 175, 176, and 177; or Column numbers 179, 176, and 180; or sequence numbers 182, 176, and 177; or sequence numbers 184, 176, and 177; or sequence numbers 186, 187, and 188; or sequence numbers 190, 191, and 192; or sequence numbers 194, 195, and 196; or sequence numbers 198, 199, and 200; or sequence numbers 201, 202, and 203; or sequence numbers 205, 206, and 207; or sequence numbers 410, 6, and 7; or sequence numbers 413, 6, and 7; or sequence numbers 5, 416, and 7;or SEQ ID NOs. 5, 419, and 7; or SEQ ID NOs. 426, 6, and 7; or SEQ ID NOs. 5, 431, and 7; or SEQ ID NOs. 434, 6, and 7; or SEQ ID NOs. 437, 6, and 7; or SEQ ID NOs. 5, 6, and 452; or SEQ ID NOs. 5, 6, and 455; or SEQ ID NOs. 607, 608, and 609; or SEQ ID NOs. 610, 611, and 612; or SEQ ID NOs. 671, 2, and 3; or SEQ ID NOs. 672, 2, and 3; or SEQ ID NOs. 673, 6, and 7; or SEQ ID NOs. 674, 6, and 7; or SEQ ID NOs. 1, 2, and 713; or SEQ ID NOs. 5, 6, and 714; or include SEQ ID NOs. 674, 164, and 7; or SEQ ID NOs. 710, 6, and 7; or SEQ ID NOs. 5, 6, and 715; or SEQ ID NOs. 674, 712, and 7; or SEQ ID NOs. 711, 6, and 7; or SEQ ID NOs. 673, 6, and 741; or SEQ ID NOs. 673, 6, and 742; or SEQ ID NOs. 673, 6, and 743; or SEQ ID NOs. 673, 431, and 741; or SEQ ID NOs. 673, 431, and 742; or SEQ ID NOs. 673, 431, and 743; or SEQ ID NOs. 673, 6, and 7; or SEQ ID NOs. 674, 6, and 7.

[0182] In certain embodiments, the present invention is (i) One or more VHH molecules of formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and (ii) With respect to a conjugate compound comprising one or more oligonucleotides, The VHH molecules described above bind to TfR on the surface of both muscle cells and nervous system cells, and the VHH molecules described above are as follows: 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; SEQ ID NOs: 17, 2, and 3; or SEQ ID NOs: 19, 2, and 3; or SEQ ID NOs: 1, 21, and 3; or SEQ ID NOs: 1, 23, and 3; or SEQ ID NOs: 1, 2, and 25; or SEQ ID NOs: 1, 2, and 27; or SEQ ID NOs: 1, 2, and 29; or SEQ ID NOs: 1, 2, and 31; or SEQ ID NOs: 1, 2, and 33; or SEQ ID NOs: 67, 2, and 3; or SEQ ID NOs: 69, 2, and 3; or SEQ ID NOs: 1, 71, and 3; or SEQ ID NOs: 1, 73, and 3; or SEQ ID NOs: 1, 75, and 3; or SEQ ID NOs: 1, 2, and 77; or SEQ ID NOs: 1, 2, and 79; or SEQ ID NOs: 1, 2, and 81; or SEQ ID NOs: 1, 2, and 83; or SEQ ID NOs: 1, 2, and 85; or SEQ ID NOs: 392, 2, and 3; or SEQ ID NOs: 1, 113, and 3; or SEQ ID NOs: 1, 115, and 3; or SEQ ID NOs. 1, 2, and 117; or SEQ ID NOs. 1, 2, and 119; or SEQ ID NOs. 1, 2, and 121; or SEQ ID NOs. 1, 2, and 123; or SEQ ID NOs. 125, 2, and 3; or SEQ ID NOs. 17, 73, and 3; or SEQ ID NOs. 17, 128, and 3; or SEQ ID NOs. 5, 160, and 7; or SEQ ID NOs. 5, 162, and 7; or SEQ ID NOs. 5, 164, and 7; or SEQ ID NOs. 5, 166, and 7; or SEQ ID NOs. 9, 169, and 11; or SEQ ID NOs. 9, 171, and 11; or SEQ ID NOs. 17 5, 176, and 177; or sequence numbers 179, 176, and 180; or sequence numbers 182, 176, and 177; or sequence numbers 184, 176, and 177; or sequence numbers 186, 187, and 188; or sequence numbers 190, 191, and 192; or sequence numbers 194, 195, and 196; or sequence numbers 198, 199, and 200; or sequence numbers 201, 202, and 203; or sequence numbers 205, 206, and 207; or sequence numbers 410, 6, and 7; or sequence numbers 413, 6, and 7;or SEQ ID NOs. 5, 416, and 7; or SEQ ID NOs. 5, 419, and 7; or SEQ ID NOs. 426, 6, and 7; or SEQ ID NOs. 5, 431, and 7; or SEQ ID NOs. 434, 6, and 7; or SEQ ID NOs. 437, 6, and 7; or SEQ ID NOs. 5, 6, and 452; or SEQ ID NOs. 5, 6, and 455; or SEQ ID NOs. 607, 608, and 609; or SEQ ID NOs. 610, 611, and 612; or SEQ ID NOs. 671, 2, and 3; or SEQ ID NOs. 672, 2, and 3; or SEQ ID NOs. 673, 6, and 7; or SEQ ID NOs. 674, 6, and 7; or SEQ ID NOs. 1, 2, and 713; or SEQ ID NOs. 5, 6, and 714; or sequence number The conjugate includes numbers 674, 164, and 7; or SEQ ID NOs. 710, 6, and 7; or SEQ ID NOs. 5, 6, and 715; or SEQ ID NOs. 674, 712, and 7; or SEQ ID NOs. 711, 6, and 7; or SEQ ID NOs. 673, 6, and 741; or SEQ ID NOs. 673, 6, and 742; or SEQ ID NOs. 673, 6, and 743; or SEQ ID NOs. 673, 6, and 744; or SEQ ID NOs. 673, 431, and 741; or SEQ ID NOs. 673, 431, and 742; or SEQ ID NOs. 673, 431, and 743; or SEQ ID NOs. 673, 6, and 7; or SEQ ID NOs. 674, 6, and 7; the conjugate is administered intracerebral, intraventricular, or intrathecally. Since the conjugate of the present invention is functionally delivered to both muscle cells and nerve cells after topical CNS (IT or ICV) administration, and therefore can be used to address and treat muscle and neuromuscular disorders not only by systemic administration but also by topical CNS (IT or ICV) administration, such topical (IT or ICV) CNS administration is considered particularly interesting by the inventors.

[0183] In other specific embodiments, the present invention relates to VHH-oligonucleotide conjugates, preferably VHH-siRNA or VHH-ASO conjugates, as defined herein, which are administered intracerebral, intraventricular, or intrathecally for use in preventing or treating muscle diseases preferably selected from myopathy, cardiomyopathy, muscular dystrophy (such as DMD, BMD, FSHD, Pompe disease, or familial hypertrophic cardiomyopathy), or neuromuscular disorders such as spinal muscular atrophy, amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, multiple sclerosis, or Huntington's disease.

[0184] The further compound to which the VHH molecule of the present invention is conjugated may be a different VHH or a non-VHH molecule. At least one further target molecule, agent, or compound may be any molecule, agent, or compound, such as a half-life extension moiety, a stabilizing group, or a scaffold, a therapeutic (i.e., active) compound, a pharmaceutical or drug, a diagnostic agent, an imaging molecule, a tracer, etc., or a vehicle containing such therapeutic, diagnostic, or imaging compound.

[0185] In certain embodiments, a chimeric agent (i.e., a conjugate) may comprise further compounds of both types, namely (i) a half-life extension moiety, a stabilizing group, or a scaffold, and (ii) a therapeutic, diagnostic, or imaging compound, or a vehicle containing the same.

[0186] The therapeutic compound is selected from, for example, peptides, polypeptides, proteins, antibodies, nucleic acids, and any fragments thereof. In a preferred embodiment, the therapeutic compound is a nucleic acid molecule as described below.

[0187] Examples of target conjugate molecules, drugs, or compounds include, without limitation, any chemical components, such as small chemical molecules (e.g., chelating agents, antibiotics, antivirals, immunomodulators, anti-cancer 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 coding genes; inhibitory nucleic acids, such as ribozymes, antisense oligonucleotides (ASOs), interfering nucleic acids (siRNA), etc.; small activated RNA (saRNA), mRNA, whole genome or part thereof; plasmids, etc.); lipid (nano) particles, cell-derived vesicles (CDVs), such as exosomes, viruses, markers, or tracers. In general, “target molecule, drug, or compound” can be any drug (active) component, whether chemical, biochemical, natural, or synthetic. Generally, the expression "chemical small molecule, drug, or compound" refers to a molecule intended for pharmaceutical purposes with a maximum molecular weight of 1000 daltons, typically between 300 and 700 daltons.

[0188] The 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.

[0189] Conjugate compounds are typically pharmaceuticals (small molecule drugs, nucleic acids, or polypeptides, such as antibodies or their fragments) or imaging agents suitable for the treatment or detection of muscle or neuromuscular diseases, such as myopathy, cardiomyopathy, muscular dystrophy (DMD, BMD, FSHD, Pompe disease, or familial hypertrophic cardiomyopathy), neuromuscular diseases (ALS, SMA, MS, CMT, or HD), or cancers of the muscle (such as rhabdomyosarcoma or leiomyosarcoma).

[0190] Furthermore, the chimeric drug may include, in addition to or instead of, the target compound described above, a half-life extending moiety or stabilizing group in order to extend the half-life of the VHH or conjugate in plasma. Thus, a particular chimeric drug in the present invention comprises (i) at least one VHH molecule, e.g., multiple VHH molecules, (ii) a half-life extending moiety or stabilizing group, (iii) the target compound, typically a therapeutic, diagnostic, or imaging compound, and optionally (iv) a vehicle in any order.

[0191] In the specific embodiments described herein, the compound of interest is a group that simultaneously enables the stabilization of the VHH molecule of the present invention and / or extends its half-life in plasma. The half-life extending portion or stabilizing group may be any group known to have a certain half-life in plasma (e.g., at least several hours) and to be substantially free of harmful biological activity. Examples of such half-life extending portions or stabilizing groups include, for example, antibodies or fragments thereof, e.g., Fc fragments of immunoglobulins, VHH molecules or variants thereof, preferably VHH molecules conjugated to albumin, large human serum proteins, e.g., albumin, HSA, or IgG, or PEG molecules.

[0192] In certain embodiments, the conjugate in the present invention comprises a low molecular weight organicalbumin moiety, a half-life extending moiety, or a stabilizing group that binds to albumin with low micromolar affinity and thus progressively releases the conjugate from albumin, thereby improving the pharmacokinetic profile of the compound of interest. Such low molecular weight organicalbumin moieties include, for example, fragments of Evans blue (EB) dye, fatty acids and their derivatives, such as a C16 group, and a 4-(p-iodophenyl)butyryl (PIB) group.

[0193] In other specific embodiments, the conjugate in the present invention comprises a half-life extension moiety or stabilizing group which is human IgG1 or IgG4, preferably an Fc fragment of IgG1. Such conjugates have the general formula VHH-hFc-siRNA or VHH-hFc-ASO.

[0194] In further specific embodiments, the conjugate in the present invention comprises a half-life extension moiety or stabilizing group that is an Fc homodimer or heterodimer.

[0195] In other embodiments, the conjugate in the present invention comprises a half-life extension moiety or stabilizing group which is a modified Fc fragment homodimer or heterodimer of IgG1 or IgG4 in which effector function is suppressed or eliminated and / or the half-life is extended. In further specific embodiments, the conjugate in the present invention comprises a modified Fc fragment of IgG1, for example, a deglycosylated Fc fragment of IgG1 due to the N297 mutation.

[0196] In further specific embodiments, the conjugates in the present invention include modified Fc fragments of IgG1, which are fragments of Fc variants having symmetric or asymmetric amino acid modifications (i.e., in one or two arms of the Fc dimer) selected from deletions, insertions, reversals, or substitutions, or combinations thereof, such as amino acid substitutions in L234A and L235A (i.e., LALA mutations). Such Fc modifications enable modulation, reduction, or elimination of Fc effector functions such as modulation of Fc receptor interactions, FcγR binding, antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cell-mediated cytotoxicity (CDC), or modulation of glycosylation.

[0197] In further specific embodiments, the conjugate in the present invention comprises a modified Fc fragment that is a fragment of IgG1 containing mutations at residue positions N434, E380, M252, I253, S254, T256, or H433, or combinations thereof. Specific examples of such mutations are E380A, M252Y, S254T, T256E, H433K, N434A, or N434F.

