Transferrin receptor binding molecules, conjugates thereof and uses thereof for preventing or treating muscle diseases

Abstract

The present invention relates to Camelidae heavy chain variable domain only (VHH) molecules that bind to TfR and their use for, for example, transporting molecules of therapeutic interest into muscle cells (typically under pathological conditions).

Description

Technical Field

[0001] This invention relates to a transferrin receptor (TfR) binding molecule, a coupling compound comprising such a TfR binding molecule, and its uses. More specifically, this invention relates to a camelid heavy chain variable domain (VHH) molecule that binds to TfR on the surface of muscle cells and its use in a preventative or therapeutic context, such as for the transport of nucleic acid molecules, particularly oligonucleotide therapeutic agents, into muscle. Background Technology

[0002] In the muscular system, muscle tissue is divided into three distinct types: skeletal muscle, cardiac muscle, and smooth muscle tissue. Each type of muscle tissue in the human body has its unique structure and specific function. Muscle diseases are often associated with muscle weakness or dysfunction, which can lead to life-threatening complications. Numerous instances of such diseases have been characterized, including various forms of muscular dystrophy and neuromuscular disorders. Muscular dystrophy is a group of inherited diseases that progressively cause muscle weakness, leading to increased disability. Various types of muscular dystrophy exist, each involving eventual loss of strength and possible deformities. These diseases include Duchenne muscular dystrophy (DMD), Benedict's muscular dystrophy (BMD), faciescapular muscular dystrophy (FSHD), Pompe disease, familial hypertrophic cardiomyopathy, and others. Many muscle diseases are single-gene disorders associated with gain-of-function or loss-of-function mutations, which may have dominant or recessive phenotypes. For example, activating mutations leading to muscle diseases have been identified in genes encoding ion channels, structural proteins, metabolic proteins, and signal transduction proteins. For example, DMD is caused by mutations in the DMD gene located on the short arm (p) (Xp21.2) of the X chromosome. Despite advances in understanding the genetic etiology of muscle diseases, effective treatment options remain very limited.

[0003] Neuromuscular disorders affect the nerves that control voluntary muscles and transmit sensory information back to the brain. They represent multi-layered abnormalities, are often incurable, and lead to death from complete muscle wasting and atrophy. Neuromuscular disorders can affect the peripheral nervous system (PNS), muscle tissue, and the central nervous system (CNS), and include spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), peroneal muscular atrophy (CMT), multiple sclerosis (MS), Huntington's disease (HD), and others.

[0004] Currently, no oligonucleotides have been developed for treating muscle disorders that act on muscle tissue after local CNS administration (e.g., intrathecal or lateral ventricle). Although some oligonucleotides have been proposed to act on the CNS manifestations of such muscle disorders after local CNS administration, the lack of accompanying delivery to muscle tissue necessitates additional systemic administration to address peripheral muscle manifestations (Ait Benichou et al., 2022; Bizot et al., 2020). RNA therapeutics are a novel class of drugs engineered to use RNA-based molecules to prevent and / or treat specific diseases. These include a diverse group of oligonucleotide-based drugs, such as antisense oligonucleotides (ASO), small interfering RNA (siRNA), short hairpin RNA (shRNA), double-stranded RNA (dsRNA), microRNA (miRNA), etc., which can be engineered to selectively interact with biological targets in disease contexts where small molecule-based drugs or monoclonal antibodies cannot currently address or properly resolve the condition. They represent promising therapeutic options for modulating gene expression and addressing mutations in several pathological conditions, such as muscle disorders. Furthermore, RNA-based therapeutics have the potential to modulate entire disease pathways, potentially offering better treatment options to target the pathophysiological mechanisms of various disorders, which could lead to better patient outcomes. In addition, many RNA-based therapeutics have been approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA), and a growing number of therapies are in various stages of clinical trials, demonstrating the effectiveness of such RNA therapies for a wide range of diseases. However, the anionic charge of therapeutic RNAs and their sensitivity to RNases present in the bloodstream and tissues make it difficult for them to efficiently enter cells and exert their effects independently. Delivery remains a core challenge for the therapeutic application of RNA therapeutics. Available delivery systems are used to deliver RNA therapeutics to their target cells. Various strategies have been developed. These strategies include lipid particles (LNPs) and direct conjugation with delivery agents such as cholesterol. In recent years, significant progress has been made using N-acetylgalactosamine (GalNAc) as a targeting ligand for the liver-specific desialyl glycoprotein receptor (Nair JK et al.; Tai, W, Debacker et al.). In fact, GalNAc-siRNA conjugates have been shown to successfully silence genes expressed in the liver. However, there is a need for effective strategies for preventing or treating muscle diseases. Summary of the Invention

[0005] This invention relates to specific VHH molecules that advantageously target transferrin receptor (TfR) in muscle tissue and their use for transporting various therapeutic agents into muscle. More specifically, the invention provides conjugates comprising such VHH molecules, optimized for directing to muscle and mediating the efficient functional delivery of oligonucleotide therapeutic agents to muscle tissue. This invention demonstrates that the conjugates of this invention can efficiently accumulate in muscle and deliver conjugated therapeutic agents within muscle tissue, thus making them suitable for the highly effective prevention and treatment of muscle diseases.

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

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

[0008] A particular object of the present invention is a coupling 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 muscle cells, cardiomyocytes, or muscle cancer cells.

[0010] According to the present invention, the preferred VHH molecule binds to TfR with an affinity (Kd) of 0.01 nM to 4 µM, 0.01 nM to 2500 nM, 0.01 nM to 1000 nM, 0.01 nM to 500 nM, 0.01 nM to 100 nM, 0.1 nM to 4 µM, 0.1 nM to 2500 nM, 0.1 nM to 1000 nM, 0.1 nM to 500 nM, or 0.1 nM to 100 nM.

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

[0012] In one particular aspect, the VHH molecule according to the invention comprises: - CDR1, which comprises a sequence selected from SEQ ID NO: 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, which comprises a sequence selected from SEQ ID NO: 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 - CDR3, which contains sequences selected from SEQ ID NO: 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 another specific aspect, the VHH molecule according to the invention comprises SEQ ID NO: 1, 2 and 3; or SEQ ID NO: 5, 6 and 7; or SEQ ID NO: 9, 10 and 11; or SEQ ID NO: 13, 14 and 15; SEQ ID NO: 17, 2 and 3; or SEQ ID NO: 19, 2 and 3; or SEQ ID NO: 1, 21 and 3; or SEQ ID NO: 1, 23 and 3; or SEQ ID NO: 1, 2 and 25; or SEQ ID NO: 1, 2 and 27; or SEQ ID NO: 1, 2 and 29; or SEQ ID NO: 1, 2 and 31; or SEQ ID NO: 1, 2 and 33; or SEQ ID NO: 67, 2 and 3; or SEQ ID NO: 69, 2 and 3; or SEQ ID NO: 1, 71 and 3; or SEQ ID NO: 1, 73 and 3; or SEQ ID NO: 1, 75 and 3; or SEQ ID NO: 1, 2 and 77; or SEQ ID NO: 1, 2 and 77; or SEQ ID NO: 1, 2 and 77; or SEQ ID NO: 1, 2 and 31; or SEQ ID NO: 1, 2 and 33; or SEQ ID NO: 67, 2 and 3; or SEQ ID NO: 69, 2 and 3; or SEQ ID NO: 1, 71 and 3; or SEQ ID NO: 1, 73 and 3; or SEQ ID NO: 1, 75 and 3; or SEQ ID NO: 1, 2 and 77; or SEQ ID NO: 1, 2 and 77; or SEQ ID NO: 1, 2 and 31; SEQ ID NO: 1, 2 and 79; or SEQ ID NO: 1, 2 and 81; or SEQ ID NO: 1, 2 and 83; or SEQ ID NO: 1, 2 and 85; or SEQ ID NO: 392, 2 and 3; or SEQ ID NO: 1, 113 and 3; or SEQ ID NO: 1, 115 and 3; or SEQ ID NO: 1, 2 and 117; or SEQ ID NO: 1, 2 and 119; or SEQ ID NO: 1, 2 and 121; or SEQ ID NO: 1, 2 and 123; or SEQ ID NO: 125, 2 and 3; or SEQ ID NO: 17, 73 and 3; or SEQ ID NO: 17, 128 and 3; or SEQ ID NO: 5, 160 and 7; or SEQ ID NO: 5, 162 and 7; or SEQ ID NO: 5, 164 and 7; or SEQ ID NO: 5, 166 and 7; or SEQ ID NO: 9, 169 and 11; or SEQ ID SEQ ID NO: 9, 171 and 11; or SEQ ID NO: 175, 176 and 177; or SEQ ID NO: 179, 176 and 180; or SEQ ID NO: 182, 176 and 177; or SEQ ID NO: 184, 176 and 177; or SEQ ID NO: 186, 187 and 188; or SEQ ID NO: 190, 191 and 192; or SEQ ID NO: 194, 195 and 196; or SEQ ID NO: 198, 199 and 200; or SEQ ID NO: 201, 202 and 203; or SEQ ID NO: 205, 206 and 207; or SEQ ID NO: 410, 6 and 7;Or SEQ ID NO: 413, 6 and 7; or SEQ ID NO: 5, 416 and 7; or SEQ ID NO: 5, 419 and 7; or SEQ ID NO: 426, 6 and 7; or SEQ ID NO: 5, 431 and 7; or SEQ ID NO: 434, 6 and 7; or SEQ ID NO: 437, 6 and 7; or SEQ ID NO: 5, 6 and 452; or SEQ ID NO: 5, 6 and 455; or SEQ ID NO: 607, 608 and 609; or SEQ ID NO: 610, 611 and 612; or SEQ ID NO: 671, 2 and 3; or SEQ ID NO: 672, 2 and 3; or SEQ ID NO: 673, 6 and 7; or SEQ ID NO: 674, 6 and 7; or SEQ ID NO: 1, 2 and 713; or SEQ ID NO: 5, 6 and 714; or SEQ ID SEQ ID NO: 674, 164 and 7; or SEQ ID NO: 710, 6 and 7; or SEQ ID NO: 5, 6 and 715; or SEQ ID NO: 674, 712 and 7; or SEQ ID NO: 711, 6 and 7; or SEQ ID NO: 673, 6 and 741; or SEQ ID NO: 673, 6 and 742; or SEQ ID NO: 673, 6 and 743; or SEQ ID NO: 673, 6 and 744; or SEQ ID NO: 673, 431 and 741; or SEQ ID NO: 673, 431 and 742; or SEQ ID NO: 673, 431 and 743; or SEQ ID NO: 673, 6 and 7; or SEQ ID NO: 674, 6 and 7.

[0014] In another specific aspect, the VHH molecule comprises an amino acid sequence selected from any one of SEQ ID NO: 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 amino acid sequence optionally comprises a tag and / or a linker.

[0015] In another specific aspect, the VHH molecule comprises an amino acid sequence selected from any one of SEQ ID NO: 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 another specific aspect, the VHH molecule comprises an amino acid sequence selected from any one of SEQ ID NO: 213, 216-271, 274, 275, 675, 676, and 701. In another specific aspect, the VHH molecule comprises an amino acid sequence selected from any one of SEQ ID NO: 215 and 285-299. In another specific aspect, the VHH molecule comprises an amino acid sequence selected from any of SEQ ID NO: 613-615. In another specific aspect, the VHH molecule is humanized and is preferably selected from SEQ ID NO: 87-92, 130-149, 152-154, 236-241, 252-271, 273-275, 752-765, and 773-786.

[0016] In another particular aspect, the oligonucleotides according to the invention are selected from any single-stranded or double-stranded oligonucleotides, such as small interfering RNA (siRNA), small activating RNA (saRNA), gapmer, antisense oligonucleotide (ASO), shRNA, miRNA, aptamer RNA, and bridging nucleic acid (BNA).

[0017] Another object of the present invention relates to a coupling compound, wherein the VHH molecule is coupled to the oligonucleotide directly or via at least one coupling head by covalent or nonvalent binding. The coupling compound may also contain at least one additional compound.

[0018] In one particular aspect, the additional compound is a half-life-extending moiety or stabilizing group or scaffold, such as an antibody or a fragment thereof (e.g., an Fc fragment), a VHH molecule, PEG, serum albumin, and a serum albumin-binding moiety, preferably an Fc fragment. Preferably, the Fc fragment is an Fc heterodimer comprising an Fc modified with the sequence of SEQ ID NO: 664 on the club arm and an Fc modified with the sequence of SEQ ID NO: 665 on the acetabular arm.

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

[0020] The VHH molecules, conjugates, and pharmaceutical compositions of the present invention can be administered via any conventional route, preferably parenteral, systemic, intravenous, intramuscular, subcutaneous, intracerebral, intraventricular, or intrathecal administration. The present invention can be used in any mammal, particularly in human subjects. It is suitable for treating any muscular or neuromuscular disease, such as myopathy, cardiomyopathy, muscular dystrophy (e.g., DMD, BMD, FSHD, Pompe disease, or familial hypertrophic cardiomyopathy), neuromuscular disease (e.g., ALS, SMA, MS, HD, CMT), or cancers of the muscle (e.g., rhabdomyosarcoma or leiomyosarcoma). Attached Figure Description

[0021] Figure 1 VHH-oligonucleotide conjugates (e.g., B8-siSOD1m, C5-siSOD1m, B8-) are shown. MALAT1 -ASO, B8-siSOD1h or C5-siSOD1h) and free VHH (B8 or C5) compete with the fluorescent reference TfR-binding VHH for concentration-dependent binding and epigenetic binding affinity (Ki / app) of mouse, rhesus monkey or human TfR expressed by mouse Neuro-2A cells, CHO cells stably expressing rhesus monkey TfR or human MCF-7 cells, respectively.

[0022] Figure 2 This study demonstrates that, upon free uptake of the following substances in human MCF-7 cells or MIA PaCa-2 cells, concentration-dependent downregulation of mRNA or lncRNA levels was observed: (A) targeting human... SOD1 TfR-binding VHH-siRNA conjugates (VHH-siSOD1h, where VHH is B8, C5, or C5V8) or non-binding conjugates (C5neg-siSOD1h) of mRNA, or (B) targeting humans and mice. MALAT-1 TfR-binding VHH-ASO conjugates of long non-coding RNAs (VHH- MALAT1 -ASO, where VHH is B8).

[0023] Figure 3 This study demonstrates the potential for downregulation of the mRNA or lncRNA of TfR-binding VHH-oligonucleotide conjugates after free uptake in the mouse Neuro-2A cell line. (A) Figure 3 A shows the results obtained using either the TfR-binding VHH-siRNA conjugate (VHH-siSOD1m, where VHH is C5) or the non-binding conjugate (C5neg-siSOD1m). SOD1 mRNA levels were downregulated in a concentration-dependent manner. (B) Figure 3 B shows the use of targeted mice. SOD1Various examples of VHH-siRNA conjugates with TfR binding to mRNA (i.e., VHH-siSOD1m, VHH-thiol-Mal-siSOD1m, or VHH-ΔHis-siSOD1m, wherein VHH is or contains C5, B8, B8h1, C5V1, C5V13, C5h18, C5h19, or C5V7) were obtained. SOD1 Downregulation of mRNA levels, and such Figure 1 The epigenetic binding affinity (Ki / app) of the test conjugate to TfR, as evaluated on mouse Neuro-2A cells, is shown in Figure (C). Figure 3 C shows the use of targeted humans and mice. MALAT-1 VHH- of long non-coding RNA MALAT1 -ASO conjugates (VHH- MALAT1 -ASO, where VHH is obtained from B8) MALAT-1 Downregulation of lncRNA levels.

[0024] Figure 4 This demonstrates the effects of a single systemic (intravenous IV or subcutaneous SC) administration of a VHH-siRNA conjugate (where VHH is C5 or B8) in wild-type C57Bl / 6 mice. SOD1 Muscle-specific downregulation of mRNA levels.

[0025] Figure 5 This demonstrates the effect of a single subcutaneous administration of a TfR-binding VHH-siSOD1 conjugate (where VHH is B8) in wild-type C57Bl / 6 mice. SOD1 TfR levels at the mRNA level were downregulated specifically and muscle-specifically.

[0026] Figure 6 This study demonstrates the effects of a single subcutaneous administration of a TfR-bound VHH-siSOD1m conjugate (where VHH is B8) on skeletal muscle (gastrocnemius) or myocardium in wild-type C57Bl / 6 mice. SOD1 dose-dependent downregulation of mRNA levels.

[0027] Figure 7 This demonstrates the murine effects in muscle tissues (e.g., gastrocnemius, diaphragm, and heart tissue) following a single subcutaneous (SC) administration of a TfR-bound VHH-siSOD1m conjugate (where VHH is B8) in wild-type C57Bl / 6 mice. SOD1 The time process of mRNA downregulation.

[0028] Figure 8The concentration-dependent binding and epigenetic binding affinity (Ki / app) values ​​of the TfR-binding VHH-hFc-siSOD1 conjugate (where VHH is C5 or B8) competing with the fluorescent reference TfR-binding VHH for mouse and human TfR expressed in mouse Neuro-2A and human MCF-7 cells, respectively, are shown.