[0198] In other specific embodiments, the conjugate in the present invention comprises a modified Fc fragment that is a fragment of IgG1 containing mutations at residue positions E233, L234, L235, G236, G237, S239, D265, D270, P329, A327, A330, or combinations thereof. Specific examples of such mutations are E233P, L234A, L234V, L235A, ΔG236, G237A, S239A, D265A, D265N, D270N, D270A, A327G, P329A, P329G, A330S, or P331S.

[0199] Also, the conjugate in the present invention may comprise a modified Fc fragment that includes any combination of the above-described mutations, optionally further combined with a LALA mutation.

[0200] In other specific embodiments, the conjugate in the present invention may comprise a modified Fc fragment that is a fragment of IgG4 containing mutations at residue position L248, or L235, or combinations thereof. Specific examples of such mutations are L248E, L235A, or L235E.

[0201] In further specific embodiments, the conjugate in the present invention comprises a half-life extension moiety or a stabilizing group that is an albumin protein or an albumin-binding moiety.

[0202] VHH can be conjugated at the N-terminus or C-terminus of the half-life extension portion or the stabilizing group, or both thereof. When the half-life extension portion or stabilizing group is an Fc fragment, the conjugation is typically by gene fusion. The resulting protein can be monomeric or polymeric, depending on the properties of the half-life extension portion or stabilizing group. In the case of an Fc fragment, the fusion protein Fc-VHH or VHH-Fc can form homodimers or heterodimers.

[0203] In this regard, in certain embodiments, the VHH molecule of the present invention is conjugated to at least one oligonucleotide, i.e., one or more oligonucleotides (e.g., any single-stranded or double-stranded oligonucleotide, e.g., small interfering RNA (siRNA), small activating RNA (saRNA), gapmer, antisense oligonucleotide (ASO), shRNA, miRNA, aptamer RNA, or cross-linked nucleic acid (BNA), preferably ASO or siRNA) by a half-life extension moiety or stabilizing group, such as an Fc fragment of human IgG1 or IgG4, and the Fc-VHH or VHH-Fc is a homodimer or heterodimer.

[0204] For example, heterodimeric VHH-Fc fusions may be prepared using the "knob-into-hole" or "KiH" technique, containing VHH at the N-terminus (or N-ter) of one arm ("knob" arm) of a human IgG1-derived Fc dimer (hFc), and inserting a transglutaminase (TGase) enzyme-specifically recognized tag sequence (i.e., Q-tag) at the C-terminus (or C-ter) of the other arm ("hole" arm) of the Fc dimer, which may be site-specifically modified at the Q-tag to introduce an azide linker. The resulting heterodimeric VHH-hFc-Q-tag-azide intermediate can be conjugated to an alkyne-siRNA using a copper-free click reaction to produce a VHH-hFc-siRNA conjugate with a stable linker (described in Example 6 and shown in Figure 14). In certain embodiments, the conjugate in the present invention comprises a VHH-Fc heterodimer, where Fc comprises a T366W mutation in the "knob" arm of the heterodimer, and / or T366S, L368A, and Y407V mutations in the "hole" arm of the heterodimer.

[0205] In other specific embodiments, the conjugate in the present invention comprises a VHH-Fc heterodimer, where Fc comprises T366W, L234A, and L235A mutations in the "knob" arm of the heterodimer, and / or T366S, L368A, Y407V, L234A, and L235A mutations in the "hole" arm of the heterodimer.

[0206] In other specific embodiments, the conjugate in the present invention comprises a VHH-Fc heterodimer, where Fc comprises T366W, L234A, and L235A mutations in the "knob" arm of the heterodimer, and T366S, L368A, Y407V, L234A, and L235A mutations in the "hole" arm of the heterodimer. In this regard, the VHH-Fc heterodimer may comprise a modified Fc having the sequence of SEQ ID NO: 664 in the "knob" arm, and a modified Fc having the sequence of SEQ ID NO: 665 in the "hole" arm. Specific examples of such VHH-Fc heterodimers containing SEQ ID NOs: 664 and 665 are described in Example 6, and in Figures 8, 9, and 10.

[0207] All of the mutations mentioned above are determined according to the standard Kabat system for numbering the amino acid residues of antibodies.

[0208] In the conjugate compounds of the present invention, coupling can be performed by any acceptable bonding means, taking into account the chemical properties, steric hindrance, and number of the components to be conjugated. Therefore, coupling can be performed by one or more covalent, ionic, hydrogen, hydrophobic, or van der Waals bonds, which are cleavable or incapable in a physiological medium or intracellularly, preferably cleavable, especially when the present invention is used in a context where at least one activator is delivered to a muscle site. Furthermore, coupling can be performed at various reactive groups, particularly at one or more terminals and / or at one or more internal or lateral reactive groups. Coupling can also be performed using genetic engineering.

[0209] A strong interaction is required between the VHH and the different transporter to prevent dissociation of the VHH and the different transporter before the conjugate reaches its site of action (i.e., the muscle site). For this reason, the preferred coupling in the present invention is a covalent coupling, although non-covalent couplings may also be used. The compound of interest can be coupled to the VHH at one of its terminals (N-terminus or C-terminus) or at a side chain in one of the constituent amino acids of the sequence (Majumdar S. and Siahaan TJ., “Peptide-mediated targeted drug delivery” Med Res Rev., 2012 May;32(3):637-58). The compound of interest can be coupled to the VHH directly or indirectly via a linker or spacer. Means of covalent chemical coupling with or without linkers include conventional bioconjugation techniques, such as those selected from bifunctional or polyfunctional materials containing alkyl, aryl, thiol, azide, alkyne, nucleotide, or peptide groups, derived from esters, aldehydes, or alkyl or aryl acids, anhydrides, sulfhydryl or carboxyl groups, or groups derived from cyanogen bromide or cyanogen chloride, carbonyl diimidazole, succinimide ester, or sulfonic acid halides. This may further include specific enzymatic conjugation, for example, via a bacterial transglutaminase that catalyzes the amide group transfer of glutamine, provided that glutamine is inserted into a specific tag.

[0210] In certain embodiments, coupling (or conjugation) is involved in gene fusion. Such strategies can be used when the coupling molecule is a peptide or polypeptide. In this case, the nucleic acid molecule encoding VHH fused with the molecule is prepared and expressed in any suitable expression system to produce the conjugate. A general structure of the VHH-oligoconjugate of the present invention is shown in Figure 13, and example strategies for conjugating the VHH of the present invention into a molecule or scaffold are disclosed in Figure 14.

[0211] In other specific embodiments, coupling (or conjugation) is carried out using thiol / maleimide chemical reaction techniques. To bring about this reaction, VHH is fused at the C-terminus of a further cysteine-containing peptide sequence. In particular, the further peptide tag is typically (GGGGS)[C]"HHHHHH" (SEQ ID NO: 399), where the parentheses indicate a Gly linker used as a spacer and the quotation marks indicate a 6His tag used for purification purposes.

[0212] Since VHH contains only cysteine ​​involved in disulfide crosslinking, any additional cysteine ​​introduced into the tag will be the only one that is chemically reactive with maleimide. This enables specific conjugation of VHH with the maleimide derivatization molecule of interest. This reaction proceeds in two steps. First, since additional cysteine ​​in the tag may be partially involved in disulfide crosslinking during the preparation process (formation of disulfide bonds with other VHH-(GGGGS)[C]"HHHHHH" or free cysteine), it is necessary to smoothly reduce VHH-GGGGS[C]HHHHHH. Therefore, the first step is based on partial reduction using a weak reducing agent such as 2-MEA (2-mercaptoethanol), TCEP (tris(2-carboxyethyl)phosphine), or DTT (dl-1,4-dithiothreitol). In the second step, VHH-GGGGS[C]HHHHHH is reacted with the target maleimide functionalized molecule at a pH in the range of 6.5 to 7.5 to form a covalently stably linked VHH-molecular conjugate. These two steps can be performed sequentially or together in situ.

[0213] In other specific embodiments, coupling (or conjugation) is carried out by an enzymatic reaction. In particular, site-directed conjugation of VHH can be performed using a 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) that is specifically recognized by Tgase and (ii) an amino-functionalized donor substrate. In this regard, the inventors have developed a specific tag sequence (referred to as the "Q tag") that is recognized by Tgase and can be used to couple the VHH of the present invention with any molecule of interest that is either a chemical drug or a pharmaceutical agent, or with a heterobifunctional linker for further conjugation with a chemical drug or pharmaceutical agent. For this purpose, the VHH is prepared by gene fusion such that the following tags are added in tandem (typically to its C-terminus): first optionally a trialanine linker, then optionally a His tag, then optionally a low-molecular-weight triglycine linker, and finally a Q tag. The triglycerin linker aims to separate the Q tag, allowing Tgase to access glutamine more effectively, while the His tag aims to facilitate the purification of VHH and its further functionalized types.

[0214] The common conjugation strategies being developed are convergent synthesis based on processes that include the following: (1) To further conjugate the target molecule, the reactive moiety is introduced into Q-tagged glutamine fused to VHH. For this purpose, a heterobifunctional conjugation linker having two different reactive ends, one of which is a suitable primary amine group for Tgase and the other being an orthogonal reactive moiety, can be processed by Tgase. Typical examples of such orthogonal reactive groups include azides, restricted alkynes such as DBCO (dibenzocyclooctin) or BCN (bicyclo[6.1.0]nonine), tetrazines, TCO (trans-cyclooctene), free or protected thiols, maleimides, etc. (2) A reactive group complementary to the one incorporated into the Q tag of VHH is introduced into the target molecule. Typical examples of such orthogonal reactive groups include azides, restricted alkynes such as DBCO or BCN, tetrazines, TCOs, free or protected thiols, maleimides, etc. (3) These complementary reactive groups conjugate both the functionalized VHH and the molecule.

[0215] Furthermore, a method for coupling two molecules via a Tgase coupling reaction using a Q tag as defined above is described herein. A further object of the present invention is a VHH containing a Q tag. A further object of the present invention is a linker such as a Gly linker and a VHH molecule containing a Q tag.

[0216] The preferred VHH of the present invention has the following structure: VHH-linker-His m -Linker-LQR, where VHH is any VHH molecule, 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 m is 6 or 8.

[0217] In certain embodiments, the present invention relates to a conjugate comprising at least one VHH covalently linked to at least one chemical component. Preferred variants of such a conjugate comprise one VHH and one chemical component.

[0218] In other specific embodiments, the present invention relates to a conjugate comprising at least one nucleic acid and at least one VHH covalently linked thereto. The nucleic acid may be an antisense oligonucleotide ("ASO"), ribozyme, aptamer, siRNA, etc. Preferred variants of such conjugates include one VHH and one nucleic acid molecule. In preferred embodiments, the nucleic acid is an oligonucleotide (i.e., a short DNA or RNA molecule, also referred to herein as "oligo" or "oligomer") selected from any single-stranded or double-stranded oligonucleotide that can specifically bind to a target mRNA and thereby regulate gene expression in a cell, e.g., small interfering RNA (siRNA), small activating RNA (saRNA), gapmer, antisense oligonucleotide (ASO), shRNA, miRNA, aptamer RNA, or cross-linked nucleic acid (BNA), etc. Preferably, such oligonucleotide is an ASO or siRNA that can regulate (repress or inhibit or activate) the expression of a target gene.

[0219] The conjugate in the present invention is complementary to the target gene and, in certain embodiments, can suppress or inhibit the expression of the target gene by at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. More specifically, the conjugate in the present invention can suppress or inhibit the expression of the target gene by about 40% to about 80%.

[0220] Accordingly, in certain embodiments, the present invention relates to a conjugate compound comprising at least one VHH covalently linked to any single-stranded or double-stranded oligonucleotide, selected from, for example, small interfering RNA (siRNA), small activating RNA (saRNA), gapmers, antisense oligonucleotides (ASOs), shRNA, miRNA, aptamer RNA, or cross-linked nucleic acids (BNA), which preferably binds to TfR on the surface of muscle cells and specifically binds to target mRNA, thereby regulating gene expression in the cell.