[0029] Figure 9 This demonstrates the targeting of humans or rodents. SOD1 After the TfR-binding VHH-hFc-siSOD1 conjugate of mRNA (where VHH is C5 or B8) was freely taken up in (A) human mammary MCF-7 cells and (B) mouse Neuro-2A cells, respectively, the human or mouse cells... SOD1 mRNA levels were downregulated in a concentration-dependent manner.

[0030] Figure 10 The study shows the muscle-specific and dose-dependent downregulation of murine SOD1 mRNA levels in wild-type C57B l / 6 mice following (A) a single intravenous administration of the TfR-binding VHH-hFc-siSOD1m conjugate (where VHH is C5) or (B) a single subcutaneous administration of the TfR-binding VHH-hFc-siSOD1m-5'VP conjugate (where VHH is C5), as well as the estimated ED50 value and maximum downregulation effect (Max KD) in muscle tissue.

[0031] Figure 11 It shows in hTfR1 + / + -KI mice showed that after a single subcutaneous (SC) administration of a TfR-bound VHH-siSOD1m conjugate (where VHH is C5 or B6), the mice... SOD1 Muscle-specific downregulation of mRNA levels was observed in any test samples analyzed after treatment with unconjugated siSOD1m. No downregulation of mouse SOD1 mRNA levels was observed; both hTfR-binding conjugates induced similar potent and muscle-tissue-selective effects, with approximately 70-80% knockdown in the gastrocnemius and diaphragm, approximately 40-50% knockdown in the myocardium, and no effect in the liver or lungs.

[0032] Figure 12 This demonstrates the effect of a single intravenous administration of a TfR-bound VHH-siSOD1h conjugate (where VHH is C5) into various muscle tissues of the olive baboon, such as the gastrocnemius, quadriceps, or tibialis anterior muscles. SOD1 The time process of mRNA downregulation.

[0033] Figure 13The following is shown: (A) the overall structure of the VHH-oligonucleotide conjugate of the present invention, comprising i) an oligonucleotide moiety, which may be any oligonucleotide-based drug such as single-stranded ASO or double-stranded siRNA, ii) VHH, and iii) a linker moiety, which may consist of a half-life extension entity, wherein the oligonucleotide and VHH are linked to the half-life extension entity at two different sites; and (BE) detailed structures of the linkers for some conjugates evaluated in the experimental section, such as VHH-siRNA conjugate (B), VHH-hFc-siRNA conjugate (C), VHH-thiol-Mal-siRNA (D), or VHH-ASO conjugate (E).

[0034] Figure 14 Examples of conjugation strategies for generating VHH-oligonucleotide (A) or heterodimeric VHH-hFc-oligonucleotide (B) conjugates with stable linkers are shown. This general conjugation strategy involves a pooled synthesis using parallel modifications: (i) modifying the VHH (A) or heterodimeric VHH-hFc (B) to introduce, for example, an azido linker with site specificity; and (ii) modifying the oligonucleotide to introduce, for example, a constrained alkyne group complementary to the azido functional group. In the final step, a copper-free click reaction is preferably used to link the homofunctionalized azido-VHH (A) or VHH-hFc-azido (B) to the alkyne-oligonucleotide precursor to generate VHH-oligonucleotide or VHH-hFc-oligonucleotide conjugates with stable linkers.

[0035] Figure 15 The study demonstrated downregulation of murine SOD1 mRNA levels in muscle tissue of B-hTfR mice following a single intraventricular (ICV) administration of a TfR-bound VHH-siSOD1 conjugate (where VHH is B8h1 or B8V32) compared to either a lipophilic C16 (palmitic acid)-siSOD1 conjugate or a non-bound C5neg-siSOD1 conjugate; VHH-siSOD1m-5'VP, single ICV administration in hTfR1+ / + mice, 7 days.

[0036] Figure 16This study demonstrates the downregulation of murine SOD1 mRNA levels in muscle tissue of B-hTfR mice following a single subcutaneous (SC) administration of a TfR-binding VHH-hFc-siSOD1 conjugate (where 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 the conjugates to human TfR for each test was assessed using surface plasmon resonance; VHH-hFc-siSOD1m-5'VP, 4.5 mpk equivalent siRNA, single SC in hTfR1+ / + mice, 14 days.

[0037] Figure 17 This study demonstrates the downregulation of murine SOD1 mRNA levels in muscle tissue of B-hTfR mice following multiple intravenous boluses (Q2D x3) of a TfR-binding VHH-hFc-siSOD1 conjugate (where VHH is B8V40, B8V32, B8V31, or B8V31h5) at 1.5 mg / kg (siRNA molar equivalent). The binding affinity of each conjugate to human and rhesus / cynomolgus monkey TfR was assessed using surface plasmon resonance; VHH-hFc-siSOD1m-5'VP, 1.5 mpk equivalent siRNA, IV Q2D x3, 14 days in B-hTfR mice. Detailed Implementation

[0038] This invention provides novel transferrin receptor (TfR) binders that can be used to deliver molecules, such as therapeutic agents, imaging agents, or diagnostic agents, into muscle. More specifically, this invention discloses modified VHH molecules that bind to TfR in muscle tissue and cells, and their uses. Therefore, targeting TfR with drugs is particularly suitable for treating any muscle or neuromuscular disease, such as myopathy, cardiomyopathy, muscular dystrophy (e.g., DMD, BMD, FSHD, Pompe disease, or familial hypertrophic cardiomyopathy), and neuromuscular diseases (e.g., ALS, SMA, MS, HD, or CMT).

[0039] TfR participates in the transport of iron to cells and organs via its ligand, transferrin (Tf). This receptor has been shown to be highly expressed in the brain endothelium (Jefferies et al.; Pardridge et al.), although it is also abundant in blood cells and the lungs (Chan and Gerhardt). This receptor has been used to deliver pharmacological agents across the blood-brain barrier (Johnsen et al.; WO2012075037; WO2016208695; WO2020144233 et al.). Furthermore, TfR has been described as playing an important role in muscle development (Ying Li et al.). In addition to its role in muscle development, TfR plays a crucial role in iron homeostasis in muscle tissue and systemic metabolism (Ying Li et al.; Barrientos T. et al.; Xu W. et al.). TfR is involved in the incorporation of iron transported by its transferrin ligand and in the regulation of cell growth (Neckers and Trepel 1986; Ponka and Lok 1999).

[0040] There are two types of transferrin receptors: TfR1 and the homologous receptor TfR2, which is primarily expressed in the liver. In the context of this invention, the term TfR is used to refer to a TfR1 homolog. TfR is a type II homodimeric transmembrane glycoprotein composed of two identical 90 kDa subunits linked by two disulfide bonds (Jing and Trowbridge 1987; McClelland et al., 1984). Each monomer has a short cytoplasmic N-terminal domain of 61 amino acids containing the YTRF (tyrosine-threonine-arginine-phenylalanine) internalization motif, a hydrophobic transmembrane segment of 27 amino acids, and a wide C-terminal extracellular domain of 670 amino acids containing a trypsin cleavage site and a transferrin binding site (Aisen, 2004). Each subunit can bind transferrin molecules. The extracellular domain has one O-glycosylation site and three N-glycosylation sites, the latter being particularly important for the proper folding and transport of the receptor to the cell surface (Hayes et al., 1997). Palmitoylation sites are also present in the intracellular domain, which may anchor the receptor and allow for its endocytosis (Alvarez et al., 1990; Omary and Trowbridge, 1981). In addition, intracellular phosphorylation sites are present, the function of which is uncertain and do not play a role in endocytosis (Rothenberger et al., 1987).

[0041] The TfR receptor is expressed at high levels by highly proliferating cells (both healthy and tumor cells) (Gatter et al., 1983). Numerous studies have shown that TfR expression levels are higher in cancer cells compared to healthy cells. Therefore, pathology in diseases 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's lymphoma (Habeshaw et al., 1983) shows increased TfR expression, which is associated with tumor grade, disease stage, or prognosis. Therefore, targeting TfR with drugs may be suitable for cancer treatment, particularly for treating muscle cancers such as rhabdomyosarcoma and leiomyosarcoma.

[0042] Using purified membrane preparations from cells expressing high levels of hTfR and mTfR, the inventors generated and selected VHH molecules, particularly those binding both human and non-human TfR. They also showed that these VHH molecules, when fused with oligonucleotides such as siRNA or ASO, retain TfR-binding capacity and efficient delivery in vivo, in muscle cells and other cell types. The VHH molecules exhibit appropriate levels of affinity and specificity upon TfR binding for proper endocytosis. Therefore, the present invention provides novel TfR-binding molecules that represent valuable agents for targeting drugs to muscle. More specifically, the inventors showed that conjugates of the present invention comprising VHH molecules and oligonucleotides such as siRNA bind TfR in various muscle tissues, and in WT mice expressing murine TfR1 or in humanized mice expressing human TfR1. hTfR1 + / + In vivo downregulation of target mRNAs in mice and non-human primates. For example, the TfR-binding conjugate according to the invention induces potent muscle tissue-selective action, achieving over 70% mRNA knockdown in the gastrocnemius, diaphragm, and cardiac muscle. Receptor-mediated transcytosis (RMT), a physiological process involving the binding of a ligand to a receptor expressed on the organ / tissue, is included in strategies evaluated for delivering RNA therapeutics to their target organs / tissues. Interestingly, a single systemic administration of the TfR-binding VHH-siRNA conjugate induced potent and durable downregulation of target mRNA levels in all muscle tissues tested in mice and non-human primates (e.g., gastrocnemius, quadriceps femoris, and tibialis anterior), with 60% knockdown observed in non-human primates lasting for more than three months.

[0043] Overall, the inventors have demonstrated the ability of the TfR-binding VHH-siRNA conjugate to mediate TfR-dependent binding, functional uptake, and persistent in vivo downregulation of target mRNAs in rodents / NHPs / humans at low therapeutic doses in skeletal and cardiac muscle tissues.

[0044] Furthermore, the inventors have demonstrated that when conjugated to an RNA therapeutic agent (e.g., siRNA or ASO) via an antibody or antibody fragment scaffold (e.g., human IgG1-Fc region), the VHH molecule according to the invention retains its TfR binding capacity in vitro and has demonstrated potent muscle-selective targeting and functional uptake properties in vivo by low-dose systemic administration (e.g., intravenous (IV) or subcutaneous (SC) administration).

[0045] The inventors also demonstrated that the conjugates of the present invention exhibited strong functional delivery potential in muscle tissue of mice expressing hTfR at low doses. Interestingly, the inventors observed that several VHH variants and conjugates showed very similar binding affinity (less than 2-fold difference) between human and non-human primate (rhesus / cynomolgus monkey) TfR, thus allowing for easier translation from preclinical settings in non-human primates to clinical studies in humans.

[0046] Surprisingly, the inventors have demonstrated for the first time that conjugates according to the invention can also be functionally delivered to muscle tissue following local CNS administration (e.g., intracerebral, intraventricular (ICV), or intrathecal (IT) administration), even at doses similar to those used for muscle delivery via systemic administration. Therefore, the TfR-binding VHH-oligonucleotide conjugates according to the invention have the potential to address muscle and neuromuscular disorders not only through systemic administration but also through local CNS administration. Furthermore, the inventors have demonstrated for the first time that the TfR-binding VHH-oligonucleotide conjugates according to the invention can be used to simultaneously target various disordered muscle components (particularly muscle cells) and neuronal components (particularly nervous system cells), such as muscle diseases preferably selected from myopathy, cardiomyopathy, muscular dystrophy (e.g., DMD, BMD, FSHD, Pompe disease, or familial hypertrophic cardiomyopathy), or neuromuscular disorders such as spinal muscular atrophy, amyotrophic lateral sclerosis, peroneal muscular atrophy, multiple sclerosis, or Huntington's disease, because the conjugates according to the invention are functionally delivered to both muscle and nervous system tissues after local CNS (IT or ICV) administration, and are able to simultaneously bind TfRs on the surface of both muscle cells and nervous system cells after local CNS administration. Therefore, the conjugates according to the invention can be used to address and treat muscle and neuromuscular disorders not only through systemic administration but also through local CNS (IT or ICV) administration.

[0047] Therefore, one object of the present invention relates to a VHH molecule, wherein the VHH molecule binds to both human and non-human (e.g., non-human primate (NHP) or rodent, such as rat or mouse) TfR. Preferably, the VHH binds to muscle tissue expressing TfR. The present invention also relates to conjugates comprising such VHH, their preparation, compositions comprising them, and their uses.

[0048] definition

[0049] Unless otherwise defined herein, all scientific and technical terms used in connection with this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0050] As used herein, the term “treatment” refers to any action aimed at improving a patient’s health condition, such as treating, stopping, preventing, and blocking a disease such as a muscle disease, neuromuscular disease, or cancer of the muscle, or at least one symptom of said muscle disease, neuromuscular disease, or cancer. It refers to both curative and / or preventative treatment of a muscle disease, neuromuscular disease, or cancer. In the context of cancer, this particularly includes alleviating symptoms, reducing inflammation, inhibiting cancer cell growth, and / or reducing tumor size. For example, in the case of cancer, responses to treatment include reduction of cachexia, increased survival time, prolonged time before tumor progression, reduction of tumor mass, reduction of tumor burden, and / or time before tumor metastasis, time before tumor recurrence, tumor response, complete response, partial response, stable disease, progressive disease, progression-free survival, and prolonged overall survival, each of which is measured, for example, by standards established by the American Cancer Institute and the U.S. Food and Drug Administration for the approval of new drugs (Johnson et al., J. Clin. Oncol., 2009; 21(7): 1404-1411).

[0051] The "therapeutic effective dose" described herein refers to the dose that produces a therapeutic effect for a given condition and administration regimen. It is typically the average dose of an active substance administered to significantly improve some symptoms associated with a disease or pathological condition. For example, in the treatment of cancers of the muscle (such as rhabdomyosarcoma or leiomyosarcoma) or cancers of other tissues, or pathologies, lesions, or disorders affecting the muscle (such as muscular or neuromuscular diseases), a dose of an active substance that reduces, prevents, delays, eliminates, or stops one of the causes or symptoms of said disease or disorder would be therapeutically effective. A "therapeutic effective dose" of an active substance does not necessarily cure a muscle disease or disorder, but will provide treatment for such disease or disorder, thereby delaying, hindering, or preventing its onset, or alleviating its symptoms, or altering its duration, such as reducing its severity, or accelerating the patient's recovery.

[0052] The term "VHH molecule" as used in this article corresponds to the variable region of a naturally occurring, heavy-chain-only Camelidae antibody that lacks a light chain. VHHs have a very small molecular weight, approximately 12-15 kDa. They contain a single-chain molecule and can bind to their homologous antigens using a single domain. The antigen-binding surface of a VHH is typically more convex (or protruding) than that of a conventional antibody, which is usually flat or concave. More specifically, a VHH consists of four framework regions (or FRs) and three complementarity-determining regions (or CDRs). The sequences and structures of the framework regions are defined as conserved, while the complementarity-determining regions exhibit high variability in sequence content and structural conformation, participating in antigen binding and providing antigen specificity. Compared to conventional human antibody VHs, several amino acids are substituted in the FR2 region and the complementarity-determining regions (CDRs) of VHHs. For example, highly conserved hydrophobic amino acids (e.g., Val42, Gly49, Leu50, and / or Trp52) in the FR2 region are typically replaced with hydrophilic amino acids (Phe42, Glu49, Arg50, Gly52) to make the overall structure more hydrophilic and contribute to high stability, solubility, and anti-aggregation properties. The VHH molecule according to the invention is a polypeptide comprising (or consisting of, or substantially consisting of) an antigen-binding domain of a heavy-chain-only antibody (HcAb). The term “and / or” as used herein should be considered as specifically disclosing each of the two specified features or components, regardless of the presence of the other. For example, “A and / or B” should be considered as specifically disclosing (i) A, (ii) B, and (iii) each of A and B, as if each were described separately.

[0053] A designation without a specific number can refer to one or more designations (e.g., “VHH molecule” can mean one or more VHH molecules), unless the context clearly describes one designation or more than one designation.

[0054] VHH molecules

[0055] To generate VHH molecules with suitable properties, the inventors tested over 2000 TfR-binding VHHs from a VHH library generated by immunization of alpacas with a TfR immunogen. After analyzing the binding and specificity of these clones, the inventors further selected approximately 450 clones, sequenced them all, and compared them. Other VHHs with controlled / improved binding properties were generated through mutagenesis or humanization. The relevant domains and sequences of preferred VHHs are provided in the experimental section and sequence listing. The properties of the VHHs and their conjugates are also described in the experimental section.

[0056] The VHH molecule of the present invention typically comprises or is composed of the following formula: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, Where FRn represents the framework region and CDRn represents the complementarity determination region.

[0057] In one particular embodiment, the VHH molecule of the present invention comprises a CDR1 domain, the CDR1 domain comprising components selected from SEQ ID NO. NO: 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 amino acid sequences or variants thereof, or consisting of said amino acid sequences or variants thereof, said variants having at least 60%, particularly at least 65%, 70%, or 75%, for example at least 80% or 85%, preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity with any of said sequences over their entire length (preferred percentages of identity for a particular sequence are preferably percentages corresponding to an integer number of amino acids), and retaining TfR binding capacity. The preferred VHH molecule of the present invention contains a CDR1 domain having an amino acid sequence selected from SEQ ID NO: 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 a variant having several amino acid modifications, for example, at least three amino acid modifications, preferably at most three or two amino acid modifications, and in certain cases at most one amino acid modification. The “identity %” between amino acid (or nucleic acid) sequences can be determined by techniques known per se in the art. Typically, the identity percentage (%) between two nucleic acid or amino acid sequences is determined using a computer program such as the GAP provided in the GCG package (Program Manual for the Wisconsin Package, 8th edition, August 1996, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman, SB and Wunsch, CD, (1970), Journal of Molecular Biology, 48, 443-453). The identity percentage between two sequences represents the identity over the entire length of the sequence.As described above, the preferred identity percentage of a specific sequence is preferably a percentage corresponding to an integer number of amino acids in both the reference sequence and its variants (the reference sequence is, for example, SEQ ID NO: 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 identified herein, such as ...426, 434 or 437, 607, 610, 671-674, 710, 711 or any other reference sequence identified herein, such as SEQ ID NO: 1, 5, 9, 13, 17, 19, 67, 69, 426, 434 or 437, 607, 610, 671-674, NO: 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, 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).

[0058] Specific examples of the VHH molecule of the present invention include the CDR1 sequence, which comprises or is substantially composed of SEQ ID NO: 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 to 674, 710 or 711.

[0059] In another specific embodiment, the VHH molecule of the present invention includes a CDR2 domain, the CDR2 domain comprising components selected from SEQ ID NO. NO: 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 a variant thereof, or composed of said amino acid sequence or a variant thereof, said variant having at least 60%, particularly at least 65%, 70%, or 75%, for example at least 80% or 85%, preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity with any of said sequences over its entire length, and retaining TfR binding ability. The preferred VHH molecule of the present invention contains a CDR2 domain having an amino acid sequence selected from SEQ ID NO: 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 having several amino acid modifications, for example at least 3 amino acid modifications, preferably at most 3 or 2 amino acid modifications, and in certain cases at most 1 amino acid modification variants.

[0060] Specific examples of the VHH molecule of the present invention include a CDR2 sequence, which comprises or is substantially composed of SEQ ID NO: 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.

[0061] In another specific embodiment, the VHH molecule of the present invention includes a CDR3 domain, said CDR3 domain comprising a number selected from SEQ ID NO: 3, 7, 11, 15, 25, 27, 29, 31, 33, 77, 79, 81, 83, 85, 117, 119, 121, 123, 177, 180, The amino acid sequence of 188, 192, 196, 200, 203, 207, 452, 455, 609, 612, 713-715 or 741-744 or a variant thereof, or consisting of the amino acid sequence or a variant thereof, wherein the variant has at least 60%, particularly at least 65%, 70% or 75%, for example at least 80% or 85%, 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 with any of the sequences in its entire length, and retains TfR binding ability. The preferred VHH molecule of the present invention contains a CDR3 domain having an amino acid sequence selected from SEQ ID NO: 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 having several amino acid modifications, for example at least 3 amino acid modifications, preferably at most 3 or 2 amino acid modifications, and in certain cases at most 1 amino acid modification variants.

[0062] Specific examples of the VHH molecule of the present invention include the CDR3 sequence, which comprises or is substantially composed of SEQ ID NO: 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.

[0063] In another specific embodiment, the VHH molecule of the present invention comprises: - A CDR1 domain comprising, or consisting of, an amino acid sequence selected from SEQ ID NO: 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 a variant thereof, or consisting of, said amino acid sequence or a variant thereof, said variant having at least 60%, particularly at least 65%, 70%, or 75%, for example at least 80% or 85%, preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, more preferably at least 95% amino acid identity with any of said sequences over their entire length; and - A CDR2 domain comprising, or consisting of, an amino acid sequence selected from SEQ ID NO: 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 a variant thereof, or consisting of, said amino acid sequence or a variant thereof, said variant having at least 60%, particularly at least 65%, 70%, or 75%, for example at least 80% or 85%, preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, more preferably at least 95% amino acid identity with any of said sequences over their entire length; and - CDR3 domain, the CDR3 domain containing elements selected from SEQ ID The amino acid sequence of NO: 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 a variant thereof, or consisting of said amino acid sequence or a variant thereof, said variant having at least 60%, particularly at least 65%, 70% or 75%, for example at least 80% or 85%, preferably at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, more preferably at least 95% amino acid identity with any of said sequences over their entire length. The VHH has TfR binding capability.

[0064] In a preferred embodiment, the VHH molecule of the present invention comprises: - A CDR1 domain having an amino acid sequence selected from the following: SEQ ID NO: 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 variants thereof having at most 3, 2 or 1 amino acid modifications; and - A CDR2 domain having an amino acid sequence selected from the following: SEQ ID NO: 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 variants thereof having at most 3, 2, or 1 amino acid modifications; and - CDR3 domain, the CDR3 domain having an amino acid sequence selected from the following: SEQ ID NO: 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 variants thereof having at most 3, 2 or 1 amino acid modifications.

[0065] In a more preferred embodiment, the VHH molecule of the present invention comprises CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 domains respectively comprise the following sequences or variants thereof as defined above, or consist of said sequences or variants: - SEQ ID NO: 1, 2 and 3; or - SEQ ID NO: 17, 2 and 3; or - SEQ ID NO: 19, 2 and 3; or - SEQ ID NO: 67, 2 and 3; or - SEQ ID NO: 69, 2 and 3; or - SEQ ID NO: 1, 21 and 3; or - SEQ ID NO: 1, 23 and 3; or - SEQ ID NO: 1, 71 and 3; or - SEQ ID NO: 1, 73 and 3; or - SEQ ID NO: 1, 75 and 3; or - SEQ ID NO: 1, 2 and 25; or - SEQ ID NO: 1, 2 and 27; or - SEQ ID NO: 1, 2 and 29; or - SEQ ID NO: 1, 2 and 31; or - SEQ ID NO: 1, 2 and 33; or - SEQ ID NO: 1, 2 and 77; or - SEQ ID NO: 1, 2 and 79; or - SEQ ID NO: 1, 2 and 81; or - SEQ ID NO: 1, 2 and 83; or - SEQ ID NO: 1, 2 and 85; or - SEQ ID NO: 5, 6 and 7; or - SEQ ID NO: 9, 10 and 11; or - SEQ ID NO: 13, 14 and 15; or - SEQ ID NO: 392, 2 and 3; or - SEQ ID NO: 1, 113 and 3; or - SEQ ID NO: 1, 115 and 3; or - SEQ ID NO: 1, 2 and 117; or - SEQ ID NO: 1, 2 and 119; or - SEQ ID NO: 1, 2 and 121; or - SEQ ID NO: 1, 2 and 123; or - SEQ ID NO: 125, 2 and 3; or - SEQ ID NO: 17, 73 and 3; or - SEQ ID NO: 17, 128 and 3; or - SEQ ID NO: 5, 160 and 7; or - SEQ ID NO: 5, 162 and 7; or - SEQ ID NO: 5, 164 and 7; or - SEQ ID NO: 5, 166 and 7; or - SEQ ID NO: 9, 169 and 11; or - SEQ ID NO: 9, 171 and 11; or - SEQ ID NO: 175, 176 and 177; or - SEQ ID NO: 179, 176 and 180; or - SEQ ID NO: 182, 176 and 177; or - SEQ ID NO: 184, 176 and 177; or - SEQ ID NO: 186, 187 and 188; or - SEQ ID NO: 190, 191 and 192; or - SEQ ID NO: 194, 195 and 196; or - SEQ ID NO: 198, 199, and 200; or - SEQ ID NO: 201, 202 and 203; or - SEQ ID NO: 205, 206 and 207; or - SEQ ID NO: 410, 6 and 7; or - SEQ ID NO: 413, 6 and 7; or - SEQ ID NO: 5, 416 and 7; or - SEQ ID NO: 5, 419 and 7; or - SEQ ID NO: 426, 6 and 7; or - SEQ ID NO: 5, 431 and 7; or - SEQ ID NO: 434, 6 and 7; or - SEQ ID NO: 437, 6 and 7; or - SEQ ID NO: 5, 6 and 452; or - SEQ ID NO: 5, 6 and 455; or - SEQ ID NO: 607, 608, and 609; or - SEQ ID NO: 610, 611 and 612; or - SEQ ID NO: 671, 2 and 3; or - SEQ ID NO: 672, 2 and 3; or - SEQ ID NO: 673, 6 and 7; or - SEQ ID NO: 674, 6 and 7; or - SEQ ID NO: 1, 2 and 713; or - SEQ ID NO: 5, 6 and 714; or - SEQ ID NO: 674, 164 and 7; or - SEQ ID NO: 710, 6 and 7; or - SEQ ID NO: 5, 6 and 715; or - SEQ ID NO: 674, 712 and 7; or - SEQ ID NO: 711, 6 and 7; or - SEQ ID NO: 673, 6 and 741; or - SEQ ID NO: 673, 6 and 742; or - SEQ ID NO: 673, 6 and 743; or - SEQ ID NO: 673, 6 and 744; or - SEQ ID NO: 673, 431 and 741; or - SEQ ID NO: 673, 431 and 742; or - SEQ ID NO: 673, 431 and 743; or - SEQ ID NO: 673, 6 and 7; or - SEQ ID NO: 674, 6 and 7; Preferably, the variant has at most 3, 2 or 1 amino acid modifications.

[0066] In another preferred embodiment, the VHH molecule of the present invention comprises CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 domains are C5 domains or variants thereof (as listed in Table 1), and each comprises, or consists of, the following sequences or variants thereof as defined above: - SEQ ID NO: 1, 2 and 3; or - SEQ ID NO: 13, 14 and 15; or - SEQ ID NO: 17, 2 and 3; or - SEQ ID NO: 19, 2 and 3; or - SEQ ID NO: 67, 2 and 3; or - SEQ ID NO: 69, 2 and 3; or - SEQ ID NO: 1, 21 and 3; or - SEQ ID NO: 1, 23 and 3; or - SEQ ID NO: 1, 71 and 3; or - SEQ ID NO: 1, 73 and 3; or - SEQ ID NO: 1, 75 and 3; or - SEQ ID NO: 1, 2 and 25; or - SEQ ID NO: 1, 2 and 27; or - SEQ ID NO: 1, 2 and 29; or - SEQ ID NO: 1, 2 and 31; or - SEQ ID NO: 1, 2 and 33; or - SEQ ID NO: 1, 2 and 77; or - SEQ ID NO: 1, 2 and 79; or - SEQ ID NO: 1, 2 and 81; or - SEQ ID NO: 1, 2 and 83; or - SEQ ID NO: 1, 2 and 85; or - SEQ ID NO: 392, 2 and 3; or - SEQ ID NO: 1, 113 and 3; or - SEQ ID NO: 1, 115 and 3; or - SEQ ID NO: 1, 2 and 117; or - SEQ ID NO: 1, 2 and 119; or - SEQ ID NO: 1, 2 and 121; or - SEQ ID NO: 1, 2 and 123; or - SEQ ID NO: 125, 2 and 3; or - SEQ ID NO: 17, 73 and 3; or - SEQ ID NO: 17, 128 and 3; or - SEQ ID NO: 671, 2 and 3; or - SEQ ID NO: 672, 2 and 3; or - SEQ ID NO: 1, 2 and 713; Preferably, the variant has at most 3, 2 or 1 amino acid modifications.

[0067] In another preferred embodiment, the VHH molecule of the present invention comprises CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 domains are B6 domains (as listed in Table 1), each comprising, or consisting of, the following sequences or variants thereof as defined above: - SEQ ID NO: 9, 10 and 11; or - SEQ ID NO: 9, 169 and 11; or - SEQ ID NO: 9, 171 and 11, Preferably, the variant has at most 3, 2 or 1 amino acid modifications.

[0068] In one particular embodiment, the VHH molecule of the present invention is a B6 or variant thereof that targets the top domain of hTfR and contains CDR1, CDR2, and CDR3, wherein the CDRs respectively contain the following sequences or variants thereof as defined above, or consist of said sequences or variants: - SEQ ID NO: 9, 10 and 11; or - SEQ ID NO: 9, 169 and 11; or - SEQ ID NO: 9, 171 and 11; or - SEQ ID NO: 175, 176 and 177; or - SEQ ID NO: 179, 176 and 180; or - SEQ ID NO: 182, 176 and 177; or - SEQ ID NO: 184, 176 and 177; Preferably, the variant has at most 3, 2 or 1 amino acid modifications.

[0069] In another specific embodiment, the VHH molecule of the present invention is a VHH molecule that targets the top domain of hTfR and includes CDR1, CDR2, and CDR3 (as listed in Table 1), wherein each CDR contains the following sequence or a variant thereof as defined above, or is composed of said sequence or variant: - SEQ ID NO: 9, 10 and 11; or - SEQ ID NO: 9, 169 and 11; or - SEQ ID NO: 9, 171 and 11; or - SEQ ID NO: 175, 176 and 177; or - SEQ ID NO: 179, 176 and 180; or - SEQ ID NO: 182, 176 and 177; or - SEQ ID NO: 184, 176 and 177; or - SEQ ID NO: 186, 187 and 188; or - SEQ ID NO: 190, 191 and 192; or - SEQ ID NO: 194, 195 and 196; or - SEQ ID NO: 198, 199, and 200; or - SEQ ID NO: 201, 202 and 203; or - SEQ ID NO: 205, 206 and 207, Preferably, the variant has at most 3, 2 or 1 amino acid modifications.

[0070] In another preferred embodiment, the VHH molecule of the present invention comprises CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 domains are B8 domains or variants thereof (as listed in Table 1), and each comprises, or consists of, the following sequences or variants thereof as defined above: - SEQ ID NO: 5, 6 and 7; or - SEQ ID NO: 5, 160 and 7; or - SEQ ID NO: 5, 162 and 7; or - SEQ ID NO: 5, 164 and 7; or - SEQ ID NO: 5, 166 and 7; or - SEQ ID NO: 410, 6 and 7; or - SEQ ID NO: 413, 6 and 7; or - SEQ ID NO: 5, 416 and 7; or - SEQ ID NO: 5, 419 and 7; or - SEQ ID NO: 426, 6 and 7; or - SEQ ID NO: 5, 431 and 7; or - SEQ ID NO: 434, 6 and 7; or - SEQ ID NO: 437, 6 and 7; or - SEQ ID NO: 5, 6 and 452; or - SEQ ID NO: 5, 6 and 455; or - SEQ ID NO: 673, 6 and 7; or - SEQ ID NO: 674, 6 and 7; or - SEQ ID NO: 5, 6 and 714; or - SEQ ID NO: 674, 164 and 7; or - SEQ ID NO: 710, 6 and 7; or - SEQ ID NO: 5, 6 and 715; or - SEQ ID NO: 674, 712 and 7; or - SEQ ID NO: 711, 6 and 7; or - SEQ ID NO: 673, 6 and 741; or - SEQ ID NO: 673, 6 and 742; or - SEQ ID NO: 673, 6 and 743; or - SEQ ID NO: 673, 6 and 744; or - SEQ ID NO: 673, 431 and 741; or - SEQ ID NO: 673, 431 and 742; or - SEQ ID NO: 673, 431 and 743; or - SEQ ID NO: 673, 6 and 7; or - SEQ ID NO: 674, 6 and 7; Preferably, the variant has at most 3, 2 or 1 amino acid modifications.

[0071] In another specific embodiment, the VHH molecule of the present invention cross-reacts with human, mouse, and / or non-human primate species, as detailed in Tables 5 and 6 below. The preferred VHH molecule of the present invention comprises the FR domain as defined below.

[0072] In one particular embodiment, the FR1 domain comprises, or consists of, the sequence or variant thereof as shown in SEQ ID NO: 35, which has at least 58% amino acid identity with the sequence over its entire length, such as 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 80% amino acid identity: EVQLVESGGGLVQPGGSLKLSCAAS (SEQ ID NO: 35). More preferably, the bolded amino acid residues are present, and the variation occurs only at other positions.

[0073] In one specific implementation, E in position 1 can be replaced by Q.

[0074] In one specific implementation, V in position 5 can be replaced by Q.

[0075] In one specific implementation, E in position 6 can be replaced by Q.

[0076] In one specific implementation, G in position 10 can be replaced with K or A.

[0077] In one specific implementation, L in position 11 can be replaced by V or E.

[0078] In one specific implementation, P in position 14 can be replaced by A.

[0079] In one specific implementation, G in position 16 can be replaced by D.

[0080] In another specific implementation, K in position 19 can be replaced by R.

[0081] In another specific embodiment, A in position 23 can be replaced by V or T.

[0082] In another specific embodiment, S in position 25 can be replaced by D.

[0083] More preferably, referring to this sequence, the FR1 contains at most four amino acid modifications in the non-bold amino acid residues, even more preferably at most three, and even more preferably at most two. In a preferred embodiment, the amino acid modification is R at position 19.

[0084] In another specific embodiment, the FR1 has an amino acid sequence selected from any of the amino acid sequences listed below: EVQLVESGGGVVQPGGSLKLSCVAS (SEQ ID NO: 36); EVQLVESGGGVVQPGGSLRLSCAAS (SEQ ID NO: 37); EVQLVESGGGLVQPGGSLRLSCTAS (SEQ ID NO: 38); or EVQLVESGGGEVQPGGSLKLSCVAS (SEQ ID NO: 39); or a variant thereof.