[0221] In a preferred embodiment, the present invention is (i) One or more VHH molecules that bind to TfR on the surface of muscle cells, CDR1 containing sequences selected from sequence numbers 1, 5, 9, 13, 17, 19, 67, 69, 125, 175, 179, 182, 184, 186, 190, 194, 198, 201, 205, 392, 410, 413, 426, 434, 437, 607, 610, 671-674, 710, and 711, and / or CDR2 containing sequences selected from sequence numbers 2, 6, 10, 14, 21, 23, 71, 73, 75, 113, 115, 128, 160, 162, 164, 166, 169, 171, 176, 187, 191, 195, 199, 202, 206, 416, 419, 431, 608, 611, and 712, and / or One or more VHH molecules containing a CDR3 containing a sequence selected from sequence numbers 3, 7, 11, 15, 25, 27, 29, 31, 33, 77, 79, 81, 83, 85, 117, 119, 121, 123, 177, 180, 188, 192, 196, 200, 203, 207, 452, 455, 609, 612, 713-715, and 741-744, as well as, (ii) The present invention relates to a conjugate compound comprising one or more oligonucleotides selected from any single-stranded or double-stranded oligonucleotides, such as small interfering RNA (siRNA), small activating RNA (saRNA), gapmers, antisense oligonucleotides (ASOs), shRNA, miRNA, aptamer RNA, or cross-linked nucleic acids (BNAs), which can specifically bind to a target mRNA and thereby reduce its expression level in cells.

[0222] In other preferred embodiments, the present invention is (i) Binds to TfR on the surface of muscle cells, and Sequence IDs 1, 2, and 3; or Sequence IDs 5, 6, and 7; or Sequence IDs 9, 10, and 11; or Sequence IDs 13, 14, and 15; Sequence IDs 17, 2, and 3; or Sequence IDs 19, 2, and 3; or Sequence IDs 1, 21, and 3; or Sequence IDs 1, 23, and 3; or Sequence IDs 1, 2, and 25; or Sequence IDs 1, 2, and 27; or Sequence IDs 1, 2, and 29; or Sequence IDs 1, 2, and 31; or Sequence IDs 1, 2, and 33; or Sequence ID number Sequence numbers 67, 2, and 3; or Sequence numbers 69, 2, and 3; or Sequence numbers 1, 71, and 3; or Sequence numbers 1, 73, and 3; or Sequence numbers 1, 75, and 3; or Sequence numbers 1, 2, and 77; or Sequence numbers 1, 2, and 79; or Sequence numbers 1, 2, and 81; or Sequence numbers 1, 2, and 83; or Sequence numbers 1, 2, and 85; or Sequence numbers 392, 2, and 3; or Sequence numbers 1, 113, and 3; or Sequence numbers 1, 115, and 3; or Sequence numbers 1, 2, and 117; or Sequence numbers 1, 2, and 119; or SEQ ID NOs: 1, 2, and 121; or SEQ ID NOs: 1, 2, and 123; or SEQ ID NOs: 125, 2, and 3; or SEQ ID NOs: 17, 73, and 3; or SEQ ID NOs: 17, 128, and 3; or SEQ ID NOs: 5, 160, and 7; or SEQ ID NOs: 5, 162, and 7; or SEQ ID NOs: 5, 164, and 7; or SEQ ID NOs: 5, 166, and 7; or SEQ ID NOs: 9, 169, and 11; or SEQ ID NOs: 9, 171, and 11; or SEQ ID NOs: 175, 176, and 177; or SEQ ID NOs: 179, 176, and 180 ; or SEQ ID NOs. 182, 176, and 177; or SEQ ID NOs. 184, 176, and 177; or SEQ ID NOs. 186, 187, and 188; or SEQ ID NOs. 190, 191, and 192; or SEQ ID NOs. 194, 195, and 196; or SEQ ID NOs. 198, 199, and 200; or SEQ ID NOs. 201, 202, and 203; or SEQ ID NOs. 205, 206, and 207; or SEQ ID NOs. 410, 6, and 7; or SEQ ID NOs. 413, 6, and 7; or SEQ ID NOs. 5, 416, and 7; or SEQ ID NOs. 5, 419, and 7;or SEQ ID NOs. 426, 6, and 7; or SEQ ID NOs. 5, 431, and 7; or SEQ ID NOs. 434, 6, and 7; or SEQ ID NOs. 437, 6, and 7; or SEQ ID NOs. 5, 6, and 452; or SEQ ID NOs. 5, 6, and 455; or SEQ ID NOs. 607, 608, and 609; or SEQ ID NOs. 610, 611, and 612; or SEQ ID NOs. 671, 2, and 3; or SEQ ID NOs. 672, 2, and 3; or SEQ ID NOs. 673, 6, and 7; or SEQ ID NOs. 674, 6, and 7; or SEQ ID NOs. 1, 2, and 713; or SEQ ID NOs. 5, 6, and 714; or SEQ ID NOs. 674, 164, and One or more VHH molecules, including 7; or SEQ ID NOs. 710, 6, and 7; or SEQ ID NOs. 5, 6, and 715; or SEQ ID NOs. 674, 712, and 7; or SEQ ID NOs. 711, 6, and 7; or SEQ ID NOs. 673, 6, and 741; or SEQ ID NOs. 673, 6, and 742; or SEQ ID NOs. 673, 6, and 743; or SEQ ID NOs. 673, 6, and 744; or SEQ ID NOs. 673, 431, and 741; or SEQ ID NOs. 673, 431, and 742; or SEQ ID NOs. 673, 431, and 743; or SEQ ID NOs. 673, 6, and 7; or SEQ ID NOs. 674, 6, and 7; and; (ii) The present invention relates to a conjugate compound comprising one or more oligonucleotides selected from any single-stranded or double-stranded oligonucleotides, such as small interfering RNA (siRNA), small activating RNA (saRNA), gapmers, antisense oligonucleotides (ASOs), shRNA, miRNA, aptamer RNA, or cross-linked nucleic acids (BNAs), which can specifically bind to a target mRNA and thereby regulate gene expression in a cell.

[0223] In a preferred embodiment, the conjugate of the present invention (i) binds to TfR on the surface of muscle cells, and SEQ ID NOs: 4, 8, 12, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86-92, 114, 116, 118, 120, 122, 124 , 126, 127, 129~149, 152~159, 161, 163, 165, 167, 168, 170, 172~174, 178, 181, 183, 185, 189, 193, 197, 204, 208, 213~271, 273~299, 393, 411~412, 414~415, 417~418, 420~425, 427~4 (ii) comprising at least one VHH molecule including or consisting of any of 30, 432-433, 435-436, 438-451, 453-454, 456-457, 691-709, or 745-786, and (ii) at least one oligonucleotide selected from any single-stranded or double-stranded oligonucleotide capable of specifically binding to a target mRNA and thereby regulating gene expression in a cell, such as small interfering RNA (siRNA), small activating RNA (saRNA), gapmers, antisense oligonucleotides (ASOs), shRNA, miRNA, aptamer RNA, or cross-linked nucleic acids (BNA).

[0224] In other preferred embodiments, the conjugate of the present invention is (i) C5 or a variant thereof (listed in Table 1), which is an interspecies VHH molecule that binds to human, non-human primate (NHP), and mouse TfR on the surface of muscle cells, and SEQ ID NOs: 4, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86-92, 114, 116, 118, 120, 122, 124, 126, 127, 129, or 130-149, 153-154, 213, 216-271, 274-275, 393, (ii) comprising one or more VHH molecules comprising or consisting of any of 675, 676, 679, 680, 692, or 701, and (ii) one or more oligonucleotides selected from any single-stranded or double-stranded oligonucleotides that can specifically bind to a target mRNA and thereby regulate gene expression in a cell, such as small interfering RNA (siRNA), small activating RNA (saRNA), gapmers, antisense oligonucleotides (ASOs), shRNA, miRNA, aptamer RNA, or cross-linked nucleic acids (BNA).

[0225] In other preferred embodiments, the conjugate of the present invention comprises (i) at least one VHH molecule which is a humanized variant of C5 (listed in Table 1) including or consisting of any of SEQ ID NOs: 87-92, 130-149, 153-154, 236-241, 252-271, or 274-275; and (ii) at least one oligonucleotide, selected from any single-stranded or double-stranded oligonucleotide that can specifically bind to a target mRNA and thereby regulate gene expression in a cell, such as small interfering RNA (siRNA), small activating RNA (saRNA), gapmers, antisense oligonucleotides (ASOs), shRNA, miRNA, aptamer RNA, or cross-linked nucleic acids (BNA).

[0226] In other preferred embodiments, the conjugate of the present invention comprises (i) one or more VHH molecules that bind to the apical domain of TfR, preferably the apical domain of TfR1, on the surface of muscle cells and are B6 or a variant thereof (listed in Table 1), comprising or consisting of any of SEQ ID NOs: 12, 168, 170, 172-174, 178, 181, 183, 185, 215, 285-299; and (ii) one or more oligonucleotides selected from any single-stranded or double-stranded oligonucleotides that can specifically bind to a target mRNA and thereby regulate gene expression in the cell, such as small interfering RNA (siRNA), small activating RNA (saRNA), gapmers, antisense oligonucleotides (ASOs), shRNA, miRNA, aptamer RNA, or cross-linked nucleic acids (BNA).

[0227] In other preferred embodiments, the conjugate of the present invention comprises (i) one or more VHH molecules that bind to the apical domain of TfR, preferably the apical domain of TfR1, on the surface of muscle cells, and comprising or including any of SEQ ID NOs: 12, 168, 170, 172-174, 178, 181, 183, 185, 189, 193, 197, 204, 208, 215, 285-299, or 691; and (ii) one or more oligonucleotides selected from any single-stranded or double-stranded oligonucleotides that can specifically bind to a target mRNA and thereby regulate gene expression in the cell, such as small interfering RNA (siRNA), small activating RNA (saRNA), gapmers, antisense oligonucleotides (ASOs), shRNA, miRNA, aptamer RNA, or cross-linked nucleic acids (BNA).

[0228] In another preferred embodiment, the conjugate of the present invention is (i) any of SEQ ID NOs: 8, 152, 155-159, 161, 163, 165, 167, 214, 273, 276-284, 411, 412, 414, 415, 417, 418, 420-425, 427-430, 432, 433, 435, 436, 438-451, 453, 454, 456, 457, 677, 678, 681, 682, 693-700, 702-709, 745-786, preferably SEQ ID NOs: 677, 678, 681, 682, 752-765, or 773-786 (ii) one or more VHH molecules which are B8 or a variant thereof (listed in Table 1) and (ii) one or more oligonucleotides which are any single-stranded or double-stranded oligonucleotides which can specifically bind to a target mRNA and thereby regulate gene expression in a cell, selected from, for example, small interfering RNA (siRNA), small activating RNA (saRNA), gapmers, antisense oligonucleotides (ASOs), shRNA, miRNA, aptamer RNA, or cross-linked nucleic acids (BNAs).

[0229] Specific examples of conjugation methods used to conjugate VHH molecules to oligonucleotides include a direct thiol-maleimide chemical reaction by introducing an unpaired cysteine ​​to the C-terminus of VHH, or an indirect SPAAC reaction (strain-enhanced alkyne-assisted click) via a preliminary site-directed enzyme-assisted reaction in VHH to introduce a suitable component for a click reaction. Various linkers, which may be stable or cleavable, can be used between the two partners, VHH and the oligonucleotide. Examples of cleavable linkers include disulfides, enzyme-unstable peptide linkers such as valine-citrulline or phenylalanine-lysine dipeptides, or pH-unstable linkers such as hydrazones.

[0230] In certain embodiments, VHH molecules and oligonucleotides are linked together, preferably using a copper-free click reaction, to produce a VHH-oligoconjugate having a stable or cleavable linker.

[0231] In other specific embodiments, the present invention relates to a conjugate comprising a VHH covalently linked to a peptide. The peptide may be an active molecule, bait, tag, ligand, etc. A preferred variant of such a conjugate comprises one VHH and one peptide.

[0232] In other embodiments, the present invention relates to a conjugate comprising VHH covalently linked to nanoparticles and / or liposomes, such as lipid particles or nanoparticles ("LNPs"). The nanoparticles and / or liposomes can be loaded with or functionalized with activators. Preferred variants of such conjugates include multiple VHH molecules coupled to each nanoparticle or liposome.

[0233] In further embodiments, the conjugate comprises an antibody or fragment thereof coupled with one or more VHH molecules as a half-life extension portion or stabilizing group. Typically, the VHH molecule is coupled to the C-terminus or N-terminus of the heavy chain or light chain, or both, or to the C-terminus or N-terminus of the Fc fragment. In a preferred embodiment, the VHH molecule is coupled to the N-terminus of Fc. In a more preferred embodiment, the VHH molecule is coupled to the C-terminus of Fc.

[0234] In other specific embodiments, 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 embodiment, the VHH molecule is coupled to either the C-terminus or the N-terminus of the chain. Preferably, it is coupled to the N-terminus.