[0085] Other specific examples of FR1 of the VHH molecule according to the present invention are provided below (see also Table 3): EVQLVESGGGVVQPGGSLKLSCAAS (SEQ ID NO: 331), EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 332), EVQLVESGGGVVQPGGSLRLSCAAD (SEQ ID NO: 333), EVQLVESGGGVVQPGGSLRLSCVAS (SEQ ID NO: 400), QVQLVQSGGGLVQAGGSLTLSCTAS (SEQ ID NO: 334), EVQLVESGGGLVQAGGSLRLSCTAS (SEQ ID NO: 335), QVQLVQSGGGLVQPGGSLRLSCAAS (SEQ ID NO: 336), EVQLVESGGGLVQAGDSLRLSCTAS (SEQ ID NO: 337), QVQLVQSGGGLVQAGGSLLRLSCAAS (SEQ ID NO: 338), EVQLVQSGGGLVQAGGSLLRLSCAAS (SEQ ID NO: 339), EVQLVESGGGLVQPGESLRLSCTAS (SEQ ID NO: 340), EVQLVESGGGLVQPGGSLRLSCVSS (SEQ ID NO: 341), EVQLVESGGGLVQAGDSLRLSCAAS (SEQ ID NO: 619), or VQLVESGGRLVQAGGSLRLSCTAS (SEQ ID NO: 620).

[0086] In one particular embodiment, the VHH molecule of the present invention comprises an FR2 domain comprising, or consists of, the sequence shown below (SEQ ID NO: 40) or a variant thereof, said sequence or variant 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 with said sequence over its entire length: MRWYRQAPGKQRELVAT (SEQ ID NO: 40). More preferably, said bold amino acid residues are present, and the variation occurs only at other positions.

[0087] In one specific implementation, M in position 1 can be replaced by I or V.

[0088] In one specific implementation, R in position 2 can be replaced by G, H, or S.

[0089] In one specific implementation, Y in position 4 can be replaced by F or V.

[0090] In one specific implementation, R in position 5 can be replaced by G.

[0091] In one specific implementation, Q in position 6 can be replaced by R or E.

[0092] In one specific implementation, A in position 7 can be replaced by R.

[0093] In one specific implementation, G in position 9 can be replaced by I or E.

[0094] In one specific implementation, Q at position 11 can be replaced by E, G, I, or D.

[0095] In one specific implementation, R in position 12 can be replaced by L.

[0096] In one specific implementation, E in position 13 can be replaced by N or H.

[0097] In one specific implementation, L in position 14 can be replaced by F, W, S, or Q.

[0098] In one specific implementation, V in position 15 can be replaced by Q or I.

[0099] In one specific implementation, A in position 16 can be replaced by M or S.

[0100] In one specific implementation, T in position 17 can be replaced by G or S.

[0101] More preferably, referring to this sequence, the FR2 contains up to 6 amino acid modifications in the non-bold amino acid residues, even more preferably up to 5, up to 3, or even more preferably up to 2 amino acid modifications. In a preferred embodiment, the amino acid modification is V at position 4 and / or G at position 11 and L at position 12 and / or W at position 14 and / or S at position 16 and / or G at position 17.

[0102] In one particular embodiment, the VHH molecule of the present invention contains at least one of the following amino acids in the FR2 domain: Phe42, Glu49, Arg50 or Gly52 (according to IMGT number).

[0103] In another specific embodiment, the FR2 has an amino acid sequence selected from any of the amino acid sequences listed below: IRWYRQAPGKQREFVAG (SEQ ID NO: 41); MRWYRQAPGKQREWVAG (SEQ ID NO: 42); MGWFRRAPGKERELVAS (SEQ ID NO: 43); VRWYRQRPGKQREWVAG (SEQ ID NO: 44); or a variant thereof.

[0104] Other specific examples of FR2 of the VHH molecule according to the present invention are provided below (see also Table 3): IRWVRQAPGKGLEWVAG (SEQ ID NO: 342), IRWYRQAPGKGLEFVAG (SEQ ID NO: 343), IRWVRQAPGKGLEFVAG (SEQ ID NO: 344), IRWYRQAPGKGREFVAG (SEQ ID NO: 345), IRWVRQAPGKQREFVAG(SEQ ID NO:346), IRWYRQAPGKGLEWVAG(SEQ ID NO:347), MRWYRQAPGKGLEWVAG(SEQ ID NO:348), MRWYGQAPGKQREWVAG(SEQ ID NO:349), MRWYREAPGKQREWVAG(SEQ ID NO:350), MRWYRQAPIKQREWVAG(SEQ ID NO:351), MRWYRQAPGKIREWVAG(SEQ ID NO:352), MGWFRRAPGKERNLVAS(SEQ ID NO:353), MGWFRRAPGKERESVAS(SEQ ID NO:354) MGWFRRAPGKERELQAS(SEQ ID NO:355), MGWFRRAPEKERELVAS(SEQ ID NO:356) MGWFRRAPGKDRELVAS(SEQ ID NO:357) MSWVRQAPGKGRELVAS(SEQ ID NO:358), MGWFRRAPGKERELIAS(SEQ ID NO:359), LAWHRQIPGKEREWGUG(SEQ ID NO:360), MAWHRQAPGKERLWVAG(SEQ ID NO:361), VGWYRQAPGEQRVLVAH(SEQ ID NO:362), MGWFRQAPGKEREFVAA(SEQ ID NO:363), MGWYRQAPGKQRELVAV(SEQ ID NO:364), MGWFRQTPGKEREFVAA(SEQ ID NO:365), MRWYRQAPGKQREQVAG(SEQ ID NO:458), MRWYRQAPGKQREFVAG(SEQ ID NO:459), MRWYRQAPGKQRHWVAG (SEQ ID NO: 460), MIWYRQAPGKQREWVAG (SEQ ID NO: 461), MEWYRQAPGKQREWVAG (SEQ ID NO: 462), MRWYRQAPGKQREWVAA (SEQ ID NO: 463), MRWYRQAPGKQREWVAK (SEQ ID NO: 464), IGWFRQAPGKEREKVSC (SEQ ID NO: 621), MHWFRQAPGKEREFVGA (SEQ ID NO: 622), IRWYSQAPGKQREFVAG (SEQ ID NO: 716), MRWYRQAPGKQREWVSG (SEQ ID NO: 720), MRWYRQAPGKQLEWVAG (SEQ ID NO: 787), MRWYRQAPGKGREWVAG (SEQ ID NO: 788), MRWYRQAPGKGLEWVSG (SEQ ID NO: 789), or MRWYRQAPGKGREWVSG (SEQ ID NO: 790).

[0105] In one particular embodiment, the VHH molecule of the present invention comprises an FR3 domain comprising, or consists of, the sequence shown below (SEQ ID NO: 45) or a variant thereof, wherein the variant has 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%, preferably at least 70% amino acid identity with the sequence: YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC (SEQ ID NO: 45). More preferably, the bolded amino acid residues are present, and the variation occurs only at other positions.

[0106] In one specific implementation, Y in position 1 can be replaced by N.

[0107] In one specific implementation, Y in position 2 can be replaced by A.

[0108] In one specific implementation, A in position 3 can be replaced by P or I.

[0109] In one specific implementation, D in position 4 can be replaced by S or N.

[0110] In one specific implementation, A in position 17 can be replaced by S.

[0111] In one specific implementation, K in position 29 can be replaced by R.

[0112] In one specific implementation, P in position 30 can be replaced with A.

[0113] More preferably, referring to the sequence, the FR3 contains up to 7 amino acid modifications in the non-bold amino acid residues, even more preferably up to 6, up to 3, or even more preferably up to 2 amino acid modifications.

[0114] In another specific embodiment, the FR3 has an amino acid sequence selected from any of the amino acid sequences listed below: NYADSMKGRFTISRDNTKNAVYLQIDSLKPEDTAVYYC (SEQ ID NO: 46); NYPDSAKGRFTISRDNAKNTVYLQIDSLKPEDTAVYYC (SEQ ID NO: 47); YAISSVKGRFTISRDNAENTVFLQMNSLKPDDTAVYYC (SEQ ID NO: 48); or NYPDSMKGRFTISRDNAKNTVYLQINSLKSEDTAVYYC (SEQ ID NO: 49); or a variant thereof.

[0115] Other specific examples of FR3 of the VHH molecule according to the present invention are provided below (see also Table 3): NYADSMKGRFTISRDNTKNALYLQIDSLRPEDTAVYYC (SEQ ID NO: 366), NYADSVKGRFTISRDNTKNTLYLQIDSLRPEDTAVYYC (SEQ ID NO: 367), NYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYC(SEQ ID NO:368), NYADSVKGRFTISRDNTKNTLYLQINSLRPEDTAVYYC(SEQ ID NO:369), NYADSMKGRFTISRDNTKNTLYLQMNSLRPEDTAVYYC(SEQ ID NO:370), NYADSVKGRFTISRDNAKNTLYLQIDSLRPEDTAVYYC(SEQ ID NO:371), NYADSVKGRFTISRDNTKNTLYLQMNSLRPEDTAVYYC(SEQ ID NO:372), NYADSVKGRFTISRDNTKNALYLQMNSLRPEDTAVYYC(SEQ ID NO:373), NYADSVKGRFTISRDNTKNTLYLQMDSLRPEDTAVYYC(SEQ ID NO:374), NYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC(SEQ ID NO:375), NYADSVKGRFTISRDNAKNAVYLQMNSLRPEDTAVYYC(SEQ ID NO:376), NYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYC(SEQ ID NO:377), NYADSMKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYC(SEQ ID NO:378), NYPDSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYC(SEQ ID NO:379), YYADGMRGRFTISRDNSENTVSLQMNNLKPEDTAVYYC(SEQ ID NO:380), YYANSMKERFTISRDNAQNTVSLQISSLKPEDTAVYYC(SEQ ID NO:381), YYADGMKGRFTISRDNAENTVSLQINSLKPEDTAIYYC(SEQ ID NO:382), YYADSSVKGRFTISRDNAENTVSLQMNSLKPEDTAVYYC(SEQ ID NO:383), SYRDSVKGRFTISRDNAKNTVFLQMNSLEPEDTGVYYC(SEQ ID NO:384), SYADSVKGRFTISRDDAKNTVYLQMDNLTPEDTAVYFC(SEQ ID NO:385), EYKDSVKGRFTISRDNARNTIYLEMKNLKPEDTAIYYC(SEQ ID NO:386), DYADGVMGRFTISRNSALNTVYLQMDSLKSTDTGVYVC(SEQ ID NO:387), KYGDSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYC(SEQ ID NO:388), TYADSVKGRFTISRDNAKNTVYLQMNSLEPTDTAVYYC(SEQ ID NO:389), YYADSVKGRFTISRDTVKDMVYLQMNSLKPEDTAVYYC(SEQ ID NO:623), EYADSVKGRFTISRDNAKSTVYLQMNNLKPEDTAVYYC(SEQ ID NO:624), VYPDSAKGRFTISRDNAKNTVYLQIDSLKPEDTAVYYC(SEQ ID NO:625), FYPDSAKGRFTISRDNAKNTVYLQIDSLKPEDTAVYYC(SEQ ID NO:626), NYADSMKGRLTISRDNTKNAVYLQIDSLKPEDTAVYYC(SEQ ID NO:717), NYPDIAKGRFTISRDNAKNTVYLQIDSLKPEDTAVYYC(SEQ ID NO:718), NYPDSAKGRFTISEDNAKNTVYLQIDSLKPEDTAVYYC(SEQ ID NO:719), NYPDSVKGRFTISRDNAKNTAYLQMNSLRAEDTAVYYC (SEQ ID NO: 791), NYPDSAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYC (SEQ ID NO: 792), NYPDSAKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYC (SEQ ID NO: 793), NYPDSAKGRFTISRDNSKNTVYLQMDSLRPEDTAVYYC (SEQ ID NO: 794), or NYPDSAKGRFTISRDNAKNTVYLQMDSLRPEDTAVYYC (SEQ ID NO: 795).

[0116] In one particular embodiment, the VHH molecule of the present invention comprises an FR4 domain comprising, or consists of, the sequence shown below (SEQ ID NO: 50) or a variant thereof, said sequence or variant 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%, preferably at least 90% amino acid identity with said sequence: WGQGTQVTVSS (SEQ ID NO: 50). More preferably, said bold amino acid residues are present, and the variation occurs only at other positions.

[0117] More preferably, referring to this sequence, the FR4 contains at most four amino acid modifications in the non-bold amino acid residues, even more preferably at most three, and even more preferably at most two amino acid modifications. A specific illustrative example of the FR4 sequence is SEQ ID NO: 50.

[0118] Other specific examples of FR4 according to the present invention are as follows: WGQGTLVTVSS (SEQ ID NO: 390), WGKGTQVTVSS (SEQ ID NO: 391), or WGRGTQVTVSS (SEQ ID NO: 670).

[0119] Specific examples of the TfR-binding VHH molecules of the present invention are molecules comprising or composed of any of the amino acid sequences selected from SEQ ID NO: 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 SEQ ID NO: 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 sequence (when x is 0, as detailed in Table 1 below).

[0120] Other examples of TfR-binding VHH molecules of the present invention are those comprising the following SEQ ID NO: 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, 19 7, 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, amino acid sequences or molecules composed thereof (as listed in Table 1 below; in these examples, when x equals 1, each VHH molecule contains the specific tag sequence of SEQ ID NO: 51 below: AAAEQKLISEEDLNGAAHHHHHHGS).

[0121] In one particular embodiment, the VHH of the present invention is humanized. For humanization, one or more FR and / or CDR domains may be modified by replacing one or more amino acids.

[0122] In this regard, in one particular embodiment, the VHH is humanized by selected modifications (e.g., amino acid substitutions) to 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., by reference to any of SEQ ID NOs: 35-39, 331, 332, 400, or any variants thereof as defined herein). Therefore, specific examples of such humanized FR1 include SEQ ID NOs: 37, 331, 400, where K19 and / or V23 are modified to 19R and 23A, respectively.

[0123] Another humanized position in FR1 is 11L, so a specific instance of such humanized FR1 includes SEQ ID NO: 332, in which V11 is modified to 11L, and in which K19 and V23 are modified to 19R and 23A, respectively.

[0124] In another particular embodiment, the VHH is humanized by selected modifications of the FR2 domain. Typical humanization sites in FR2 are selected from 1M, 2S or 2H, 4V, 11G, 12L, 14W or combinations thereof (by reference, for example, any of SEQ ID NO: 40-44, 342-348 or any variant thereof as defined herein).

[0125] Therefore, a specific example of such humanized FR2 includes SEQ ID NO: 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.

[0126] Another specific example of the humanized FR2 includes SEQ ID NO: 342, in which Y4, Q11, R12 and F14 are modified to 4V, 11G, 12L and 14W, respectively.

[0127] Another specific example of the humanized FR2 includes SEQ ID NO: 343, in which Q11 and R12 are modified to 11G and 12L, respectively.

[0128] Another specific example of the humanized FR2 includes SEQ ID NO: 344, in which Y4, Q11 and R12 are modified to 4V, 11G and 12L, respectively.

[0129] Another specific instance of the humanized FR2 includes SEQ ID NO: 345, where Q11 is modified to 11G.

[0130] Another specific instance of the humanized FR2 includes SEQ ID NO: 346, where Y4 is modified to 4V.

[0131] Another specific example of the humanized FR2 includes SEQ ID NO: 347, in which Q11, R12 and F14 are modified to 11G, 12L and 14W, respectively.

[0132] Another specific example of the humanized FR2 includes SEQ ID NO: 348, in which Q11 and R12 are modified to 11G and 12L, respectively.

[0133] Another specific example of the humanized FR2 includes SEQ ID NO: 787, in which R12, L14 and T17 are modified to 12L, 14W and 17G, respectively.

[0134] Another specific example of the humanized FR2 includes SEQ ID NO: 788, in which Q11, L14 and T17 are modified to 11G, 14W and 17G, respectively.

[0135] Another specific example of the humanized FR2 includes SEQ ID NO: 789, in which Q11, R12, A16 and T17 are modified to 11G, 12L, 16S and 17G, respectively.

[0136] Another specific example of the humanized FR2 includes SEQ ID NO: 790, in which Q11, A16 and T17 are modified to 11G, 16S and 17G, respectively.

[0137] In another specific embodiment, the VHH is humanized by selected modifications of the FR3 domain. Typical humanization positions in the FR3 are selected from 6V, 17A or S, 20T, 21L, 25M, 26N, 29R, 30A, and any combination thereof (by reference, for example, any of SEQ ID NO: 45-49, 366-379, or any variant thereof as defined herein). Thus, a specific example of such humanized FR3 includes SEQ ID NO: 46, wherein 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.

[0138] Another specific example of the humanized FR3 includes SEQ ID NO: 366, in which V21 and K29 ​​are modified to 21L and 29R, respectively.

[0139] Another specific example of such humanized FR3 includes SEQ ID NO: 367, in which M6, A20, V21 and K29 ​​are modified to 6V, 20T, 21L and 29R, respectively.

[0140] Another specific example of the humanized FR3 includes SEQ ID NO: 368, in which M6, T17, A20, V21, I25, D26 and K29 ​​are modified to 6V, 17A, 20T, 21L, 25M, 26N and 29R, respectively.