[0235] The present invention also relates to a method for preparing conjugate compounds as defined above, characterized by comprising the step of coupling VHH with a molecule or scaffold, preferably by a chemical, biochemical, or enzymatic pathway, or by genetic engineering.

[0236] In the chimeric drug of the present invention, when multiple VHHs are present, they may have similar or different binding specificities.

[0237] Nucleic acids, vectors, and host cells Further aspects of the present invention relate to a VHH as defined above, or a nucleic acid encoding that conjugate (when 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 a mixture of the two. It may contain modified nucleotides, for example, modified linkages, modified purine or pyrimidine bases, or modified sugars. It may be prepared by any method known to those skilled in the art, including chemical synthesis, recombination, and / or mutagenesis. The nucleic acid in the present invention may be inferred from the amino acid sequence of the VHH molecule in the present invention, and codon usage may be adapted depending on the host cell to which the nucleic acid is 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 of Sambrook et al.

[0238] Specific examples of such nucleic acid sequences include sequences containing any one of sequence numbers 301-329, 52-64, 95-110, 469-606, 646-663, 683-690, 721-738, or 796-837, either without the tag code portion or optionally containing the tag code portion of sequence number 330, and their complementary sequences. The corresponding domains encoding CDR1, CDR2, and CDR3 are underlined in Table 2 below. The tag code portion of sequence number 330 is shown in bold in Table 2.

[0239] The present invention also relates to vectors containing such nucleic acids, which are optionally under the control of regulatory sequences (e.g., promoters, terminators, etc.). Vectors may be plasmids, viruses, cosmids, phagemids, artificial chromosomes, etc. In particular, vectors may contain nucleic acids of the present invention operably ligated to regulatory regions, i.e., regions containing one or more regulatory sequences. Optionally, vectors may contain multiple nucleic acids of the present invention operably ligated to multiple regulatory regions. The term "regulatory sequence" means a nucleic acid sequence necessary for the expression of a coding region. Regulatory sequences may be endogenous or heterologous. Well-known regulatory sequences currently used by those skilled in the art would be preferred. Such regulatory sequences include, but are not limited to, promoters, signal peptide sequences, and transcription terminators. The term "operably ligated" means a configuration in which the regulatory sequence is positioned appropriately relative to the coding sequence so that the regulatory sequence controls the expression of the coding region.

[0240] The present invention further relates to the use of nucleic acids or vectors in the present invention for transforming, introducing genes into, or transducing host cells, or for producing compositions comprising pharmaceutical compositions. The present invention also provides host cells comprising one or more nucleic acids and / or one or more vectors of the present invention. The term “host cell” also includes any offspring of the parent host cell that are not identical to the parent host cell due to mutations that occurred during replication. Suitable host cells may be prokaryotes (e.g., bacteria) or eukaryotes (e.g., yeast, plants, insects, or mammalian cells). Examples for a specific description of such cells include Escherichia coli strains, CHO cells, Saccharomyces strains, plant cells, sf9 insect cells, etc.

[0241] use The VHH molecule of the present invention can bind to TfR, and therefore the molecule can be targeted / delivered to TfR-expressing muscle cells. In the context of the present invention, binding is preferably specific, and binding to TfR occurs with higher affinity than binding to any other antigen of the same species. In certain embodiments, the VHH molecule of the present invention specifically binds to human TfR1. In other specific embodiments, the VHH molecule of the present invention binds to human and non-human primate TfR1. In other specific embodiments, the VHH molecule of the present invention binds to human and rodent TfR1. In other specific embodiments, the VHH molecule of the present invention binds to human TfR1, non-human primate TfR1, and rodent (e.g., mouse or rat) TfR1. In other specific embodiments, the VHH molecule binds to human, non-human primate, and mouse receptors with substantially similar affinity.

[0242] Accordingly, the present invention relates to a method for targeting / delivering a compound to / via TfR-expressing cells or organs, comprising coupling the compound to at least one VHH of the present invention.

[0243] The present invention further relates to the use of VHH as defined above as a vector for transporting compounds to / through TfR-expressing muscle cells.

[0244] The present invention also relates to the use of VHH as defined above for preparing drugs or pharmaceuticals that can target muscle sites.

[0245] The present invention also relates to a method for enabling or improving the delivery of a compound of interest to a muscle site, which includes coupling a molecule to the VHH of the present invention.

[0246] As described above in this specification, the VHH of the present invention can be used to transport or deliver any compound, such as chelating agents, small molecule drugs, amino acids, peptides, polypeptides, proteins, lipids, nucleic acids, viruses, liposomes, exosomes, and preferably nucleic acids, to muscles.

[0247] The vehicle may be used to transport or deliver the conjugate (including VHH), such as a virus, virus-like particles (VLP), cell-derived vesicles (CDV), exosome, lipid vehicle, or polymer vehicle, preferably lipid nanoparticles (LNP), micelles, or liposomes.

[0248] The present invention also relates to pharmaceutical compositions, particularly diagnostic or therapeutic compositions, characterized by comprising at least one VHH or conjugate compound, which is bound to or not bound to a vehicle, or present or not present on a vehicle, for example, in the context of a diagnostic composition, a VHH-drug conjugate as defined above, and one or more pharmaceutically acceptable supports, carriers, or excipients.

[0249] The present invention also relates in particular to diagnostic compositions, characterized by comprising VHH or conjugate compounds that are bound to or not bound to a vehicle, or present or not present on a vehicle, such as VHH-diagnostic agents or medical imaging agent conjugate compounds as defined above.

[0250] The conjugate can be used in any pharmaceutically acceptable salt form. The expression "pharmaceutically acceptable salt" refers, for example, non-limitingly, to pharmaceutically acceptable base or acid addition salts, hydrates, esters, solvates, precursors, metabolites, or stereoisomers, the vector or conjugate loaded with at least one substance of interest. The expression "pharmaceutically acceptable salt" refers to a non-toxic salt that can generally be prepared by reacting a free base with a suitable organic or inorganic acid. These salts retain the biological efficacy and properties of the free base. Typical examples of such salts include water-soluble and water-insoluble salts, such as acetate, N-methylglucamine ammonium, amsonate (4,4-diaminostilbene-2,2'-disulfonate), benzenesulfonate, benzoate, bicarbonate, bisulfate, tartrate, borate, hydrobromide, bromide, butyrate, camsylate, carbonate, hydrochloride, chloride, citrate, clavulanate, dihydrochloride, diphosphate, edetate, calcium edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycolyl arsanilate, hexafluorophosphate, hexylresorcinate, hydravamin, hydroxynaphthoate, iodide, isothionate, and lactate. This includes lactobionates, laurates, malates, maleates, mandelates, mesylates, methyl bromides, methylnitrates, methyl sulfates, mucinates, napsylates, nitrates, 3-hydroxy-2-naphthoates, oleates, oxalates, palmitates, pamoates (1,1-methylene-bis-2-hydroxy-3-naphthoates or embonates), pantothenates, phosphates, picrates, polygalacturonates, propionates, p-toluenesulfonates, salicylates, stearates, basic acetates, succinates, sulfates, sulfosalicylates, suramates, tannates, tartrates, theoclates, tosylates, triethiodides, trifluoroacetates, and valersates.

[0251] The compositions of the present invention advantageously comprise a pharmaceutically acceptable carrier or excipient. The pharmaceutically acceptable carrier can be selected from conventionally used carriers according to each administration mode. The compound may be in solid, semi-solid, or liquid form, depending on the intended administration mode. For solid compositions, such as tablets, pills, powders, or granules that are not combined or contained in gelatin capsules, the active substance may be combined with (a) diluents, such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and / or glycine; (b) lubricants, such as silica, talc, stearic acid, its magnesium or calcium salts, and / or polyethylene glycol; (c) binders, such as magnesium silicate and aluminum silicate, starch paste, gelatin, tragacanth gum, methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone; (d) disintegrants, such as starch, agar, alginic acid or its sodium salts, or effervescent mixtures; and / or (d) adsorbents, colorants, flavorings, and sweeteners. Excipients may include, for example, mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and analogs of medicinal substances. For semi-solid compositions, such as suppositories, the excipient may be, for example, an emulsion or an oily suspension, or a polyalkylene glycol, such as a polypropylene glycol-based compound. Liquid compositions, especially those to be included 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, dextrose aqueous solution, glycerol, ethanol, oil, and analogs thereof.

[0252] The composition or conjugate of the present invention can be administered by any suitable route, and not limited to, parenteral routes, such as in the form of formulations that can be injected via subcutaneous (SC), intravenous (IV), or intramuscular (IM) routes, or intracerebral (IC), intraventricular (ICV), or intrathecal (IT) routes; or orally (or per os) routes, such as coated tablets or uncoated tablets, gelatin capsules, powders, pellets, suspensions, or oral solutions (one such form for oral administration may have immediate release or long-term or delayed release); or rectally, such as in the form of suppositories; or topically, such as in the form of patches, pomades, or gels; particularly transdermally, such as intranasal, translingual, or intraocular routes, such as in aerosols and sprays. Preferably, the VHH or conjugate of the present invention, or a composition containing such VHH or conjugate, is administered intravenously or subcutaneously.

[0253] The pharmaceutical composition typically comprises an effective dose of the VHH or conjugate of the present invention. The “therapeutic effective dose” of the conjugate of the present invention is, for example, about 1 nmole to about 500 nmoles (nmole / kg) per kilogram of body weight of the subject to whom the conjugate is administered, preferably about 10 nmole / kg to about 500 nmole / kg. It is understood that the “therapeutic effective dose” in a person will depend on a variety of factors, including, in particular, the activity / efficacy of the active substance, the time of its administration, the route of its administration, its toxicity, its excretion rate and metabolism, drug combinations / interactions, and the severity of the disease (or disorder) being treated on a preventive or curative basis, as well as the patient’s age, weight, overall health status, sex and / or diet.

[0254] Depending on the substance being coupled, the conjugates and compositions of the present invention can be used to image, diagnose, prevent and / or treat pathological conditions or disorders affecting muscle, such as any muscle disease or neuromuscular disease, preferably, for example, myopathy, cardiomyopathy, muscular dystrophy (DMD, BMD, FSHD, Pompe disease, or familial hypertrophic cardiomyopathy, etc.), neuromuscular diseases (ALS, SMA, MS, CMT, or HD, etc.), and cancers of muscle (rhabdomyosarcoma, leiomyosarcoma, etc.). The VHH of the present invention has the ability to target TfR-expressing cells, particularly muscle cells (such as skeletal muscle cells, cardiomyocytes, or muscle cancer cells as described herein), and / or to pass through the muscle cell membrane. TfRs are abundant in muscle compared to different organs such as bone marrow, placenta, and the digestive tract.

[0255] In this regard, the present invention relates to the use of pharmaceutical conjugates or pharmaceutical compositions (particularly therapeutic compositions) as described herein above for the prevention or treatment of muscular pathologies or disorders, such as, not limited to, any muscular or neuromuscular diseases.

[0256] In the context of the present invention, muscle disease is a muscle disease or neuromuscular disease preferably selected from myopathy, cardiomyopathy, muscular dystrophy (such as DMD, BMD, FSHD, Pompe disease, or familial hypertrophic cardiomyopathy), neuromuscular disease (such as ALS, SMA, MS, CMT, or HD), or cancer of muscle (such as rhabdomyosarcoma or leiomyosarcoma). Preferred muscle diseases are selected from myopathy, cardiomyopathy, DMD, BMD, FSHD, Pompe disease, and familial hypertrophic cardiomyopathy. Preferred neuromuscular diseases are selected from ALS, SMA, MS, CMT, and HD.

[0257] The present invention also relates to VHH, conjugates, or pharmaceutical compositions as described herein above for use in the treatment of muscle pathologies or disorders, such as myopathy, cardiomyopathy, muscular dystrophy (DMD, BMD, FSHD, Pompe disease, or familial hypertrophic cardiomyopathy, etc.), neuromuscular diseases (ALS, SMA, MS, CMT, or HD, etc.), or cancers of the muscle (rhabdomyosarcoma or leiomyosarcoma, etc.).

[0258] The present invention also relates to VHH, conjugates, or pharmaceutical compositions as defined above for use in the treatment of pathological conditions affecting muscles, for example, not limited to muscle diseases or neuromuscular diseases, such as myopathy, cardiomyopathy, muscular dystrophy (DMD, BMD, FSHD, Pompe disease, or familial hypertrophic cardiomyopathy, etc.), neuromuscular diseases (ALS, SMA, MS, CMT, or HD, etc.), or cancers of the muscle (rhabdomyosarcoma or leiomyosarcoma, etc.).

[0259] The present invention also relates to VHH, conjugates, or pharmaceutical compositions as defined above for use in imaging, diagnosis, prevention, and / or treatment of muscle diseases or neuromuscular diseases, such as myopathy, cardiomyopathy, muscular dystrophy (DMD, BMD, FSHD, Pompe disease, or familial hypertrophic cardiomyopathy, etc.), neuromuscular diseases (ALS, SMA, MS, CMT, or HD, etc.), or cancers of muscle (rhabdomyosarcoma or leiomyosarcoma, etc.).

[0260] In certain embodiments, the present invention also relates to VHH, conjugates, or pharmaceutical compositions as defined above for use in the prevention and / or treatment of muscle diseases or neuromuscular diseases, such as myopathy, cardiomyopathy, muscular dystrophy (DMD, BMD, FSHD, Pompe disease, or familial hypertrophic cardiomyopathy, etc.), neuromuscular diseases (ALS, SMA, MS, CMT, or HD, etc.), wherein the VHH described above binds to TfR on the surface of both muscle cells and CNS cells.

[0261] In other specific embodiments, the present invention also relates to VHH, conjugates, or pharmaceutical compositions as defined above for use in the prevention and / or treatment of muscle diseases or neuromuscular diseases, such as myopathy, cardiomyopathy, muscular dystrophy (DMD, BMD, FSHD, Pompe disease, or familial hypertrophic cardiomyopathy, etc.), neuromuscular diseases (ALS, SMA, MS, CMT, or HD, etc.), the VHH, conjugates, or compositions being administered intracerebral, intraventricular, or intrathecally.

[0262] In other specific embodiments, the present invention also relates to VHH, conjugates, or pharmaceutical compositions as defined above for use in the prevention and / or treatment of muscle diseases or neuromuscular diseases, such as myopathy, cardiomyopathy, muscular dystrophy (DMD, BMD, FSHD, Pompe disease, or familial hypertrophic cardiomyopathy, etc.), neuromuscular diseases (ALS, SMA, MS, CMT, or HD, etc.), wherein the VHH, conjugates, or compositions are administered intracerebral, intraventricular, or intrathecally, and the VHH described above binds to TfR on the surface of both muscle cells and CNS cells.

[0263] Furthermore, the present invention relates to VHHs, conjugates, or pharmaceutical compositions as defined 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 delivery of recombinant (e.g., replication-deficient or attenuated) viruses used in gene therapy, such as adenoviruses, adeno-associated viruses, lentiviruses, retroviruses, or virus-like particles, to any TfR-rich muscle tissue. Coupling with a virus or VLP can be performed, for example, by coupling to the viral capsid protein.

[0264] The present invention also relates to a method for preventing or treating any of the above-mentioned conditions or diseases by administering the VHH, conjugate, or composition of the present invention to a subject requiring administration.

[0265] The present invention also relates to the use of the VHH, conjugate, or composition of the present invention for the manufacture of a pharmaceutical product for treating any of the above-mentioned conditions or diseases.

[0266] The following examples are included solely to illustrate specific aspects and embodiments of the present invention and are not intended to limit the scope of the claims set forth herein. [Examples]

[0267] Example 1: Measurement of VHH binding properties for hTfR, mTfR, and rhTfR The binding properties of VHH with affinity for TfR in various species (i.e., humans (h), mice (m), and rhesus monkeys (rh)) were tested using flow cytometry, and apparent affinity (K) was determined. d app The following measurements were taken in a 96-well plate: 2 × 10⁻⁶ 5 All experiments were performed using cell / wells with shaking at 4°C. CHO cell lines expressing EGFP-fused TfR, or CHO wild-type cells, were saturated with 2% PBS / BSA solution for 30 minutes to avoid nonspecific binding, and then incubated with increasing concentrations of purified VHH for 1 hour. After one wash in 2% PBS / BSA, cells were incubated with anti-6His tagged antibody (mouse) for 1 hour, washed twice in 2% PBS / BSA, and incubated with Alexa647 conjugate anti-mouse secondary antibody for 1 hour. After the final two washes in 2% PBS / BSA, cells were fixed by incubation in 2% PBS / PFA for 15 minutes, washed once in PBS, and finally resuspended in PBS. Fluorescence levels were assessed using an Attune NxT flow cytometer (Thermo Fisher Scientific). Experimental data were fitted using nonlinear fitting in GraphPad Prism software to determine apparent K d The number of units has been determined.

[0268] In control conditions, where cells were incubated with control VHH D12 or C5neg, no nonspecific labeling was present. All VHHs tested induced concentration-dependent changes in the signal, confirming binding to the target receptor. Labeling of CHO wild-type control cells was not detected with any of the tested VHHs (not shown). Below, Table 5 shows the apparent K obtained for the tested VHHs. d This summarizes the findings. Most VHHs exhibit interspecies reactivity, with apparent K in the range of 0.1 nM to 4 μM. d We then bound to all three human, mouse, and rhesus monkey TfRs, revealing strong interest and diversity in the constructed VHH libraries.

[0269] [Table 5] JPEG2026521069000001.jpg222158JPEG2026521069000002.jpg229158JPEG2026521069000003.jpg156158

[0270] Furthermore, the binding properties of some VHHs were evaluated using surface plasmon resonance (SPR) experiments. The external domains of human, mouse, and rhesus monkey TfRs (with GeneBank numbers NM_003234.2, NM_011638, and NC_041755.1, respectively) were fused to the N-terminus of mouse IgG1 Fc fragments, and recombinant proteins were prepared and purified in-house. The interaction between VHHs and receptors was tested using a Biacore T200 (GE Healthcare). Receptors were immobilized directly or to previously immobilized anti-mouse IgG on an HC1500M or CM5 sensor chip (Xantec). VHHs were injected into flow cells using single-cycle or multi-cycle kinetics. Table 6 below summarizes the binding properties of the tested VHHs. These results further demonstrate the interspecies reactivity and diversity of binding parameters of VHHs.

[0271] [Table 6] JPEG2026521069000004.jpg229160JPEG2026521069000005.jpg229160JPEG2026521069000006.jpg229160JPEG2026521069000007.jpg130160

[0272] Example 2: Oligonucleotide sequence and its conjugation with TfR-bound VHH The in vitro and in vivo experiments described in the examples below were performed using the chemically modified siRNAs or gapmer ASO sequences listed in Table 7 below. The RNAi-competent siRNA sequences used in the following examples target ubiquitous superoxide dismutase 1 (SOD1) mRNA in either mouse and rat (siSOD1m) as described in International Publication No. 2019 / 217459, or human and non-human primate (NHP, including Papio anubis, Macaca mulatta, and Macaca fascicularis) (siSOD1h) adapted from siSOD1m to match SOD1 mRNA from human and non-human primates (NHP). The single-stranded gapmer, a ribonuclease H-competent antisense oligonucleotide (ASO) used in the following examples, targets MALAT-1, a ubiquitously expressed long non-coding RNA (lncRNA) that is preferentially abundant in nuclear speckles and regulates post-transcriptional RNA processing. This is an adaptation of Tripathi et al.'s method by introducing the modification "mc" (i.e., 2'-O-methoxyethyl-5-methylcytidine; 2'MOE meC) instead of 2'-MOE-C (i.e., 2'-O-methoxyethyl-cytidine) in the sequence of SEQ ID NO: 405 in Table 7 below.

[0273] [Table 7] JPEG2026521069000008.jpg81166

[0274] siSOD1m is a double-stranded siRNA targeting mouse and rat superoxide dismutase 1 (SOD1) mRNA. siSOD1h is a double-stranded siRNA targeting human and non-human primates (including NHP, Papio anubis, Macaca mulatta, and Macaca fascicularis). hMALAT1-ASO is a gapmer antisense oligonucleotide (ASO) targeting human MALAT-1 long non-coding RNA. Lowercase letters indicate 2'-O-methyl(2'-OMe) glycosphagnum modifications to adenosine, cytidine, guanosine, or uridine nucleotides, respectively. Italicized uppercase letters indicate 2'-deoxy-2'-fluoro(2'F) glycosphagnum modifications to adenosine, cytidine, guanosine, or uridine nucleotides, respectively. • indicates a phosphorothioate (PS) nucleoside bond. VP indicates a vinyl phosphonate. L indicates a linker (conjugation handle). dN indicates a deoxynucleotide. Italicized lowercase letters indicate 2'-O-methoxyethyl (2'-MOE) glycosphagia modification of adenosine, guanosine, or thymidine nucleotides. mc indicates 2'-O-methoxyethyl-5-methylcytidine (2'MOE meC).

[0275] Oligonucleotides are purchased from Horizon Discovery Biosciences or GeneLink and have a conjugation handle, such as a hexylaminolinker, introduced at the 3' end of the sense strand of the siRNA double strand or the 5' end of the gapmer ASO. The overall conjugation strategy involved convergent synthesis, such as the strategy described in International Publication 2020 / 144233, consisting of (i) VHH or VHH-hFc heterodimers with site-specific introduction of an azide linker, for example, and (ii) parallel modification of oligonucleotides with a constrained alkyne group complementary to the azide functional group, for example. In the final step, both the functionalized azide-VHH or VHH-hFc-azide and the alkyne-oligo precursor are linked together, preferably using a copper-free click reaction, to produce a VHH-oligo or heterodimer VHH-hFc-oligo conjugate with a stable linker (see Figure 14). The inventors have utilized other methods to produce VHH-oligoconjugates using either cleavable disulfide linkers or other types of linkers such as long-chain PEG linkers, or other conjugation chemical reactions such as the thiol-maleimide chemical reaction (see Figure 13).

[0276] Example 3: Evaluation of the binding affinity of VHH-oligoconjugate to mTfR, rhTfR, and hTfR in viable cells The binding affinity of the VHH-oligoconjugate to mTfR, rhTfR, and hTfR was evaluated using competitive assays in viable cells expressing the target receptor, namely mouse neuroblastoma Neuro-2a cells (N2a), CHO cells genetically engineered by the inventors to stably express rhesus macaque TfR fused with eGFP protein at the C-terminus (CHO-rhTfR-GFP cells), or human ductal carcinoma MCF-7 cells. MCF-7 and N2a cells were grown in Dulbecco's modified Eagle medium (DMEM) GlutaMAX supplemented with 10% v / v FBS at 37°C in 5% CO2. CHO-rhTfR-GFP cells were grown in Ham F12 GlutaMAX supplemented with 10% v / v FBS at 37°C in 5% CO2. Two days prior to the experiment, cells were seeded in 96-well plates at a density of 50,000 cells / well. On the day of the experiment, free VHH or VHH-oligoconjugates diluted to various concentrations were co-incubated at 37°C for 3 hours in DMEM (MCF-7 and N2a cells) supplemented with 1% bovine serum albumin (BSA) or Ham F12 (CHO-rhTfR-GFP cells) supplemented with 1% BSA, along with subsaturated reference fluorescent compound C5-Alexa680 (100 nM in N2a and CHO-rhTfR-GFP cell lines, 10 nM in MCF-7 cells) at various concentrations in each cell line. After treatment, cells were washed with D-PBS, incubated with trypsin / EDTA at 37°C for 5 minutes, and then dissociated by adding cold medium (4°C) to suppress trypsin activity. The resuspended cells were transferred to a 96-deep-well plate (V-bottom) containing 1% fetal bovine serum (FBS) / 0.02% sodium azide in phosphate-buffered saline (PBS), and then centrifuged at 2000 rpm at 4°C for 5 minutes. After removing the supernatant, 5 mM EDTA and 4% paraformaldehyde (PFA) (v / v 1:1) in D-PBS were added to the wells, and the cells were fixed at room temperature for 15 minutes. The PFA was removed by centrifugation (3000 rpm, 4°C, 5 minutes), and the cells were resuspended in D-PBS with 5 mM EDTA.Subsequently, the A680-related fluorescence signal of the cells was quantified using an Attune NxT flow cytometer (Thermo Fisher Scientific) equipped with Attune NxT v3.1.2. The experimental data (experimental triplicates) were fitted using nonlinear fitting in GraphPad Prism software to obtain the apparent K. i The inhibition constant was determined. The graph in Figure 1 shows a representative inhibition curve, and the table shows the mean ± SD of at least two independent experiments.

[0277] All VHH-oligoconjugates tested (e.g., B8-siSOD1m, C5-siSOD1m, B8-siSOD1h, C5-siSOD1h, B8-MALAT1-ASO) had K levels similar to or lower than free VHH. i The study demonstrated effective interspecies binding to TfR by inducing concentration-dependent inhibition of the reference compound's binding and uptake in human, rhesus monkey, and mouse TfR-expressing cells (Figure 1).