[0141] Another specific example of the humanized FR3 includes SEQ ID NO: 369, in which M6, A20, V21, D26 and K29 ​​are modified to 6V, 20T, 21L, 26N and 29R, respectively.

[0142] Another specific example of the humanized FR3 includes SEQ ID NO: 370, in which A20, V21, I25, D26 and K29 ​​are modified to 20T, 21L, 25M, 26N and 29R, respectively.

[0143] Another specific example of the humanized FR3 includes SEQ ID NO: 371, in which M6, T17, A20, V21 and K29 ​​are modified to 6V, 17A, 20T, 21L and 29R, respectively.

[0144] Another specific example of the humanized FR3 includes SEQ ID NO: 372, in which M6, A20, V21, I25, D26 and K29 ​​are modified to 6V, 20T, 21L, 25M, 26N and 29R, respectively.

[0145] Another specific example of the humanized FR3 includes SEQ ID NO: 373, in which M6, V21, I25, D26 and K29 ​​are modified to 6V, 21L, 25M, 26N and 29R, respectively.

[0146] Another specific example of the humanized FR3 includes SEQ ID NO: 374, in which M6, A20, V21, I25 and K29 ​​are modified to 6V, 20T, 21L, 25M and 29R, respectively.

[0147] Another specific example of the humanized FR3 includes SEQ 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.

[0148] Another specific example of the humanized FR3 includes SEQ ID NO: 376, in which M6, T17, I25, D26 and K29 ​​are modified to 6V, 17A, 25M, 26N and 29R, respectively.

[0149] Another specific example of the humanized FR3 includes SEQ ID NO: 377, in which M6, T17, A20, V21, I25 and D26 are modified to 6V, 17A, 20T, 21L, 25M and 26N, respectively.

[0150] Another specific example of the humanized FR3 includes SEQ ID NO: 378, in which T17, A20, V21, I25, D26 and K29 ​​are modified to 17A, 20T, 21L, 25M, 26N and 29R, respectively.

[0151] Another specific example of the humanized FR3 includes SEQ ID NO: 379, in which M6, I25, D26 and K29 ​​are modified to 6V, 25M, 26N and 29R, respectively.

[0152] Another specific example of the humanized FR3 includes SEQ ID NO: 791, in which Y1, A3 and K29 ​​are modified to 1N, 3P and 29R, respectively.

[0153] Another specific example of the humanized FR3 includes SEQ ID NO: 792, in which Y1, A3, V6, A17, K29 and P30 are modified to 1N, 3P, 6A, 17S, 29R and 30A, respectively.

[0154] Another specific example of the humanized FR3 includes SEQ ID NO: 793, in which Y1, A3, V6, K29 and P30 are modified to 1N, 3P, 6A, 29R and 30A, respectively.

[0155] Another specific example of the humanized FR3 includes SEQ ID NO: 794, in which Y1, A3, A17, N26, K29, N and P30 are modified to 1N, 3P, 17S, 26D, 29R and 30A, respectively.

[0156] Another specific example of the humanized FR3 includes SEQ ID NO: 795, in which Y1, A3, N26, K29, N and P30 are modified to 1N, 3P, 26D, 29R and 30A, respectively.

[0157] Specific examples of the humanized TfR-binding VHH molecules of the present invention are molecules comprising an amino acid sequence selected from any one of SEQ ID NO: 236-241, 252-271, 273-275, 752-765 or 773-786 (see Table 1).

[0158] In another specific embodiment, the VHH molecule may also include one or more tags suitable for, for example, purification, conjugation, detection, etc. In the context of this invention, the term "tag" includes any peptide sequence attached to the polypeptide VHH molecule of this invention to facilitate easy detection or purification of the expressed protein, or identification of its binding to TfR, or for site-directed enzymatic chemistry / enzymatic conjugation purposes. The tag may be an affinity tag, an epitope tag, a site-specific conjugation tag, or a fluorescent tag.

[0159] Examples of such tags include Q-tags (which are tags containing glutamine residues inserted into the tag sequence, specifically recognized by TGase, and preferably containing or composed of the sequence LQR), myc tags (EQKLISEEDL, SEQ ID NO: 394), polyHis tags (containing 2 to 8 histidine residues, preferably 6 to 8 His residues, such as His6 (SEQ ID NO: 395) or His8 (SEQ ID NO: 396)), polyArg tags (containing 2 to 8 arginine residues), polyLys tags (containing 2 to 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, sorting enzyme tags, SNAP tags, or combinations thereof (as shown in Table 4 below).

[0160] Typically, the one or more tags are located at the C-end of the VHH.

[0161] In another specific embodiment, the VHH molecule may further include one or more connectors.

[0162] In the context of this invention, the terms "connector" and "spacer" are used interchangeably. The connector may be a peptide connector or a coupling connector. The peptide connector comprises one or more amino acid residues, typically 1 to 10 amino acid residues, for linking the VHH molecule of this invention to a tag, or between the various tags described herein, provided that the connector does not specifically bind to the target protein (i.e., TfR). The connector 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), etc., or combinations thereof. The peptide connector may be flexible (e.g., any flexible hydrophilic connector) or rigid (e.g., any α-helical rigid connector).

[0163] This peptide linker differs from coupling linkers that can be introduced between the VHH and the compound of interest, such as bifunctional or polyfunctional reagents containing alkyl, aryl, thiol, azide, alkyne, nucleotide, or peptide groups, which are coupled via esters, aldehydes or alkyl or aryl acids, acid anhydrides, thiol or carboxyl groups, groups derived from cyanogen bromide or cyanogen chloride, carbonyl diimidazoles, succinimide esters, or sulfonyl halides (as described in the “Couplings” section of this document).

[0164] As a further explanation, the VHH of the present invention may include a linker, which is preferably located at the C-terminus of the VHH. The linker may contain Gly residues or Gly repeat sequences, such as Gly repeat sequences of 2-7 Gly residues (i.e., Gly2, Gly3, Gly4, Gly5, Gly6, or Gly7, respectively). The VHH of the present invention may also contain a combination of Gly and Ser residues. Specific examples of such Gly and Ser combination connectors include: GlySerGlySer (GSGS; SEQ ID NO: 627); SerGlySerGly5 (SGSGGGGG; SEQ ID NO: 628); (Gly4Ser)n, where n is 1 to 6, such as any of SEQ ID NO: 629 to 634 (as shown in Table 4 below), preferably SEQ ID NO: 629 to 631; or any connector containing such (Gly4Ser)n sequence, such as GlyGly(Gly4Ser)3 (GGGGGGSGGGGSGGGGS; SEQ ID NO: 635).

[0165] As shown in Table 4, other specific examples of peptide linkers according to the invention include or consist of EAAAK (SEQ ID NO: 636), or include a combination of EAAAK repeat sequences with other amino acid residues, such as any one of SEQ ID NO: 637 to 641.

[0166] As shown in Table 4, other specific examples of peptide linkers according to the present invention are as follows: GG(AP)17 (SEQ ID NO: 642), ASTKGPSVFPLAP (SEQ ID NO: 643), GSAGSAAGSGEF (SEQ ID NO: 644) or KESGSVSSEQLAQFRSLD (SEQ ID NO: 645).

[0167] In one particular embodiment, the VHH of the present invention may comprise a Gly linker and a Q-tag, preferably located at the C-terminus. More specific examples of such a VHH comprise the following structure: VHH–Gly linker–Q-tag, wherein the Gly linker comprises or consists of 2-6 Gly residues; and the Q-tag comprises or consists of LQRs. For illustration, the VHH may include the following tag sequence at the C-terminus: (SEQ ID NO: 51), where a single underscore is a myc tag, a double underscore is a His6 tag (the remaining residues are linkers such as the Ala linker AAA, or residues generated by cloning).

[0168] Specific examples of such tagged TfR-binding VHH molecules of the present invention (which contain the tag sequence of SEQ ID NO: 51 at the C-terminus) are those containing the tag sequence selected from SEQ ID NO: 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, 18 3. An amino acid sequence of any one of the following: 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, or a molecule composed of said amino acid sequence.

[0169] Specific examples of the humanized TfR-binding VHH molecules of the present invention with a tag sequence are molecules containing an amino acid sequence selected from any one of SEQ ID NO: 87-92 or 130-149, 152-154 or 752-765 or composed of said amino acid sequences (see Table 1).

[0170] As another illustration, the VHH may contain the following tag sequence at the C-terminus: (SEQ ID NO: 399), where a single underscore is a spacer, bold C is a free cysteine ​​that can be used for site-directed chemical coupling, and double underscores are His tags (the remaining residues are linkers or generated by cloning).

[0171] As another illustration, the VHH of the present invention may contain a Q-tag, which preferably contains or consists of the sequence LQR, and is preferably located at the C-terminus of the VHH.

[0172] As another explanation, the VHH may contain the following tag sequence at the C-terminus (“C-ter” or “C-terminus”): GGG LQR (SEQ ID NO: 111), where the underscore is a Q-tag and the bold text is a Gly connector. Other examples are GGGGLQR (SEQ ID NO: 401), GGGGGLQR (SEQ ID NO: 402), GGGGGGLQR (SEQ ID NO: 403), and GGGGGGGLQR (SEQ ID NO: 404). In a preferred aspect of the invention, the VHH comprises the tag sequence of SEQ ID NO: 111.

[0173] In another specific embodiment, the VHH of the present invention may comprise an Ala connector, a His tag, a Gly connector, and a Q-tag. Preferably, the connector 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. More specific examples of such VHHs include the structure: VHH–Ala connector–His tag–Gly connector–Q-tag, wherein the Ala connector comprises 3 residues; the His tag comprises 2-7 His residues, preferably 6 His residues; the Gly connector comprises 2-6 Gly residues, preferably 3 residues; and the Q-tag preferably contains or is composed of LQRs.

[0174] As an illustration, the VHH may include the following tag sequence at the C-terminus: (SEQ ID NO: 112), where the underscore is the Q-tag, the bold text represents the Ala and Gly connectors, and the double underscore is the His tag. Other examples are AAAHHHHHHGGGGLQR (SEQ ID NO: 406), AAAHHHHHHGGGGGLQR (SEQ ID NO: 407), AAAHHHHHHGGGGGGLQR (SEQ ID NO: 408), and AAAHHHHHHGGGGGGGLQR (SEQ ID NO: 409). In a preferred aspect of the invention, the VHH includes the additional sequence of SEQ ID NO: 112.

[0175] As described above, specific examples of such tagged TfR-binding VHH molecules of the present invention are those comprising the following SEQ ID NO: 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, 18 3. An amino acid sequence of any one of the following: 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, or a molecule composed of said amino acid sequence.

[0176] Other specific examples of TfR-binding VHH molecules of the present invention are VHH molecules that competitively inhibit the binding of VHH as defined above to human and non-human TfR. The term "competitive inhibition" means that the VHH can reduce, inhibit, or displace the binding of the reference VHH to TfR in vitro or in vivo. Competitive assays can be performed using standard techniques such as competitive ELISA or other binding assays. Typically, a competitive binding assay involves recombinant muscle cells or membrane preparations expressing TfR (optionally bound to a solid substrate), an unlabeled test VHH (or a phage expressing it), and a labeled reference VHH (or a phage expressing it). Competitive inhibition is measured by determining the amount of labeled VHH bound in the presence of the test VHH. Typically, the test VHH is present in excess, for example, about 5 to 500 times the amount of the reference VHH. Typically, for ELISA, the test VHH is in excess by 100 times. It is considered to competitively inhibit the reference VHH when an excess of the test VHH inhibits or replaces at least 70% of the binding of the reference VHH to TfR. Preferred competitive VHH bindings share epitopes of common amino acid residues.

[0177] As shown in the experimental section, VHH molecules bind to TfR in vitro and in vivo. They exhibit sufficient affinity, with Kd values ​​of about 0.01 nM to about 4 µM, about 0.01 nM to about 2500 nM, about 0.01 nM to about 1000 nM, about 0.01 nM to about 500 nM, about 0.01 nM to about 100 nM, about 0.1 nM to about 4 µM, about 0.1 nM to about 2500 nM, about 0.1 nM to about 1000 nM, about 0.1 nM to about 500 nM, or about 0.1 nM to about 100 nM.

[0178] In the context of this invention, the term K d It refers to the following name K d app K D and / or K Deq Any of the affinity constants. More specifically, the term K d app This refers to the apparent binding affinity constant measured in cell-based assays (where indirect ligand detection is performed by flow cytometry) or enzyme-linked immunosorbent assays (ELISA) at 4°C, and corresponds to the ligand concentration at which TfR is allowed to bind at half its maximum capacity in the system. Term K D This refers to the equilibrium dissociation constant calculated from kinetic parameters, i.e., the association and dissociation rates (ki and ki, respectively) measured from the concentrations of several ligands via surface plasmon resonance (SPR) or biolayer interferometry (BLI). on and k off ), and calculate it as k off With kon The ratio. Term K Deq It refers to the equilibrium dissociation constant estimated by SPR, which corresponds to half of the ligand concentration required to induce the maximum response in an SPR or BLI system.

[0179] In one particular embodiment, the VHH molecule of the present invention binds to human and non-human primate TfRs with a Kd of about 0.01 nM to about 550 nM. Preferably, the VHH molecule of the present invention binds to human and non-human primate TfRs with a Kd of less than 500 nM, for example, about 0.01 nM to about 500 nM or about 0.1 nM to about 500 nM.

[0180] In one particular embodiment, the VHH molecule of the present invention binds to human TfR with a Kd of about 0.01 nM to about 100 nM.

[0181] In another specific embodiment, the VHH molecule of the present invention binds to rhesus monkey TfR with Kd at a concentration of about 0.01 nM to about 550 nM, preferably 0.1 nM to 500 nM.

[0182] In another specific embodiment, the VHH molecule of the present invention binds to mouse TfR at a Kd concentration of about 0.1 nM to about 4000 nM, preferably about 0.1 nM to about 2500 nM. In another specific embodiment, the conjugate according to the present invention, for example, a conjugate comprising a VHH-hFc-siRNA variant, binds to human and non-human primate TfR at a Kd concentration 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.

[0183] In yet another specific embodiment, the conjugate according to the invention, for example, comprises a conjugate containing a VHH-hFc-siRNA variant, to bind human TfR with Kd in the range of about 1 nM to about 500 nM, preferably about 150 nM to about 400 nM.

[0184] In one particular embodiment of the invention, the VHH molecule is a humanized or non-humanized C5 or a variant thereof, comprising or consisting of any of SEQ ID NO: 213, 216-271, 274, 275, 675, 676 and 701 (as listed in Table 1).

[0185] In another specific embodiment, the VHH molecule is C5 or a variant thereof, which is a cross-species VHH molecule that binds to human, non-human primate (NHP), and rodent TfR. In another specific embodiment, C5 or a variant thereof includes a tag sequence at the N- and / or C-terminus. Specific examples of such variants include or constitute any one of SEQ ID NO: 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.

[0186] In another specific embodiment, the VHH molecule is a humanized variant of C5 comprising or consisting of any one of SEQ ID NO: 236-241, 252-271, 274-275. In one specific embodiment, the humanized variant of C5 includes a tag sequence at the N- and / or C-terminus. Specific examples of such variants comprise or consist of any one of SEQ ID NO: 87-92 or 130-149, 153-154. Examples of humanized variants of C5 are the C5h20 variant comprising or consisting of SEQ ID NO: 265 or 143, or the C5V13h20 variant comprising or consisting of SEQ ID NO: 274 or 153.

[0187] In another specific embodiment, the VHH molecule of the present invention is B8 or a variant thereof (as listed in Table 1), comprising or consisting of any one of SEQ ID NO: 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. In one specific embodiment, B8 or a variant thereof includes a tag sequence at the N- and / or C-terminal ends. Specific instances of such variants include or consist of any one of SEQ ID NO: 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.

[0188] In another specific embodiment, the VHH molecule of the present invention is B8 or a variant thereof, which cross-reacts with human and primate species.

[0189] In another specific embodiment, the VHH molecule is a humanized or non-humanized B8 or a variant thereof, comprising an amino acid sequence selected from any of SEQ ID NO: 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.

[0190] In another specific embodiment, the VHH molecule of the present invention is B8 or a variant thereof, comprising or consisting of any one of SEQ ID NO: 677, 678, 681, 682, 702-709 or 693-700, 745-786, preferably any one of SEQ ID NO: 677, 678, 681, 682, 752-765 or 773-786.

[0191] In another specific embodiment, the VHH molecule is a humanized variant of B8 that comprises or consists of SEQ ID NO: 273, 152 (e.g., the B8h1 variant), 752-765, or 773-786 (e.g., the B8V31h1-5, h9, or the B8V32h1, h6, h9-14 variants). In one specific embodiment, preferred B8 variants of the invention are B8h1, B8V32, B8V31, B8V40, B8V35, B8V32h14, and B8V32h6.

[0192] In another specific embodiment, the VHH molecule of the present invention is B6 or a variant thereof (as listed in Table 1), comprising or consisting of any one of SEQ ID NO: 12, 168, 170, 172, 173, 174, 178, 181, 183, 185, 215, 285-293, and binding to TfR. B6 and its variants according to the present invention target the apical domain of TfR.