[0278] Example 4: Potential for in vitro functional delivery and gene silencing of TfR-binding VHH-oligoconjugates after free uptake in human and mouse cell lines. Human cell lines MCF-7 (ductal carcinoma cells), Igrov-1 (ovarian cancer cells), and MIA PaCa-2 cells (pancreatic cancer cells), as well as the mouse neuroblastoma cell line Neuro-2a, were grown in DMEM GlutaMAX supplemented with 10% v / v FBS at 37°C in 5% CO2. For free uptake experiments, cells were seeded at a density of 2,000 cells / well in 96-well plates. After 24 hours, the medium was removed, and the cells were further incubated for 3 days in DMEM supplemented with 1% v / v FBS, containing various concentrations of free or conjugated oligonucleotides. Subsequently, the cells were washed with D-PBS and harvested in lysis buffer using either the SuperScript® IV CellsDirect cDNA Synthesis Kit (Invitrogen) for human cell lines or the Nucleospin® RNA XS Kit (Macherey-Nagel) for N2a cells, according to the manufacturer's recommendations. For experiments using the Nucleospin® RNA XS kit, cell lysates were stored at -20°C before RNA extraction and reverse transcription was performed using the High-Capacity RNA-to-cDNA kit (Applied Biosystems). Relative RNA expression levels were quantified by RT-qPCR using TaqMan® Fast Universal PCR Master Mix (2×), the no AmpErase® UNG kit (Applied Biosystems), and commercially available TaqMan® probes for mouse SOD1, human SOD1, or human MALAT-1 genes (Applied Biosystems). Expression data were analyzed using the DDCq method (Bustin et al.), normalized to the expression of RpL13 or GAPDH reference genes based on raw quantitative cycle (Cq) values, and are shown as mRNA levels relative to untreated cells. The experimental data (experimental triplicates) are presented as mean ± standard deviation (SD), and the IC50 was determined by analyzing them using GraphPad Prism software with 3-parameter logarithmic (inhibitor) versus response nonlinear regression.Figure 2 (human cell line) and Figure 3 (mouse N2a cells) show representative inhibition curves and estimated IC50 values.

[0279] While free siSOD1h or siSOD1h conjugated to unbound C5neg VHH did not affect hSOD1 mRNA levels, all TfR-bound VHH-siSOD1h conjugates (such as C5-siSOD1h, C5V8-siSOD1h, or B8-siSOD1h, containing C5, C5V8, or B8 of the VHH molecule, respectively) showed potent, concentration-dependent hSOD1 mRNA downregulation in various human cell lines (Figure 2A and Table 1). Similar potent knockdown effects were observed after free uptake of the VHH-MALAT1-ASO conjugate in MCF-7 cells (Figure 2B). After free uptake in mouse N2a cell lines, similar specific and potent knockdown effects were observed with various VHH-siSOD1m conjugates (i.e., conjugates containing VHH molecules such as B8 or C5, or their variants such as B8h1, C5, C5V1, C5V7, C5V8, C5V13, C5h18, C5h19, as listed in Table 1) and VHH-MALAT1-ASO conjugates (containing VHH molecules such as B8, as listed in Table 1) (Figures 3A and 3B, respectively).

[0280] These results demonstrate that the VHH-oligoconjugate of the present invention effectively binds to human or mouse TfRs on the cell surface, followed by TfR-mediated endocytosis, endosomal escape, and cytosolic or nuclear delivery of active oligonucleotides, thereby enabling effective regulation of target gene expression using various oligonucleotide modalities.

[0281] Example 5: In vivo functional delivery and mRNA knockdown of TfR-binding VHH-siRNA conjugate in muscle tissue in wild-type mice The potential of the VHH-siSOD1m conjugate to mediate mouse SOD1 mRNA knockdown in muscle tissue after systemic administration in wild-type C57Bl / 6 mice was evaluated. Mice (4-8 per group) were administered either intravenously (IV bolus) or subcutaneously (SC) via injection at doses shown in Figures 4-7, using either a PBS vehicle (control), unconjugated siSOD1m, or the VHH-siSOD1m conjugate shown in the figure. Tissue samples were collected at the indicated times after injection, rapidly frozen in 10 volumes of NucleoProtect RNA stabilization reagent (Macherey-Nagel), and stored at -20°C. Frozen tissue samples were homogenized in QUIAzol lysis reagent using a Precellys Evolution tissue homogenizer (Bertin Instruments) equipped with a Cryolys Evolution cooling system. Total RNA was extracted from tissue homogenates using the rNeasy 96 QIAcube HT Kit (QIAGEN) in a QIAcube HT system. RNA samples were analyzed and quantified using the Fragment Analyzer RNA Kit (Agilent) in a Fragment Analyzer system. Relative RNA expression levels were quantified by RT-qPCR using TaqMan® Fast Universal PCR Master Mix (2×), no AmpErase® UNG Kit (Applied Biosystems), and commercially available TaqMan® probes (Applied Biosystems) for mouse SOD1 and RpL13 genes. Expression data were analyzed using the DDCq method, normalized to the expression of the RpL13 reference gene based on the raw value of the quantitative cycle (Cq) (Bustin et al.). Results were expressed as mean ± standard error of the mean (SEM) and shown as mRNA levels relative to PBS-injected control animals.When the TfR-conjugated VHH-siSOD1m conjugate was administered to wild-type mice at a low dose of 3 mg / kg (siRNA molar equivalent) via either IV or SC administration, it showed approximately 70%–80% downregulation of target SOD1 mRNA levels in the gastrocnemius muscle and approximately 60% downregulation in the myocardium (Figure 4). Little to no effect was observed in the liver or lungs (Figure 4), and the unconjugated C5neg-siSOD1m conjugate showed no effect in any tissue (Figure 5). The results shown in this example demonstrate tissue selectivity and support TfR-dependent functional delivery of these TfR-conjugated conjugates at low therapeutic doses in vivo. One dose-response study of these conjugates showed a near-maximum 80% knockdown effect at a dose of 3 mg / kg in skeletal muscle and 15 mg / kg in myocardium, with no effect at a maximum of 15 mg / kg of unconjugated siRNA (Figure 6). The ED50 for this knockdown effect was approximately 0.6 mg / kg (siRNA molar equivalent) in the gastrocnemius muscle and approximately 3 mg / kg (siRNA molar equivalent) in the myocardium. Time-course evaluation of these conjugates showed that a single dose of the TfR-bound VHH-siRNA conjugate at a low dose of 3 mg / kg was sufficient to induce a potent and long-lasting knockdown effect for more than one month (Figure 7). This demonstrates that early TfR-mediated uptake in muscle tissue allows for effective intracellular delivery of the VHH-siRNA conjugate, sustained release of active siRNA from the lysosomal compartment into the cytosol, loading into the RNA-induced silencing complex (RISC), and continuous degradation of the target mRNA molecule.

[0282] Example 6: Generation and evaluation of the potential for functional delivery and mRNA knockdown of TfR-conjugated VHH-hFc-siRNA conjugates in vitro and in vivo. To introduce a half-life extension or stabilization portion that improves the pharmacokinetic profile, TfR-conjugated VHH can be conjugated to an oligo via an antibody or antibody fragment scaffold. This strategy is illustrated herein using VHH-hFc-siRNA conjugates prepared using the same overall conjugation strategy as described for direct VHH-siRNA conjugates. Furthermore, the VHH-hFc-siRNA conjugates further include human IgG1-derived Fc dimers (hFc) having two arms called the “knob” and the “hole.” In this example, various TfR-conjugated VHH-hFc-siRNA heterodimer conjugates were prepared using the “knob-into-hole” technique. Each of these VHH-hFc-siRNAs contains a selected VHH at the N-terminus of the "knob" arm of the Fc dimer, and a tag sequence (i.e., Q-tag) specifically recognized by the transglutaminase (TGase) enzyme, inserted at the C-terminus of the "hole" arm of the Fc dimer. The VHH-hFc-siRNA heterodimers were site-specifically modified at the Q-tag to introduce an azide linker. The resulting heterodimer VHH-hFc-Q-tag-azide intermediate was conjugated to alkyne-siRNA using a copper-free click reaction to produce a VHH-hFc-siRNA conjugate with a stable linker (Figure 14). More specifically, VHH-hFc-siSOD1m and VHH-hFc-siSOD1h conjugates were prepared using the same siSOD1m and siSOD1h sequences as described in previous examples, and their biological properties in various systems were evaluated. The VHH mutants used to construct the VHH-hFc-siRNA conjugates were C5 and B8 variants (shown in Figures 8, 9, and 10). These conjugates were constructed using modified IgG1-derived Fc dimers with the sequence of SEQ ID NO: 664 in the "knob" arm and the sequence of SEQ ID NO: 665 in the "hole" arm. The linker used in the "knob" arm between VHH and hFc was GGGGSGGGGS (SEQ ID NO: 630).For example, regarding the preparation of the C5-Fc conjugate, the complete amino acid sequence used for the "knob" arm is shown in SEQ ID NO: 666, and the complete amino acid sequence used for the "whole" arm is shown in SEQ ID NO: 667. These sequences for the "knob" and "whole" arms are encoded by SEQ ID NO: 668 or 669, respectively.

[0283] Firstly, the VHH-hFc-siSOD1 conjugate showed similar or slightly higher heterologous binding affinity to mTfR and hTfR compared to free VHH (Figure 8). Secondly, in either human breast cancer MCF-7 cells (Figure 9A) or mouse neuroblastoma Neuro-2a cells (Figure 9B), the VHH-hFc-siSOD1 conjugate exhibited specific, potent, and concentration-dependent SOD1 mRNA downregulation using the same in vitro free uptake assay as described in previous examples. Thirdly, the VHH-hFc-siSOD1m conjugate demonstrated the ability to mediate mouse SOD1 mRNA knockdown in muscle tissue after intravenous (IV bolus) administration in wild-type C57Bl / 6 mice. Mice (4-8 mice per group) were administered via a single intravenous (IV bolus) injection at a dose of 0.5 or 1.5 mg / kg (siRNA molar equivalent) of either an unbound (C5neg) or TfR-bound (C5) VHH-hFc-siSOD1m conjugate to a PBS vehicle (control). Fourteen days after administration, tissue samples were collected, rapidly frozen in 10 volumes of NucleoProtect RNA stabilization reagent (Macherey-Nagel), and stored at -20°C. Frozen tissue samples were homogenized in QUIAzol lysis reagent using a Precellys Evolution tissue homogenizer (Bertin Instruments) equipped with a Cryolys Evolution cooling system. Total RNA was extracted from the tissue homogenates using the RNeasy 96 QIAcube HT kit (QIAGEN) in a QIAcube HT system. RNA samples were analyzed and quantified using the Fragment Analyzer RNA Kit (Agilent) in a Fragment Analyzer system.Relative RNA expression levels were quantified by RT-qPCR using TaqMan® Fast Universal PCR Master Mix (2×), no AmpErase® UNG kit (Applied Biosystems), and commercially available TaqMan® probes for mouse SOD1 and RpL13 genes (Applied Biosystems). Expression data were analyzed using the DDCq method, normalized to the expression of the RpL13 reference gene based on the raw value of the quantitative cycle (Cq) (Bustin et al.). Results are expressed as mean ± standard error of the mean (SEM) and shown as mRNA levels relative to PBS-injected control animals.

[0284] Downregulation of mouse SOD1 mRNA levels was little to no in any tissue analyzed after treatment with the unbound conjugate (C5neg-hFc-siSOD1m). However, treatment with the TfR-bound conjugate (C5-hFc-siSOD1m) induced a potent, muscle-tissue-selective, and specific effect, showing more than 70% knockdown in the gastrocnemius, diaphragm, or myocardium at a dose of 1.5 mg / kg, and a similar or slightly lower effect at 0.5 mg / kg, while showing no effect in the liver or lungs (Figure 10A). In particular, the ED50 of the C5-hFc-siSOD1m conjugate administered as a single IV bolus was approximately 0.16 mg / kg (siRNA molar equivalent) in the gastrocnemius muscle and approximately 0.4 mg / kg (siRNA molar equivalent) in the myocardium, which are 4-fold and 8-fold lower, respectively, than the ED50 values ​​obtained after a single SC administration of the direct C5-siSOD1m conjugate (Figure 6), demonstrating improvement due to the introduction of the half-life-extending Fc skeleton. Similarly, other conjugates containing an additional 5'-VP in the siSOD1m antisense chain (C5-hFc-siSOD1m-5'VP) were administered as single SCs at various doses in wild-type mice. A potent (over 80%) and muscle-selective KD was observed, and the ED50 was 0.1–0.6 mg / kg (siRNA molar equivalent). The results presented in this example demonstrate tissue selectivity and TfR-dependent functional delivery of these TfR-binding VHH-hFc-siRNA conjugate designs when administered as a single dose to wild-type mice at low therapeutic doses via either IV or SC.