[0193] In another specific embodiment, the VHH molecule of the present invention is a VHH molecule that binds to the top domain of TfR, preferably the top domain of TfR1, and comprises or consists 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 one specific embodiment, such VHH molecules contain a tag sequence at the N- and / or C-terminal ends. Specific examples of such variants include 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.

[0194] In another specific embodiment, the VHH molecule is B6 or a variant thereof, which contains an amino acid sequence selected from any of SEQ ID NO: 215 and 285-293.

[0195] In another specific embodiment, the VHH molecule comprises or is composed of an amino acid sequence selected from any of SEQ ID NO: 613-618. Interestingly, such VHH molecules comprising or composed of SEQ ID NO: 613-618 bind to human and non-human primate TfR, but they do not bind to mouse TfR.

[0196] Furthermore, the binding of the VHH of the present invention to the human TfR receptor does not compete with the binding of the endogenous TfR ligand transferrin, and therefore does not affect the normal function of the ligand. Conjugates generated from such VHH molecules have been further shown to bind TfR in vitro and accumulate in muscle and / or muscle cells in vivo, exhibiting endocytosis. Therefore, such VHHs represent potent agents for drug delivery or targeting of muscle.

[0197] 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.). They can be preserved as is or formulated in the presence of the substance of interest or any acceptable excipient. For chemical synthesis, commercial devices are used that can incorporate natural and non-natural amino acids (e.g., D enantiomers and residues with hydrophobicity and steric hindrance in the side chains that differ from their natural homologues (so-called foreign amino acids, i.e., non-coding amino acids)) or contain one or more peptide-like bonds (which may in particular include the embedding of methylene (-CH2-) or phosphate (-PO2-) groups, secondary amine (-NH-) or oxygen (-O-) or N-alkyl peptides). During synthesis, various chemical modifications can be introduced, such as inserting, linking, or coupling components of lipid (or phospholipid) derivatives or liposomes or nanoparticles at the N-terminal and / or C-terminal positions or on the side chains, in order to incorporate the VHHs of the present invention into lipid membranes, such as membranes of liposomes or nanoparticles consisting of one or more lipid layers or bilayers. Liposomes and nanoparticles are examples of “carriers” that can be conjugated to one or more VHH molecules of the present invention. The VHHs of the present invention can also be obtained from nucleic acid sequences encoding them, as further described below (see Table 2 and the sequence listing).

[0198] Coupled

[0199] Another object of the present invention relates to a conjugate (also referred to herein as a “chimeric agent”) comprising one or more VHH molecules as defined above, which are conjugated to at least one additional compound, particularly to at least one additional molecule, agent or compound of interest, such as to at least one oligonucleotide or scaffold of interest.

[0200] In one embodiment, the present invention relates to a coupling compound comprising: (i) One or more VHH molecules of the formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and (ii) One or more oligonucleotides, The VHH molecule binds to TfR on the surface of muscle cells, and the VHH molecule comprises: SEQ ID NO: 1, 2 and 3; or SEQ ID NO: 5, 6 and 7; or SEQ ID NO: 9, 10 and 11; or SEQ ID NO: 13, 14 and 15; SEQ ID NO: 17, 2 and 3; or SEQ ID NO: 19, 2 and 3; or SEQ ID NO: 1, 21 and 3; or SEQ ID NO: 1, 23 and 3; or SEQ ID NO: 1, 2 and 25; or SEQ ID NO: 1, 2 and 27; or SEQ ID NO: 1, 2 and 29; or SEQ ID NO: 1, 2 and 31; or SEQ ID NO: 1, 2 and 33; or SEQ ID NO: 67, 2 and 3; or SEQ ID NO: 69, 2 and 3; or SEQ ID NO: 1, 71 and 3; or SEQ ID NO: 1, 73 and 3; or SEQ ID NO: 1, 75 and 3; or SEQ ID NO: 1, 71 and 3; or SEQ ID NO: 1, 73 and 3; or SEQ ID NO: 1, 75 and 3; or SEQ ID NO: 1, 2, and 3. SEQ ID NO: 1, 2 and 77; or SEQ ID NO: 1, 2 and 79; or SEQ ID NO: 1, 2 and 81; or SEQ ID NO: 1, 2 and 83; or SEQ ID NO: 1, 2 and 85; or SEQ ID NO: 392, 2 and 3; or SEQ ID NO: 1, 113 and 3; or SEQ ID NO: 1, 115 and 3; or SEQ ID NO: 1, 2 and 117; or SEQ ID NO: 1, 2 and 119; or SEQ ID NO: 1, 2 and 121; or SEQ ID NO: 1, 2 and 123; or SEQ ID NO: 125, 2 and 3; or SEQ ID NO: 17, 73 and 3; or SEQ ID NO: 17, 128 and 3; or SEQ ID NO: 5, 160 and 7; or SEQ ID NO: 5, 162 and 7; or SEQ ID NO: 5, 164 and 7; or SEQ ID NO: 5, 166 and 7; or SEQ ID SEQ ID NO: 9, 169 and 11; or SEQ ID NO: 9, 171 and 11; or SEQ ID NO: 175, 176 and 177; or SEQ ID NO: 179, 176 and 180; or SEQ ID NO: 182, 176 and 177; or SEQ ID NO: 184, 176 and 177; or SEQ ID NO: 186, 187 and 188; or SEQ ID NO: 190, 191 and 192; or SEQ ID NO: 194, 195 and 196; or SEQ ID NO: 198, 199 and 200; or SEQ ID NO: 201, 202 and 203; or SEQ ID NO: 205, 206 and 207; or SEQ ID NO: 410, 6 and 7;Or SEQ ID NO: 413, 6 and 7; or SEQ ID NO: 5, 416 and 7; or SEQ ID NO: 5, 419 and 7; or SEQ ID NO: 426, 6 and 7; or SEQ ID NO: 5, 431 and 7; or SEQ ID NO: 434, 6 and 7; or SEQ ID NO: 437, 6 and 7; or SEQ ID NO: 5, 6 and 452; or SEQ ID NO: 5, 6 and 455; or SEQ ID NO: 607, 608 and 609; or SEQ ID NO: 610, 611 and 612; or SEQ ID NO: 671, 2 and 3; or SEQ ID NO: 672, 2 and 3; or SEQ ID NO: 673, 6 and 7; or SEQ ID NO: 674, 6 and 7; or SEQ ID NO: 1, 2 and 713; or SEQ ID NO: 5, 6 and 714; or SEQ ID SEQ ID NO: 674, 164 and 7; or SEQ ID NO: 710, 6 and 7; or SEQ ID NO: 5, 6 and 715; or SEQ ID NO: 674, 712 and 7; or SEQ ID NO: 711, 6 and 7; or SEQ ID NO: 673, 6 and 741; or SEQ ID NO: 673, 6 and 742; or SEQ ID NO: 673, 6 and 743; or SEQ ID NO: 673, 6 and 744; or SEQ ID NO: 673, 431 and 741; or SEQ ID NO: 673, 431 and 742; or SEQ ID NO: 673, 431 and 743; or SEQ ID NO: 673, 6 and 7; or SEQ ID NO: 674, 6 and 7.

[0201] In one particular embodiment, the present invention relates to a coupling compound comprising: (i) One or more VHH molecules of the formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and (ii) One or more oligonucleotides, The VHH molecule binds to TfR on the surface of muscle cells and nerve cells, and the VHH molecule comprises: SEQ ID NO: 1, 2 and 3; or SEQ ID NO: 5, 6 and 7; or SEQ ID NO: 9, 10 and 11; or SEQ ID NO: 13, 14 and 15; SEQ ID NO: 17, 2 and 3; or SEQ ID NO: 19, 2 and 3; or SEQ ID NO: 1, 21 and 3; or SEQ ID NO: 1, 23 and 3; or SEQ ID NO: 1, 2 and 25; or SEQ ID NO: 1, 2 and 27; or SEQ ID NO: 1, 2 and 29; or SEQ ID NO: 1, 2 and 31; or SEQ ID NO: 1, 2 and 33; or SEQ ID NO: 67, 2 and 3; or SEQ ID NO: 69, 2 and 3; or SEQ ID NO: 1, 71 and 3; or SEQ ID NO: 1, 73 and 3; or SEQ ID NO: 1, 75 and 3; or SEQ ID NO: 1, 71 and 3; or SEQ ID NO: 1, 73 and 3; or SEQ ID NO: 1, 75 and 3; or SEQ ID NO: 1, 21 ...1 and 3; or SEQ ID NO: 1, 71 and 3; or SEQ ID NO: 1, 71 and 3; or SEQ ID NO: 1, 2 and 77; or SEQ ID NO: 1, 2 and 79; or SEQ ID NO: 1, 2 and 81; or SEQ ID NO: 1, 2 and 83; or SEQ ID NO: 1, 2 and 85; or SEQ ID NO: 392, 2 and 3; or SEQ ID NO: 1, 113 and 3; or SEQ ID NO: 1, 115 and 3; or SEQ ID NO: 1, 2 and 117; or SEQ ID NO: 1, 2 and 119; or SEQ ID NO: 1, 2 and 121; or SEQ ID NO: 1, 2 and 123; or SEQ ID NO: 125, 2 and 3; or SEQ ID NO: 17, 73 and 3; or SEQ ID NO: 17, 128 and 3; or SEQ ID NO: 5, 160 and 7; or SEQ ID NO: 5, 162 and 7; or SEQ ID NO: 5, 164 and 7; or SEQ ID NO: 5, 166 and 7; or SEQ ID SEQ ID NO: 9, 169 and 11; or SEQ ID NO: 9, 171 and 11; or SEQ ID NO: 175, 176 and 177; or SEQ ID NO: 179, 176 and 180; or SEQ ID NO: 182, 176 and 177; or SEQ ID NO: 184, 176 and 177; or SEQ ID NO: 186, 187 and 188; or SEQ ID NO: 190, 191 and 192; or SEQ ID NO: 194, 195 and 196; or SEQ ID NO: 198, 199 and 200; or SEQ ID NO: 201, 202 and 203; or SEQ ID NO: 205, 206 and 207;Or SEQ ID NO: 410, 6 and 7; or SEQ ID NO: 413, 6 and 7; or SEQ ID NO: 5, 416 and 7; or SEQ ID NO: 5, 419 and 7; or SEQ ID NO: 426, 6 and 7; or SEQ ID NO: 5, 431 and 7; or SEQ ID NO: 434, 6 and 7; or SEQ ID NO: 437, 6 and 7; or SEQ ID NO: 5, 6 and 452; or SEQ ID NO: 5, 6 and 455; or SEQ ID NO: 607, 608 and 609; or SEQ ID NO: 610, 611 and 612; or SEQ ID NO: 671, 2 and 3; or SEQ ID NO: 672, 2 and 3; or SEQ ID NO: 673, 6 and 7; or SEQ ID NO: 674, 6 and 7; or SEQ ID NO: 1, 2 and 713; or SEQ ID NO: 5, 6 and 714; or SEQ SEQ ID NO: 674, 164 and 7; or SEQ ID NO: 710, 6 and 7; or SEQ ID NO: 5, 6 and 715; or SEQ ID NO: 674, 712 and 7; or SEQ ID NO: 711, 6 and 7; or SEQ ID NO: 673, 6 and 741; or SEQ ID NO: 673, 6 and 742; or SEQ ID NO: 673, 6 and 743; or SEQ ID NO: 673, 6 and 744; or SEQ ID NO: 673, 431 and 741; or SEQ ID NO: 673, 431 and 742; or SEQ ID NO: 673, 431 and 743; or SEQ ID NO: 673, 6 and 7; or SEQ ID NO: 674, 6 and 7, wherein the conjugate is administered intracerebral, intraventricular or intrathecal. The inventors have shown that this local (IT or ICV) CNS application is particularly interesting because the conjugate according to the invention is functionally delivered to both muscle cells and nervous system cells after local CNS (IT or IC) application, and thus can be used to address and treat muscle and neuromuscular disorders not only through systemic application but also through local CNS (IT or IC) application.

[0202] In another specific embodiment, the present invention relates to a VHH-oligonucleotide conjugate as defined herein, preferably a VHH-siRNA or VHH-ASO conjugate, wherein the conjugate is administered intracerebrally, intraventricularly, or intrathecally for the prevention or treatment of muscle diseases preferably selected from myopathy, cardiomyopathy, muscular dystrophy (e.g., DMD, BMD, FSHD, Pompe disease, or familial hypertrophic cardiomyopathy), or neuromuscular disorders such as spinal muscular atrophy, amyotrophic lateral sclerosis, peroneal muscular atrophy, multiple sclerosis, or Huntington's disease.

[0203] The additional compound coupled to the VHH molecule of the present invention can be a different VHH or a molecule that is not a VHH. The at least one additional molecule, agent, or compound of interest can be any molecule, agent, or compound, such as a half-life extension moiety, a stabilizing group or scaffold, a therapeutic (i.e., active) compound, an agent or drug, a diagnostic agent, an imaging molecule, a tracer, etc., or a carrier containing such a therapeutic, diagnostic, or imaging compound.

[0204] In one particular aspect, the chimeric agent (i.e., the conjugate) may comprise two additional compounds: i) a half-life extension portion, a stabilizing group, or a scaffold, and ii) a therapeutic, diagnostic, or imaging compound or a carrier containing them.

[0205] The therapeutic compound is selected, for example, from 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.

[0206] Examples of conjugated molecules, pharmaceuticals, or compounds of interest include, but are not limited to, any chemical entity, such as small chemical molecules (e.g., chelating agents, antibiotics, antiviral agents, immunomodulators, antitumor agents, anti-inflammatory agents, or adjuvants); peptides, polypeptides, or proteins (e.g., enzymes, hormones, cytokines, apolipoproteins, growth factors, antigens, antibodies or antibody portions, adjuvants, etc.); nucleic acids (e.g., human, viral, animal, eukaryotic, prokaryotic, plant, or synthetically derived RNA or DNA, including, for example, gene-encoding, repressive nucleic acids such as ribozymes, antisense oligonucleotides (ASO), interfering nucleic acids (siRNA), small activating RNA (saRNA), mRNA, whole genome or portions thereof, plasmids, etc.); lipid (nano) particles, cell-derived vesicles (CDVs) such as exosomes, viruses, biomarkers, or tracers, etc. Generally, the term "molecule, pharmaceutical, or compound of interest" can be any pharmaceutical (active) ingredient, whether chemical, biochemical, natural, or synthetic. Typically, the phrase "small chemical molecule, drug or compound" refers to a molecule of pharmaceutical interest with a maximum molecular weight of 1,000 Daltons, usually between 300 and 700 Daltons.

[0207] The carrier may be selected, for example, from viruses, virus-like particles (VLPs), cell-derived vesicles (CDVs), exosomes, lipid carriers, and polymer carriers, preferably lipid nanoparticles (LNPs), micelles, or liposomes.

[0208] The conjugated compound is typically an agent (e.g., a small drug, nucleic acid, or peptide, such as an antibody or fragment thereof) or imaging agent suitable for treating or detecting muscle or neuromuscular diseases such as myopathy, cardiomyopathy, muscular dystrophy (e.g., DMD, BMD, FSHD, Pompe disease, or familial hypertrophic cardiomyopathy), neuromuscular diseases (e.g., ALS, SMA, MS, CMT, or HD), or muscle cancers (e.g., rhabdomyosarcoma or leiomyosarcoma).

[0209] In addition to or as a substitute for the compound of interest, the chimeric agent may also contain a half-life-extending portion or a stabilizing group to increase the plasma half-life of the VHH or conjugate. Therefore, specific chimeric agents of the present invention comprise, in any order, i) at least one VHH, such as several VHH molecules, ii) a half-life-extending portion or a stabilizing group, iii) the compound of interest, typically a therapeutic, diagnostic, or imaging compound, and optionally iv) a carrier.

[0210] In one particular aspect described herein, the compound of interest is also a group that allows for stabilization and / or increase of the plasma half-life of the VHH molecule of the present invention. The half-life-extending portion or stabilizing group can be any group known to have a substantial plasma half-life (e.g., at least several hours) and substantially no adverse 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 bound to albumin), large human serum proteins (e.g., albumin, HAS), or IgG or PEG molecules.

[0211] In one particular embodiment, the conjugate according to the invention comprises a half-life-extending portion or stabilizing group, which is a small organic albumin moiety that binds to albumin with low micromolar affinity, thereby improving the pharmacokinetic characteristics of the compound of interest by gradually releasing the conjugate from said albumin. This small organic albumin moiety includes, for example, fragments of Evans blue (EB) dye, fatty acids and their derivatives such as C16 groups and 4-(p-iodophenyl)butyryl (PIB) groups.

[0212] In another specific embodiment, the conjugate according to the invention comprises a half-life extension portion or a stabilizing group, which is a human IgG1 or IgG4, preferably an Fc fragment of IgG1. Such a conjugate has the general formula VHH-hFc-siRNA or VHH-hFc-ASO.

[0213] In another specific embodiment, the coupling compound according to the invention comprises a half-life extension portion or a stabilizing group, which is an Fc homodimer or heterodimer.

[0214] In another embodiment, the conjugate according to the invention comprises a half-life-extending portion or stabilizing group, which is a homodimer or heterodimer of a modified Fc fragment of IgG1 or IgG4, having attenuated or eliminated effector function and / or an extended half-life. In another specific embodiment, the conjugate according to the invention comprises a modified Fc fragment of IgG1, which is a deglycosylated Fc fragment of IgG1, for example, having an N297 mutation.

[0215] In another specific embodiment, the conjugate according to the invention comprises an IgG1-modified Fc fragment, said fragment being a fragment of an Fc variant having symmetrical or asymmetrical amino acid modifications (i.e., on only one or both arms of the Fc dimer), said amino acid modifications being selected from deletions, insertions, inversions, or substitutions, or combinations thereof, such as amino acid substitutions at L234A and L235A (i.e., LALA mutations). Such Fc modifications allow for modulation of Fc receptor interactions, regulation, reduction, or elimination of Fc effector functions, such as FcγR binding, antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), or regulation of glycosylation.

[0216] In another specific embodiment, the conjugate according to the invention comprises a modified Fc fragment containing a mutated IgG1 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.

[0217] In another specific embodiment, the conjugate according to the invention comprises a modified Fc fragment containing a mutated IgG1 fragment 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.

[0218] The conjugates according to the invention may also comprise modified Fc fragments containing any combination of the above-described mutations, optionally further combined with LALA mutations.

[0219] In another specific embodiment, the conjugate according to the invention may comprise a modified Fc fragment containing a mutated IgG4 at residue position L248 or L235, or a combination thereof. Specific examples of such mutations are L248E, L235A, or L235E.

[0220] In another specific embodiment, the conjugate according to the invention comprises a half-life extension portion or a stabilizing group, which is albumin or an albumin-binding portion.

[0221] The VHH can be coupled to the N-terminus or C-terminus, or both, of the extended half-life portion or stabilizing group. When the extended half-life portion or stabilizing group is an Fc fragment, coupling is typically achieved through genetic fusion. Depending on the nature of the extended half-life portion or stabilizing group, the resulting protein can remain a monomer or polymerize. In the case of an Fc fragment, the fusion protein Fc-VHH or VHH-Fc can form a homodimer or a heterodimer.

[0222] In this regard, in one particular embodiment, the VHH molecule of the present invention utilizes the half-life extension portion or stabilizing group, such as the Fc fragment of human IgG1 or IgG4, conjugated with at least one oligonucleotide, i.e., one or more oligonucleotides, such as 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, or bridging nucleic acid (BNA), preferably ASO or siRNA, wherein Fc-VHH or VHH-Fc is a homodimer or heterodimer.

[0223] For example, heterodimer VHH-Fc fusions can be generated using a "mortar and pestle" or "KiH" technique, and VHH can be included at the N-terminus (or N-ter) of one arm ("mortar" arm) of a human IgG1-derived Fc dimer (hFc), and a tag sequence (i.e., a Q-tag) specifically recognized by transglutaminase (TGase) can be inserted into the C-terminus (or C-ter) of the other arm ("mortar" arm) of the Fc dimer. The Q-tag can be site-specifically modified to introduce an azide linker. The resulting heterodimer VHH-hFc-Q-tag-azido intermediate can be coupled with alkyne-siRNA using a copper-free click reaction to generate VHH-hFc-siRNA conjugates with stable linkers (as described in Example 6). Figure 14(as shown in the diagram). In one particular embodiment, the conjugate according to the invention comprises a VHH-Fc heterodimer, wherein Fc contains the following mutations: T366W on the "pestle" arm of the heterodimer, and / or T366S, L368A, and Y407V on the "mortar" arm of the heterodimer.

[0224] In another specific embodiment, the conjugate according to the invention comprises a VHH-Fc heterodimer, wherein Fc comprises T366W, L234A and L235A mutations on the "pestle" arm of the heterodimer and / or T366S, L368A, Y407V, L234A and L235A mutations on the "mortar" arm of the heterodimer.

[0225] In another specific embodiment, the conjugate according to the invention comprises a VHH-Fc heterodimer, wherein the Fc comprises T366W, L234A, and L235A mutations on the "pestle" arm of the heterodimer and T366S, L368A, Y407V, L234A, and L235A mutations on the "mortar" arm of the heterodimer. In this regard, the VHH-Fc heterodimer may comprise an Fc modified with the sequence of SEQ ID NO: 664 on the "pestle" arm and an Fc modified with the sequence of SEQ ID NO: 665 on the "mortar" arm. Example 6 and... Figure 8 , 9 Specific examples of such VHH-Fc heterodimers containing SEQ ID NO: 664 and 665 are described in 10 and 10.

[0226] All of the above mutations are defined according to the standard Kabat system used to number the amino acid residues in antibodies.

[0227] In the coupling compounds of this invention, coupling can be carried out through any acceptable bonding mode, taking into account the chemical properties, steric hindrance, and number of the coupled entities. Therefore, coupling can be carried out via one or more covalent bonds, ionic bonds, hydrogen bonds, hydrophobic bonds, or van der Waals bonds, which may be cleavable or incleavable in physiological media or within cells, preferably cleavable, particularly when the invention is used in scenarios involving the delivery of at least one active pharmaceutical ingredient to a muscle site. Furthermore, coupling can be carried out at various reactive groups, particularly at one or more terminal and / or one or more internal or lateral reactive groups. Coupling can also be performed using genetic engineering.

[0228] A strong interaction is required between the VHH and the different cargoes to prevent them from dissociating before the conjugate reaches its site of action (i.e., the muscle site). Therefore, the preferred conjugation of the present invention is covalent conjugation, although non-covalent conjugation can also be used. The compound of interest can be conjugated to the VHH at one of its terminals (N-terminus or C-terminus) or at a side chain of one of the amino acids constituting the sequence (Majumdar S. and Siahaan TJ., “Peptide-Mediated Targeted Drug Delivery”). Peptide-mediated targeted drug delivery (Med Res Rev., 2012 May; 32(3):637-58). Compounds of interest can be coupled directly to VHH or indirectly via a linker or spacer. Covalent chemical coupling, with or without a linker, includes classic biocoupling techniques, such as coupling with bifunctional or polyfunctional reagents selected from those containing alkyl, aryl, thiols, azides, alkynes, nucleotides, or peptide groups via esters, aldehydes or alkyl or aryl acids, anhydrides, thiols or carboxyls, groups derived from cyanogen bromide or cyanogen chloride, carbonyl diimidazoles, succinimides, or sulfonyl halides. It may also additionally include specific enzymatic coupling, such as via bacterial transglutaminase catalyzing the transamidation of glutamine, provided that the glutamine is inserted into a specific tag.

[0229] In one particular embodiment, coupling involves genetic fusion. This strategy can be used when the coupled molecule is a peptide or polypeptide. In this case, a nucleic acid molecule encoding VHH fused with the molecule is prepared and expressed in any suitable expression system to produce the conjugate. The overall structure of the VHH-oligonucleotide conjugate of the present invention is shown in [illustration details]. Figure 13 The illustrative strategy for coupling the VHH of the present invention to a molecule or scaffold is disclosed in [the document / concept]. Figure 14 middle.

[0230] In another specific embodiment, coupling is performed using a thiol / maleimide chemistry technique. For this reaction to occur, the VHH has a peptide sequence containing an additional cysteine ​​residue fused to its C-terminus. Specifically, this additional peptide tag is typically... (SEQ ID NO: 399), where the single underscore is the Gly adapter used as a spacer and the double underscore is the 6His tag used for purification purposes.

[0231] Since the VHH contains only cysteine ​​residues involved in disulfide bonds, the additional cysteine ​​introduced in the tag is the only cysteine ​​chemically reactive to maleimide. This allows the VHH to be specifically coupled to maleimide-derived molecules of interest. The reaction proceeds in two steps. First, it is necessary to smoothly reduce VHH-GGGGSCHHHH, because the additional cysteine ​​in the tag can partially participate in disulfide bonds during production (with another...). (Or free cysteine ​​forms a disulfide bond). Therefore, the first step involves partial reduction using a mild reducing agent such as 2-MEA (2-mercaptoethanol), TCEP (tris(2-carboxyethyl)phosphine), or DTT (dl-1,4-dithiothreitol). In the second step, VHH-GGGGSCHHHHHH is allowed to react with the maleimide-functionalized molecule of interest at a pH range of 6.5–7.5 to form a VHH-molecular conjugate linked covalently and stably. These two steps can be performed sequentially or together in situ.

[0232] In another specific embodiment, coupling is carried out via an enzymatic reaction. Specifically, site-specific coupling on the VHH can be performed using transglutaminase (Tgase). Tgase catalyzes the formation of a stable isopeptide bond between (i) a side chain of a glutamine residue inserted into a tag sequence (i.e., the Q-tag) 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 to any molecule of interest, whether a chemical drug or agent or a heterobifunctional linker for further coupling with a chemical drug or agent. For this purpose, the VHH is prepared by genetic fusion to tandemly add (typically at its C-terminus) the following tags: first, an optional trialanine linker, then an optional His-tag, then an optional triglycine linker, and finally the Q-tag. The triglycine linker allows the Q-tag to be separated, allowing Tgase to better access glutamine, while the His-tag is designed to facilitate the purification of VHH and its further functionalized versions.

[0233] The developed general coupling strategy is a convergence synthesis based on the following process: 1) Introducing a reactive moiety onto the glutamine in the Q-tag fused with VHH for further coupling with the molecule of interest. In this purpose, a heterobifunctional coupling head with two distinct reactive ends is allowed to be treated with Tgase: a primary amine group suitable for Tgase, and an orthorhombic reactive moiety. Representative examples of such orthorhombic and reactive groups include azides, bound alkynes such as DBCO (dibenzocyclooctyne) or BCN (bicyclo[6.1.0]nonyne), tetrazides, TCO (trans-cyclooctene), free or protected thiols, maleimides, etc.

[0234] 2) Introduce a reactive moiety on the molecule of interest that is complementary to the reactive moiety incorporated into the VHH Q-tag. Representative examples of such orthorhombic and reactive groups include azides, bound alkynes such as DBCO or BCN, tetrazines, TCO, free or protected thiols, maleimides, etc.

[0235] 3) Due to complementary reactive groups, the functionalized VHH and the molecule are coupled.

[0236] This paper also describes a method for coupling two molecules using a Q-tag as defined above via a Tgase coupling reaction. Another object of the invention is a VHH comprising a Q-tag. Yet another object of the invention is a VHH molecule comprising a linker, such as a Gly linker, and a Q-tag.

[0237] The preferred VHH of this invention has the following structure: VHH – connector – His m –Connector–LQR, where: VHH is any VHH molecule; connector is any molecular connector, such as an Ala or Gly connector (preferably the two connectors are different); m is an integer from 0 to 8, preferably m is 6 or 8.

[0238] In one particular embodiment, the present invention relates to a coupling compound comprising at least one VHH covalently linked to at least one chemical entity. Preferred variants of such coupling compounds contain one VHH and one chemical entity.

[0239] In another specific embodiment, the present invention relates to a conjugate comprising at least one VHH covalently linked to at least one nucleic acid. The nucleic acid may be an antisense oligonucleotide (“ASO”), a ribozyme, an aptamer, siRNA, etc. Preferred variants of this conjugate contain one VHH and one nucleic acid molecule. In a preferred embodiment, the nucleic acid is an oligonucleotide (i.e., a short DNA or RNA molecule, also referred to herein as an “oligonucleotide” or “oligomer”) 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, or bridging nucleic acid (BNA), which can specifically bind to a target mRNA to regulate gene expression in the cell. Preferably, this oligonucleotide is an ASO or siRNA capable of regulating (silencing, inhibiting, or activating) the expression of a target gene.

[0240] The conjugates according to the invention are complementary to the target gene, and in certain embodiments, they are capable of silencing or inhibiting the expression of the target gene, preferably at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%. More specifically, the conjugates according to the invention are capable of silencing or inhibiting the expression of the target gene by about 40% to about 80%.

[0241] Therefore, in one particular embodiment, the present invention relates to a coupling compound comprising at least one VHH that binds to TfR (preferably at the surface of muscle cells) and is covalently linked to at least one oligonucleotide, said oligonucleotide being 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, or bridging nucleic acid (BNA), which is capable of specifically binding to target mRNA, thereby regulating gene expression in cells.

[0242] In a preferred embodiment, the present invention relates to a coupling compound comprising: (i) One or more VHH molecules that bind to TfR on the surface of muscle cells and contain: - CDR1, which comprises a sequence selected from SEQ ID NO: 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, which comprises a sequence selected from SEQ ID NO: 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 - CDR3, which comprises sequences selected from SEQ ID NO: 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, and (ii) One or more oligonucleotides selected from any single-stranded or double-stranded oligonucleotides such as small interfering RNA (siRNA), small activating RNA (saRNA), gapmer, antisense oligonucleotide (ASO), shRNA, miRNA, aptamer RNA, or bridging nucleic acid (BNA) that can specifically bind to the target mRNA, thereby reducing its expression level in the cell.

[0243] In another preferred embodiment, the present invention relates to a coupling compound comprising: (i) One or more VHH molecules that bind to TfR on the surface of muscle cells and comprise: SEQ ID NO: 1, 2 and 3; or SEQ ID NO: 5, 6 and 7; or SEQ ID NO: 9, 10 and 11; or SEQ ID NO: 13, 14 and 15; SEQ ID NO: 17, 2 and 3; or SEQ ID NO: 19, 2 and 3; or SEQ ID NO: 1, 21 and 3; or SEQ ID NO: 1, 23 and 3; or SEQ ID NO: 1, 2 and 25; or SEQ ID NO: 1, 2 and 27; or SEQ ID NO: 1, 2 and 29; or SEQ ID NO: 1, 2 and 31; or SEQ ID NO: 1, 2 and 33; or SEQ ID NO: 67, 2 and 3; or SEQ ID NO: 69, 2 and 3; or SEQ ID NO: 1, 71 and 3; or SEQ ID NO: 1, 73 and 3; or SEQ ID NO: 1, 75 and 3; or SEQ ID SEQ ID NO: 1, 2 and 77; or SEQ ID NO: 1, 2 and 79; or SEQ ID NO: 1, 2 and 81; or SEQ ID NO: 1, 2 and 83; or SEQ ID NO: 1, 2 and 85; or SEQ ID NO: 392, 2 and 3; or SEQ ID NO: 1, 113 and 3; or SEQ ID NO: 1, 115 and 3; or SEQ ID NO: 1, 2 and 117; or SEQ ID NO: 1, 2 and 119; or SEQ ID NO: 1, 2 and 121; or SEQ ID NO: 1, 2 and 123; or SEQ ID NO: 125, 2 and 3; or SEQ ID NO: 17, 73 and 3; or SEQ ID NO: 17, 128 and 3; or SEQ ID NO: 5, 160 and 7; or SEQ ID NO: 5, 162 and 7; or SEQ ID NO: 5, 164 and 7; or SEQ ID NO: 5, 166 and 7; or SEQ ID SEQ ID NO: 9, 169 and 11; or SEQ ID NO: 9, 171 and 11; or SEQ ID NO: 175, 176 and 177; or SEQ ID NO: 179, 176 and 180; or SEQ ID NO: 182, 176 and 177; or SEQ ID NO: 184, 176 and 177; or SEQ ID NO: 186, 187 and 188; or SEQ ID NO: 190, 191 and 192; or SEQ ID NO: 194, 195 and 196; or SEQ ID NO: 198, 199 and 200; or SEQ ID NO: 201, 202 and 203; or SEQ ID NO: 205, 206 and 207; or SEQ ID NO: 410, 6 and 7;Or SEQ ID NO: 413, 6 and 7; or SEQ ID NO: 5, 416 and 7; or SEQ ID NO: 5, 419 and 7; or SEQ ID NO: 426, 6 and 7; or SEQ ID NO: 5, 431 and 7; or SEQ ID NO: 434, 6 and 7; or SEQ ID NO: 437, 6 and 7; or SEQ ID NO: 5, 6 and 452; or SEQ ID NO: 5, 6 and 455; or SEQ ID NO: 607, 608 and 609; or SEQ ID NO: 610, 611 and 612; or SEQ ID NO: 671, 2 and 3; or SEQ ID NO: 672, 2 and 3; or SEQ ID NO: 673, 6 and 7; or SEQ ID NO: 674, 6 and 7; or SEQ ID NO: 1, 2 and 713; or SEQ ID NO: 5, 6 and 714; or SEQ ID SEQ ID NO: 674, 164 and 7; or SEQ ID NO: 710, 6 and 7; or SEQ ID NO: 5, 6 and 715; or SEQ ID NO: 674, 712 and 7; or SEQ ID NO: 711, 6 and 7; or SEQ ID NO: 673, 6 and 741; or SEQ ID NO: 673, 6 and 742; or SEQ ID NO: 673, 6 and 743; or SEQ ID NO: 673, 6 and 744; or SEQ ID NO: 673, 431 and 741; or SEQ ID NO: 673, 431 and 742; or SEQ ID NO: 673, 431 and 743; or SEQ ID NO: 673, 6 and 7; or SEQ ID NO: 674, 6 and 7, and; (ii) One or more oligonucleotides selected from any single-stranded or double-stranded oligonucleotides such as small interfering RNA (siRNA), small activating RNA (saRNA), gapmer, antisense oligonucleotide (ASO), shRNA, miRNA, aptamer RNA, or bridging nucleic acid (BNA), which are capable of specifically binding to target mRNA to regulate gene expression in the cell.