[0285] Example 7: hTfR1 + / + In vivo functional delivery and mRNA knockdown of TfR-binding VHH-siRNA conjugates in mouse muscle tissue The ability of VHH-siSOD1m conjugates to mediate mouse SOD1 mRNA knockdown in the muscle tissue of transgenic mice constitutively expressing human TfR1 (B-hTfR1 mice, Biocytogen) was evaluated. VHH-siSOD1m conjugates containing either a PBS vehicle (control), unconjugated siSOD1m, or either interspecies rodent / NHP / human TfR1-binding VHH(C5) or human TfR1-binding only VHH(B6) were used to evaluate the ability of VHH-siSOD1m conjugates to mediate mouse SOD1 mRNA knockdown in the muscle tissue of transgenic mice constitutively expressing human TfR1 (B-hTfR1 mice, Biocytogen). + / + Mice (3-4 mice per group) were administered a single subcutaneous (SC) injection at a dose of 3 mg / kg (siRNA molar equivalent). Seven days after administration, tissue samples were collected, rapidly frozen in 10 volumes of NucleoProtect RNA stabilization reagent (Macherey-Nagel), and stored at -20°C. The frozen tissue samples were homogenized with QUIAzol lysis reagent using a Precellys Evolution tissue homogenizer (Bertin Instruments) equipped with a Cryolys Evolution cooling system. Total RNA was extracted from the tissue homogenates using the RNeasy 96 QIAcube HT kit (QIAGEN) in a QIAcube HT system. RNA samples were analyzed and quantified using the Fragment Analyzer RNA kit (Agilent) in a Fragment Analyzer system. Relative RNA expression levels were quantified by RT-qPCR using TaqMan® Fast Universal PCR Master Mix (2×), no AmpErase® UNG kit (Applied Biosystems), and commercially available TaqMan® probes for mouse SOD1 and RpL13 genes (Applied Biosystems). Expression data were analyzed using the DDCq method, normalized to the expression of the RpL13 reference gene based on the raw value of the quantitative cycle (Cq) (Bustin et al.). Results are expressed as mean ± standard error of the mean (SEM) and shown as mRNA levels relative to PBS-injected control animals.

[0286] Downregulation of mouse SOD1 mRNA levels was not observed in any of the test samples analyzed after treatment with non-conjugated siSOD1m, however, both hTfR-binding conjugates were observed in hTfR1 + / + A single subcutaneous injection of 3 mg / kg (siRNA molar equivalent) into mice induced a similar potent and muscle-tissue-selective effect, showing approximately 70-80% knockdown in the gastrocnemius and diaphragm muscles, and approximately 40-50% knockdown in the myocardium, while showing no effect in the liver or lungs (Figure 11). The results shown in this example demonstrate the interspecies capabilities of the TfR-binding VHH-siRNA conjugate in rodents / humans, inducing TfR-dependent binding, functional uptake, and targeted mRNA downregulation in vivo at low therapeutic doses in the muscle tissue of human TfR-expressing mice.

[0287] Example 8: Muscle knockdown effect of VHH-siRNA conjugate after local ICV administration in B-hTfR mice The VHH-siSOD1m conjugate was evaluated for its potential to mediate mouse SOD1 mRNA knockdown in muscle tissue after intraventricular (ICV) administration in B-hTfR mice (transgenic hTfR-ECD KI / KI mice, Biocytogen). Currently, there are no oligonucleotides under development for neuromuscular damage that act in muscle tissue after local CNS administration (e.g., intrathecal or intraventricular). These results are the first to demonstrate that the conjugate of the present invention is functionally delivered to muscle tissue after local CNS administration, and that the conjugate of the present invention can target muscle cells via two administration routes: systemic administration and local CNS administration. B-hTfR mice (2 males and 2 females per group) were given 1, 10, or 100 μg (siRNA molar equivalent) of PBS vehicle (control), unbound C5neg-siSOD1m-5'VP, or a TfR-binding VHH-siSOD1m-5'VP conjugate (B8h1-siSOD1m-5'VP, K) that has high or moderate binding affinity to hTfR.D =0.6nM;B8V32-siSOD1m-5’VP、K DA single ICV dose of 54.8 nM was injected (5-10 μL at 0.75 μL / min into the right ventricle). Seven days post-administration, tissue samples were collected from the brain region (ipsilateral hemisphere), spinal cord, liver, and kidney, rapidly frozen in 10 volumes of NucleoProtect RNA stabilization reagent (Macherey-Nagel), and stored at -20°C. The frozen tissue samples were homogenized in QUIAzol lysis reagent using a Precellys Evolution tissue homogenizer (Bertin Instruments) equipped with a Cryolys Evolution cooling system. Total RNA was extracted from the tissue homogenates using the rNeasy 96 QIAcube HT kit (QIAGEN) in a QIAcube HT system. RNA samples were analyzed and quantified using the Fragment Analyzer RNA kit (Agilent) in a Fragment Analyzer system. Relative RNA expression levels were quantified by RT-qPCR using TaqMan® Fast Universal PCR Master Mix (2×), no AmpErase® UNG kit (Applied Biosystems), and commercially available TaqMan® probes (Applied Biosystems) for the mouse SOD1, RpL13, and RpL30 genes. Expression data were analyzed using the ΔΔCq method (Bustin et al.), normalized to the expression of the RpL13 and RpL30 reference genes based on raw quantitative cycle (Cq) values ​​(multiple qPCR). Results are expressed as mean ± standard error of the mean (SEM) and shown as mRNA levels relative to PBS-injected control animals. Both TfR-binding conjugates showed potent knockdown in muscle tissue, the moderate-affinity B8V32-siSOD1m-5'VP conjugate showed no effect in excretory organs, and the high-affinity B8h1-siSOD1m-5'VP conjugate showed only slight or moderate effects in the kidneys and liver (Figure 15).The greatest effect was observed in all muscle tissues tested at a dose of 100 μg / animal, corresponding to approximately 3 mg / kg (siRNA molar equivalent), with a reduction of approximately 80%. Only a moderate effect was observed with the unbound C5neg-siSOD1-5'VP conjugate at the maximum dose tested, 100 μg, supporting the involvement of the TfR-bound VHH-siSOD1 conjugate in TfR-mediated uptake and functional delivery in muscle tissue.

[0288] These results demonstrate that the TfR-conjugated VHH-siRNA conjugate has the potential for potent functional delivery in muscle tissue after topical CNS administration, at doses similar to those used for systemic intramuscular delivery. Therefore, the VHH of the present invention has the potential to address both myopathy and neuromuscular disorders not only through systemic administration but also through CNS administration.

[0289] Example 9: Muscle knockdown effect of heterodimer VHH-hFc-siRNA conjugate after systemic administration in B-hTfR mice B-hTfR mice (n=4 per group) were administered a single subcutaneous (SC) dose of 4.5 mg / kg (siRNA molar equivalent) of the TfR-conjugated VHH-hFc-siSOD1m-5'VP conjugate, containing various VHH mutants such as C5V30, B8V31, B8V32, C5h9, C5V5, and C5h18 mutants, and were compared to PBS-injected mice (n=8). All conjugates tested exhibited a low dissociation rate k offExcept for the E8- and C5-hFc-siSOD1m-5'VP conjugates, which exhibit sub-nanomolecular binding affinity, the SOD1 metabolites bind to human TfR with affinity in the range of 150–400 nM, as assessed using SPR. Two weeks after administration, tissue samples, including muscle tissue (gastrocnemius, diaphragm, and heart), liver, and kidney, were collected, rapidly frozen in 10 volumes of NucleoProtect RNA stabilization reagent (Macherey-Nagel), and stored at -20°C until processing for qPCR quantification of SOD1 mRNA. Frozen tissue samples were homogenized in QUIAzol lysis reagent using a Precellys Evolution tissue homogenizer (Bertin Instruments) equipped with a Cryolys Evolution cooling system. Total RNA was extracted from tissue homogenates using the rNeasy 96 QIAcube HT kit (QIAGEN) in a QIAcube HT system. RNA samples were analyzed and quantified using the Fragment Analyzer RNA Kit (Agilent) in a Fragment Analyzer system. Relative RNA expression levels were quantified by RT-qPCR using TaqMan® Fast Universal PCR Master Mix (2×), no AmpErase® UNG Kit (Applied Biosystems), and commercially available TaqMan® probes (Applied Biosystems) for the mouse SOD1, RpL13, and RpL30 genes. Expression data were analyzed using the ΔΔCq method (Bustin et al.), normalized to the expression of the RpL13 and RpL30 reference genes based on raw quantitative cycle (Cq) values ​​(multiple qPCR). Results were expressed as mean ± standard error of the mean (SEM) and shown as mRNA levels relative to PBS-injected control animals. The results showed similar potent KD across all VHH variants tested in all muscle tissues, with approximately 90% KD in the gastrocnemius muscle and approximately 80% KD in the diaphragm and cardiac muscle, and no effect in the excretory organs (Figure 16).

[0290] In other experiments, B-hTfR mice (n=4 males per group) were administered three IV doses of TfR-binding VHH-hFc-siSOD1m-5'VP conjugates at 1.5 mg / kg (siRNA molar equivalent), including various VHH mutants such as B8V40, B8V31, B8V32, and B8V31h5 mutants, and compared to PBS-injected mice (n=8). The TfR-binding conjugates evaluated here were selected based on their similar binding affinity to human and non-human primate TfRs (rhesus monkey or cynomolgus monkey TfR extracellular domains) in the range of 150 nM to 1 μM (based on SPR K D (The difference was less than twice). Two weeks after the last administration, tissue samples, including muscle tissue (gastrocnemius, diaphragm, and heart), liver, and kidney, were collected as described in previous examples, rapidly frozen in 10 volumes of NucleoProtect RNA stabilization reagent (Macherey-Nagel), and stored at -20°C until processing for qPCR quantification of SOD1 mRNA. Results were expressed as mean ± standard error of the mean (SEM) and shown as mRNA levels relative to PBS-injected control animals. As previously observed after a single SC administration of 4.5 mg / kg (siRNA molar equivalent), all TfR-binding conjugates tested showed strong >80% KD in all muscle tissues tested and no effect in excretory organs (Figure 17).

[0291] These results demonstrate that the conjugate of the present invention has the potential for potent functional delivery at low doses in the muscle tissue of hTfR-expressing mice. Furthermore, several VHH variants and conjugates show very similar binding affinity (less than 2x difference) between human TfR and non-human primate (rhesus / cynomolgus) TfR, allowing for a smooth transition from preclinical settings in non-human primates to clinical studies in humans.

[0292] Example 10: In vivo functional delivery and mRNA knockdown of TfR-binding VHH-siRNA conjugate in muscle tissue of non-human primates. The ability of VHH-siSOD1h conjugates targeting the human and non-human primate (NHP) SOD1 gene to mediate SOD1 mRNA knockdown in the muscle tissue of Papio Anubis baboons was evaluated. A single intravenous dose (IV infusion, 30 minutes) of 5 mg / kg (siRNA molar equivalent) was administered to male Papio Anubis baboons (10–17 months old, 2.6–4.5 kg) using either a PBS vehicle (control) or a TfR-conjugated VHH-siSOD1h conjugate. Approximately 100 mg of gastrocnemius, quadriceps, and anterior tibialis muscle samples were collected under general anesthesia at the indicated time after administration, rapidly frozen in 10 volumes of NucleoProtect RNA stabilization reagent (Macherey-Nagel), and stored at -20°C. Frozen tissue samples were homogenized in QUIAzol lysis reagent using a Precellys Evolution tissue homogenizer (Bertin Instruments) equipped with a Cryolys Evolution cooling system. Total RNA was extracted from the tissue homogenates using the RNeasy 96 QIAcube HT kit (QIAGEN) in a QIAcube HT system. RNA samples were analyzed and quantified using the Fragment Analyzer RNA kit (Agilent) in a Fragment Analyzer system. Relative RNA expression levels were quantified by RT-qPCR using TaqMan® Fast Universal PCR Master Mix (2×), no AmpErase® UNG kit (Applied Biosystems), and commercially available TaqMan® probes (Applied Biosystems) for mouse SOD1 and RpL13 genes. Expression data were analyzed using the DDCq method, normalized to the expression of the RpL13 reference gene based on raw quantitative cycle (Cq) values ​​(Bustin et al.). Results are expressed as mean ± standard error of the mean (SEM) and shown as mRNA levels relative to PBS-injected control animals.

[0293] A single intravenous administration of the TfR-conjugated VHH-siSOD1h conjugate induced a potent and prolonged downregulation of target SOD1 mRNA levels in all muscle tissues tested compared to a control group injected with PBS, with 60% knockdown observed for more than 3 months (Figure 13).

[0294] The results presented in this example support the interspecies capabilities of TfR-binding VHH-siRNA conjugates in rodents / NHPs / humans, inducing TfR-dependent binding, functional uptake, and target mRNA downregulation in vivo at low therapeutic doses in non-human primate muscle tissue.

[0295] [Sequence List] Table 1 shows the VHH molecule of the present invention and the negative control C5. neg This is a list of the amino acid sequences of D12, the corresponding CDR1-3, and their identifiers as shown herein as sequence numbers or SEQs. [Table 1] JPEG2026521069000009.jpg215170JPEG2026521069000010.jpg226170JPEG20265210690 00011.jpg224170JPEG2026521069000012.jpg226170JPEG2026521069000013.jpg219170 JPEG2026521069000014.jpg222170JPEG2026521069000015.jpg209170JPEG20265210690 00016.jpg222170JPEG2026521069000017.jpg209170JPEG2026521069000018.jpg221170 JPEG2026521069000019.jpg225170JPEG2026521069000020.jpg223170JPEG20265210690 00021.jpg223170JPEG2026521069000022.jpg210170JPEG2026521069000023.jpg210170 JPEG2026521069000024.jpg209170JPEG2026521069000025.jpg221170JPEG20265210690 00026.jpg214170JPEG2026521069000027.jpg224170JPEG2026521069000028.jpg135170

[0296] Table 2 is a list of nucleotide sequences encoding the VHH molecule. [Table 2] JPEG2026521069000029.jpg216170JPEG2026521069000030.jpg224170JPEG2026521069000031.jpg217170JPEG20265210690 00032.jpg217170JPEG2026521069000033.jpg224170JPEG2026521069000034.jpg217170JPEG2026521069000035.jpg189170

[0297] Table 3 is a list of FR (framework region) sequences of VHH molecules. [Table 3] JPEG2026521069000036.jpg224170JPEG2026521069000037.jpg218170JPEG2026521069000038.jpg223170JPEG2026521069 000039.jpg221170JPEG2026521069000040.jpg223170JPEG2026521069000041.jpg214170JPEG2026521069000042.jpg95170

[0298] Table 4 shows examples of tag arrays and linkers. [Table 4] JPEG2026521069000043.jpg219170JPEG2026521069000044.jpg39170

[0299] References Ait Benichou et al., Gene Ther. 2022 Jan 25. pii: 10.1038 / s41434-022-00316-7. doi: 10.1038 / s41434-022-00316-7 Barrientos T. et al. (2015). “Metabolic Catastrophe in Mice Lacking Transferrin Receptor in Muscle.” EBioMedicine 2(11): 1705-1717 Bizot et al., Drugs. 2020 Jul 21. pii: 10.1007 / s40265-020-01363-3. doi: 10.1007 / s40265-020-01363-3 Bustin et al., Clin Chem. 2009 Apr;55(4):611-22. Chan and Gerhardt, Transferrin receptor gene is hyperexpressed and transcriptionally regulated in differentiating erythroid cells. J Biol Chem 1992 Apr 25;267(12):8254-9 Jefferies et al., Transferrin receptor on endothelium of brain capillaries. Nature 1984 Nov 8-14;312(5990):162-3 Debacker, A. J., et al. (2020). “Delivery of Oligonucleotides to the Liver with GalNAc: From Research to Registered Therapeutic Drug. ” Mol Ther 28(8): 1759-1771 Johnsen K.B. et al, Targeting the transferrin receptor for brain drug delivery. Prog Neurobiol. 2019 Oct;181:101665 Majumdar S. and Siahaan TJ., “Peptide-mediated targeted drug delivery”. Med Res Rev. 2012 May;32(3):637-58 Nair JK et al., Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing, J. Am. Chem. Soc. 136 (2014) 16958-16961 Needleman, S.B. and Wunsch, C.D., (1970), Journal of Molecular Biology, 48, 443-453 Pardridge et al., Human blood-brain barrier transferrin receptor. Metabolism1987 Sep;36(9):892-5 Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual, Third Edition Cold Spring Harbor Tai, W. “Current Aspects of siRNA Bioconjugate for In Vitro and In Vivo Delivery.” Molecules. 2019 Jun 13;24(12) Tripathi et al., “The nuclear-retained noncoding RNA MALAT1 regulates alternative splicing by modulating SR splicing factor phosphorylation”. Mol Cell. 2010 Sep 24;39(6):925-38 Xu W., et al. (2015). “Lethal Cardiomyopathy in Mice Lacking Transferrin Receptor in the Heart.” Cell Rep 13(3): 533-545 Ying Li et al., Transferrin receptor 1 plays an important role in muscle development and denervation-induced muscular atrophy. Neural Regen Res 2021 Jul;16(7):1308-1316

Claims

1. (i) One or more VHH molecules of formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and (ii) A conjugate compound comprising one or more oligonucleotides, The VHH molecule binds to TfR on the surface of muscle cells, and the VHH molecule is sequence number 1, 2, and 3; or sequence number 5, 6, and 7; or sequence number 9, 10, and 11; or sequence number 13, 14, and 15; sequence number 17, 2, and 3; or sequence number 19, 2, and 3; or sequence number 1, 21, and 3; or sequence number 1, 23, and 3; or sequence number 1, 2, and 25; or sequence number 1, 2, and 27; or sequence number 1, 2, and 29; or sequence number 1, 2, and 31; or sequence number 1, 2, and 33; or SEQ ID NOs: 67, 2, and 3; or SEQ ID NOs: 69, 2, and 3; or SEQ ID NOs: 1, 71, and 3; or SEQ ID NOs: 1, 73, and 3; or SEQ ID NOs: 1, 75, and 3; or SEQ ID NOs: 1, 2, and 77; or SEQ ID NOs: 1, 2, and 79; or SEQ ID NOs: 1, 2, and 81; or SEQ ID NOs: 1, 2, and 83; or SEQ ID NOs: 1, 2, and 85; or SEQ ID NOs: 392, 2, and 3; or SEQ ID NOs: 1, 113, and 3; or SEQ ID NOs: 1, 115, and 3; or SEQ ID NOs: 1, 2, and 117 ; or SEQ ID NOs. 1, 2, and 119; or SEQ ID NOs. 1, 2, and 121; or SEQ ID NOs. 1, 2, and 123; or SEQ ID NOs. 125, 2, and 3; or SEQ ID NOs. 17, 73, and 3; or SEQ ID NOs. 17, 128, and 3; or SEQ ID NOs. 5, 160, and 7; or SEQ ID NOs. 5, 162, and 7; or SEQ ID NOs. 5, 164, and 7; or SEQ ID NOs. 5, 166, and 7; or SEQ ID NOs. 9, 169, and 11; or SEQ ID NOs. 9, 171, and 11; or SEQ ID NOs. 175, 176, and 177; or sequence Numbers 179, 176, and 180; or sequence numbers 182, 176, and 177; or sequence numbers 184, 176, and 177; or sequence numbers 186, 187, and 188; or sequence numbers 190, 191, and 192; or sequence numbers 194, 195, and 196; or sequence numbers 198, 199, and 200; or sequence numbers 201, 202, and 203; or sequence numbers 205, 206, and 207; or sequence numbers 410, 6, and 7; or sequence numbers 413, 6, and 7; or sequence numbers 5, 416, and 7;or sequence numbers 5, 419, and 7; or sequence numbers 426, 6, and 7; or sequence numbers 5, 431, and 7; or sequence numbers 434, 6, and 7; or sequence numbers 437, 6, and 7; or sequence numbers 5, 6, and 452; or sequence numbers 5, 6, and 455; or sequence numbers 607, 608, and 609; or sequence numbers 610, 611, and 612; or sequence numbers 671, 2, and 3; or sequence numbers 672, 2, and 3; or sequence numbers 673, 6, and 7; or sequence numbers 674, 6, and 7; or sequence numbers 1, 2, and 713; or sequence numbers 5, 6, and 714; or sequence number Conjugate compounds comprising: numbers 674, 164, and 7; or SEQ ID NOs. 710, 6, and 7; or SEQ ID NOs. 5, 6, and 715; or SEQ ID NOs. 674, 712, and 7; or SEQ ID NOs. 711, 6, and 7; or SEQ ID NOs. 673, 6, and 741; or SEQ ID NOs. 673, 6, and 742; or SEQ ID NOs. 673, 6, and 743; or SEQ ID NOs. 673, 6, and 744; or SEQ ID NOs. 673, 431, and 741; or SEQ ID NOs. 673, 431, and 742; or SEQ ID NOs. 673, 431, and 743; or SEQ ID NOs. 673, 6, and 7; or SEQ ID NOs. 674, 6, and 7.

2. The conjugate compound according to claim 1, wherein the VHH molecule comprises an amino acid sequence selected from any one of SEQ ID NOs: 214, 273, 276-284, 412, 415, 418, 421, 423, 425, 428, 430, 433, 436, 439, 441, 443, 445, 447, 449, 451, 454, 457, 677, 678, 702-709, and 766-786.

3. The conjugate compound according to claim 1 or 2, wherein the VHH molecule comprises an amino acid sequence selected from any one of SEQ ID NOs: 213, 216-271, 274, 275, 675, 676, and 701.

4. The conjugate compound according to claim 1 or 2, wherein the VHH molecule comprises an amino acid sequence selected from any one of SEQ ID NOs. 215 and 285-299.

5. The conjugate compound according to claim 1 or 2, wherein the VHH molecule comprises an amino acid sequence selected from any one of SEQ ID NOs. 613 to 615.

6. The conjugate compound according to any one of claims 1 to 5, wherein the VHH molecule further comprises a tag and / or a linker.

7. The conjugate compound according to any one of claims 1 to 6, wherein the VHH molecule is humanized.

8. The conjugate compound according to any one of claims 1 to 7, wherein the VHH molecule is bound to TfR1 of a human, non-human primate, and / or rodent.

9. The conjugate compound according to any one of claims 1 to 8, wherein the oligonucleotide is selected from any single-stranded or double-stranded oligonucleotide, such as small interfering RNA (siRNA), small activating RNA (saRNA), gapmers, antisense oligonucleotides (ASOs), shRNA, miRNA, aptamer RNA, and cross-linked nucleic acids (BNA).

10. The conjugate compound according to any one of claims 1 to 9, wherein the muscle cell is a skeletal muscle cell, a cardiomyocyte, or a muscle cancer cell.

11. The conjugate compound according to any one of claims 1 to 10, further comprising at least one further compound, preferably a half-life extension moiety, or a stabilizing group, or a scaffold, such as an antibody or a fragment thereof (e.g., an Fc fragment), a VHH molecule, PEG, a serum albumin protein, or a serum albumin binding moiety, more preferably an Fc fragment, wherein the Fc fragment is an Fc heterodimer comprising a modified Fc having the sequence of SEQ ID NO: 664 in the knob arm and a modified Fc having the sequence of SEQ ID NO: 665 in the whole arm.

12. A pharmaceutical composition comprising a conjugate compound according to any one of claims 1 to 11, and a pharmaceutically acceptable support, carrier, or excipient.

13. A conjugate compound according to any one of claims 1 to 11, or a composition according to claim 12, for use in the treatment of a group more preferably selected muscle disease or neuromuscular disease consisting of myopathy, cardiomyopathy, muscular dystrophy (such as DMD, BMD, FSHD, Pompe disease, or familial hypertrophic cardiomyopathy), neuromuscular disease (such as ALS, SMA, MS, HD, or CMT), and cancer of muscle (such as rhabdomyosarcoma or leiomyosarcoma).

14. The conjugate compound or composition described above is administered parenterally, systemically, intravenously, intramuscularly, subcutaneously, intracerebrally, intraventricularly, or intrathecally, according to any one of claims 1 to 11, or to the composition described in claim 12, or to the conjugate compound or composition described in claim 13 for use.

15. The conjugate compound or composition according to claim 14, wherein the conjugate compound or composition is administered intracerebral, intraventricular, or intrathecally.

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