[0244] In a preferred embodiment, the conjugate of the present invention comprises: (i) at least one VHH molecule that binds to TfR on the surface of muscle cells and contains SEQ ID. NO: 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 (i) any one of or composed of 417-418, 420-425, 427-430, 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 such as small interfering RNA (siRNA), small activating RNA (saRNA), gapmer, antisense oligonucleotide (ASO), shRNA, miRNA, aptamer RNA or bridging nucleic acid (BNA), which is capable of specifically binding to target mRNA, thereby regulating gene expression in the cell.

[0245] In another preferred embodiment, the conjugate of the present invention comprises: (i) one or more VHH molecules, which are C5 or variants thereof (as listed in Table 1), are cross-species VHH molecules that bind to human, non-human primate (NHP), and mouse TfR on the surface of muscle cells, and comprise SEQ ID NO: 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, 675, 676, 679, (ii) any of 680, 692 or 701 or thereof; and (ii) one or more oligonucleotides 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 or bridging nucleic acid (BNA), which are capable of specifically binding to target mRNA to regulate gene expression in the cell.

[0246] In another preferred embodiment, the conjugate of the present invention comprises: (i) at least one VHH molecule, which is a humanized variant of C5 (as listed in Table 1), comprising or consisting of any one of SEQ ID NO: 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 such as small interfering RNA (siRNA), small activating RNA (saRNA), gapmer, antisense oligonucleotide (ASO), shRNA, miRNA, aptamer RNA or bridging nucleic acid (BNA), which is capable of specifically binding to target mRNA, thereby regulating gene expression in cells.

[0247] In another preferred embodiment, the conjugate of the present invention comprises: (i) one or more VHH molecules, which are B6 or variants thereof (as listed in Table 1), binding to the apical domain of TfR, preferably TfR1 at the surface of muscle cells, and comprising or consisting of any one of SEQ ID NO: 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 such as small interfering RNA (siRNA), small activating RNA (saRNA), gapmer, antisense oligonucleotide (ASO), shRNA, miRNA, aptamer RNA, or bridging nucleic acid (BNA), which are capable of specifically binding to target mRNA, thereby regulating gene expression in cells.

[0248] In another preferred embodiment, the conjugate of the present invention comprises: (i) one or more VHH molecules that bind to the apical domain of TfR, preferably TfR1 at the surface of muscle cells, and comprise or consist of any one of SEQ ID NO: 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 such as small interfering RNA (siRNA), small activating RNA (saRNA), gapmer, antisense oligonucleotide (ASO), shRNA, miRNA, aptamer RNA or bridging nucleic acid (BNA), which are capable of specifically binding to target mRNA, thereby regulating gene expression in cells.

[0249] In another preferred embodiment, the conjugate of the present invention comprises: (i) one or more VHH molecules, which are B8 or variants thereof (as listed in Table 1), comprising any one of SEQ ID NO: 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 ...3, 164, 165-164, 165-164, 16 IDNO: any one of 677, 678, 681, 682, 752-765 or 773-786, or composed of therein; and (ii) one or more oligonucleotides 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 or bridging nucleic acid (BNA), which are capable of specifically binding to target mRNA, thereby regulating gene expression in the cell.

[0250] Specific examples of coupling methods used to conjugate VHH molecules to oligonucleotides include direct thiol-maleimide chemistry, which involves the introduction of an unpaired cysteine ​​residue at the C-terminus of the VHH, or indirect SPAAC reactions (strain-promoted alkyne-assisted clicks), which involve the introduction of a site-specific enzyme-assisted reaction on the VHH that introduces a moiety suitable for click chemistry. Different linkers can be used between the two partner VHHs and the oligonucleotide; these can be stable or cleavable. Examples of cleavable linkers include disulfides, enzyme-labile peptide linkers such as valine-citrulline or phenylalanine-lysine dipeptides, or pH-labile linkers such as hydrazones.

[0251] In one particular implementation, the VHH molecule and the oligonucleotide are preferably linked together using a copper-free click reaction to produce a VHH-oligonucleotide conjugate with a stable or cleavable linker.

[0252] In another specific embodiment, the present invention relates to a conjugate comprising a VHH covalently linked to a peptide. The peptide may be an active molecule, a decoy, a tag, a ligand, etc. Preferred variants of such conjugates contain a VHH and a peptide.

[0253] In another embodiment, the present invention relates to a conjugate comprising VHHs covalently linked to nanoparticles and / or liposomes, such as lipid particles or nanoparticles (“LNPs”). The nanoparticles and / or liposomes may be loaded or functionalized with an active pharmaceutical agent. Preferred variants of such conjugates contain several VHH molecules coupled to each nanoparticle or liposome.

[0254] In another embodiment, the conjugate comprises an antibody or fragment thereof conjugated to one or more VHH molecules as a half-life extension portion or stabilizing group. Typically, the VHH molecule is conjugated to the C- or N-terminus of the heavy chain or light chain, or both, or to the C- or N-terminus of the Fc fragment. In a preferred aspect, the VHH molecule is conjugated to the N-terminus of the Fc. In an even more preferred aspect, the VHH molecule is conjugated to the C-terminus of the Fc.

[0255] In another specific aspect, the conjugate comprises or is composed of a single VHH molecule conjugated to an antibody fragment, which may be a heavy chain or a light chain. In this respect, the VHH molecule is indiscriminately conjugated to either the C-terminus or the N-terminus of the chain. Preferably, it is conjugated to the N-terminus.

[0256] The present invention also relates to a method for preparing a coupling compound as defined above, characterized in that the method includes a coupling step between VHH and a molecule or scaffold, preferably by a chemical, biochemical or enzymatic approach, or by genetic engineering.

[0257] In the chimeric agents of the present invention, when several VHHs are present, they may have similar or different binding specificities.

[0258] Nucleic acids, vectors, and host cells

[0259] Another aspect of the invention relates to a nucleic acid encoding a VHH or a conjugate thereof as defined above (when the conjugated 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 both. It may contain modified nucleotides, which contain, for example, modified bonds, 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 according to the invention can be deduced from the amino acid sequence of the VHH molecule according to the invention, and the codon usage can be adjusted according to the host cell in which the nucleic acid will be transcribed. These steps can be performed according to methods known to those skilled in the art, some of which are described in reference manuals such as Sambrook et al.

[0260] Specific examples of such nucleic acid sequences include sequences containing any of SEQ ID NO: 301-329, 52-64, 95-110, 469-606, 646-663, 683-690, 721-738, or 796-837, which have no tag-coding portion or include the optional tag-coding portion of SEQ ID NO: 330, and its complementary sequence. The corresponding domains encoding CDR1, CDR2, and CDR3 are shown underlined in Table 2 below. The tag-coding portion of SEQ ID NO: 330 is shown in bold in Table 2.

[0261] The present invention also relates to a vector containing such nucleic acid, optionally under the control of regulatory sequences (e.g., promoters, terminators, etc.). The vector may be a plasmid, virus, granule, phage particle, artificial chromosome, etc. In particular, the vector may contain the nucleic acid of the present invention operatively linked to a regulatory region (i.e., a region containing one or more control sequences). Optionally, the vector may contain several nucleic acids of the present invention operatively linked to several regulatory regions. The term "control sequence" refers to a nucleic acid sequence necessary for the expression of a coding region. The control sequence may be endogenous or heterologous. Well-known control sequences currently used by those skilled in the art will be preferred. Such control sequences include, but are not limited to, promoters, signal peptide sequences, and transcription terminators. The term "operatively linked" refers to a configuration in which the control sequence is placed at an appropriate position relative to the coding sequence such that the control sequence directs the expression of the coding region.

[0262] This invention also relates to the use of nucleic acids or vectors according to the invention for transforming, transfecting, or transducing host cells or for preparing compositions (including pharmaceutical compositions) for transforming, transfecting, or transducing host cells. The invention also provides a host cell comprising one or more nucleic acids of the invention and / or one or more vectors of the invention. The term "host cell" also covers any progeny of the parent host cell that differs from the parent host cell due to mutations occurring during replication. Suitable host cells can be prokaryotic (e.g., bacteria) or eukaryotic (e.g., yeast, plant, insect, or mammalian cells). Specific illustrative examples of such cells include *Escherichia coli* (E. coli). E. coli ) strains, CHO cells, yeast strains, plant cells, sf9 insect cells, etc.

[0263] use

[0264] The VHH molecule of the present invention can bind to TfR, thereby targeting / delivering the molecule to muscle cells expressing TfR. In the context of the present invention, binding is preferably specific, thus binding to TfR occurs with a higher affinity than binding to any other antigen in the same species. In one specific embodiment, the VHH molecule of the present invention specifically binds to human TfR1. In another specific embodiment, the VHH molecule of the present invention binds to human and non-human primate TfR1. In another specific embodiment, the VHH molecule of the present invention binds to human and rodent TfR1. In yet another specific embodiment, the VHH molecule of the present invention binds to human TfR1, non-human primate TfR1, and rodent (e.g., mouse or rat) TfR1. In yet another specific embodiment, the VHH molecule binds to human, non-human primate, and mouse receptors with substantially similar affinities.

[0265] Therefore, the present invention relates to a method for targeting / delivering a compound to / through cells or organs expressing TfR, the method comprising conjugating the compound to at least one VHH of the present invention.

[0266] The present invention also relates to the use of VHH as defined above as a carrier for transporting compounds to / through muscle cells expressing TfR.

[0267] The present invention also relates to the use of VHH as defined above for the preparation of drugs or agents capable of targeting muscle sites.

[0268] The present invention also relates to a method for achieving or improving the targeting of compounds of interest to muscle sites, the method comprising coupling the molecule with the VHH of the present invention.

[0269] As explained above, the VHH of the present invention can be used to transport or deliver any compound, such as chelating agents, small drugs, amino acids, peptides, polypeptides, proteins, lipids, nucleic acids, viruses, liposomes, exosomes, etc. (preferably nucleic acids), into muscles.

[0270] The carrier can be used to transport or deliver the conjugate (including VHH), such as viruses, virus-like particles (VLPs), cell-derived vesicles (CDVs), exosomes, lipid carriers or polymer carriers, preferably lipid nanoparticles (LNPs), micelles or liposomes.

[0271] The present invention also relates to a pharmaceutical composition, particularly a diagnostic or therapeutic composition, characterized in that it comprises at least one VHH or conjugate compound (e.g., a VHH-drug conjugate as defined above, in the context of a therapeutic composition) bound to or present in a carrier (or not so), and one or more pharmaceutically acceptable supports, carriers or excipients.

[0272] The present invention also particularly relates to a diagnostic composition characterized in that it comprises a VHH or coupling compound, such as a VHH-diagnostic or medical imaging agent coupling compound as defined above, which is bound to or present in a carrier (or not).

[0273] The conjugate can be used in the form of any pharmaceutically acceptable salt. The expression "pharmaceutically acceptable salt" means, for example, but not limited to, pharmaceutically acceptable base or acid addition salts, hydrates, esters, solvates, precursors, metabolites, or stereoisomers, wherein the carrier or conjugate supports at least one substance of interest. The expression "pharmaceutically acceptable salt" also refers to a non-toxic salt, which is typically prepared by reacting a free base with a suitable organic or inorganic acid. These salts retain the bioavailability and properties of the free base. Representative examples of such salts include water-soluble and water-insoluble salts, such as acetates, N-methylglucosamine ammonium, 4,4-diaminostilbene-2,2'-disulfonate, benzenesulfonate, benzoate, bicarbonate, bisulfate, tartrate, borate, hydrobromide, bromide, butyrate, camphorsulfonate, carbonate, hydrochloride, chloride, citrate, clavulanate, dihydrochloride, diphosphate, ethylenediaminetetraacetate, calcium ethylenediaminetetraacetate, ethanedisulfonate, etolate, ethanesulfonate, fumarate, gluconate, gluconate, glutamate, glycolylarsanylate, hexafluorophosphate, hexyl isophthalate, hyaluronic acid, hydroxynaphthylcarbamate, iodide, isothiocyanate. Salts, lactates, lactobionates, laurates, malates, maleates, mandelates, methanesulfonates, methyl bromide, methyl nitrates, methyl sulfates, mucilages, naphthalene sulfonates, nitrates, 3-hydroxy-2-naphthoate, oleates, oxalates, palmitates, bis(hydroxynaphthoate) (1,1-methylene-bis-2-hydroxy-3-naphthoate or emboate), pantothenates, phosphates, picrates, polygalacturonic acids, propionates, p-toluenesulfonates, salicylates, stearates, basic acetates, succinates, sulfates, sulfosalicylates, suramates, tannates, tartrates, teoclates, toluenesulfonates, triethyl iodide, trifluoroacetate, and valerianates.

[0274] The compositions of the present invention advantageously comprise a pharmaceutically acceptable carrier or excipient. The pharmaceutically acceptable carrier may be selected from classically used carriers depending on each administration method. Depending on the intended administration method, the compound may be in solid, semi-solid, or liquid form. For solid compositions, whether free or contained in gelatin capsules, such as tablets, pills, powders, or granules, the active substance may be combined with: a) diluents, such as lactose, glucose, 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 carboxymethyl cellulose, and / or polyvinylpyrrolidone; d) disintegrants, such as starch, agar, alginic acid, or their sodium salts or effervescent mixtures; and / or d) absorbents, dyes, flavorings, and sweeteners. The excipients may be, for example, mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and analogs of pharmaceutical weight. For semi-solid compositions such as suppositories, the excipients may be, for example, emulsions or oily suspensions, or based on polyalkylene glycols, such as polypropylene glycol. Liquid compositions, particularly those for injection or contained in soft capsules, can be prepared, for example, by dissolving or dispersing the active ingredient in pharmaceutically pure solvents such as water, physiological saline solution, glucose solution, glycerol, ethanol, oil, and the like.

[0275] The compositions or conjugates of the present invention can be administered via any suitable route, and in a non-limiting manner via the following routes: parenteral route, for example, in the form of a formulation that can be injected subcutaneously (SC), intravenously (IV), or intramuscularly (IM), or via intracerebral (IC), intraventricular or lateral intraventricular (ICV), or intrathecal (IT); oral route (or via the mouth), for example, in the form of coated or uncoated tablets, gelatin capsules, powders, pellets, suspensions, or oral solutions (one such form for oral administration may be immediate release or prolonged or delayed release); rectal route, for example, in the form of suppositories; local route, particularly transdermal route, for example, in the form of patches, hair oils, or gels; intranasal route, for example, in the form of aerosols and sprays; translingual route; or intraocular route. Preferably, the VHH or conjugate of the present invention, or a composition containing such VHH or conjugate, is administered via an intravenous or subcutaneous route.

[0276] The pharmaceutical compositions typically comprise 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 nanomoles to about 500 nanomoles per kilogram of the subject's body weight (nanomoles / kg), preferably about 10 nanomoles / kg to about 500 nanomoles / kg. It should be understood that, particularly for an individual, the “therapeutic effective dose” will depend on various factors, including the activity / efficacy of the active substance, the time of its administration, the route of administration, its toxicity, its elimination rate and metabolism, drug combinations / interactions and the severity of the disease (or disorder) being treated on a preventative or curative basis, as well as the patient's age, weight, overall health status, sex, and / or diet.

[0277] Depending on the conjugated substance, the conjugates and compositions of the present invention can be used for imaging, diagnosis, prevention, and / or treatment of pathologies or disorders affecting muscles, such as any muscle or neuromuscular disease, preferably, for example, myopathy, cardiomyopathy, muscular dystrophy (e.g., DMD, BMD, FSHD, Pompe disease, or familial hypertrophic cardiomyopathy), neuromuscular diseases (e.g., ALS, SMA, MS, CMT, or HD), and muscle cancers (e.g., rhabdomyosarcoma, leiomyosarcoma, etc.). The VHH of the present invention has the ability to target cells expressing TfR, particularly muscle cells (such as skeletal muscle or cardiomyocytes or muscle cancer cells as described herein) and / or cross muscle cell membranes. TfR is enriched in muscle compared to different organs such as bone marrow, placenta, and gastrointestinal tract.

[0278] In this respect, the present invention relates to the use of drug conjugates or pharmaceutical compositions (particularly therapeutic compositions) as described above for the prevention or treatment of muscle pathologies or disorders (e.g., but not limited to any muscle or neuromuscular disease).

[0279] In the context of this invention, the muscle disease is a muscular or neuromuscular disease, preferably selected from myopathy, cardiomyopathy, muscular dystrophy (e.g., DMD, BMD, FSHD, Pompe disease, or familial hypertrophic cardiomyopathy), neuromuscular disease (e.g., ALS, SMA, MS, CMT, or HD), or muscle cancer (e.g., rhabdomyosarcoma, leiomyosarcoma, etc.). 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.

[0280] The present invention also relates to VHH, conjugates or pharmaceutical compositions as described above for the treatment of muscle pathologies or disorders, such as myopathy, cardiomyopathy, muscular dystrophy (e.g., DMD, BMD, FSHD, Pompe disease or familial hypertrophic cardiomyopathy), neuromuscular diseases (e.g., ALS, SMA, MS, CMT or HD) or cancers of the muscle (e.g., rhabdomyosarcoma or leiomyosarcoma).

[0281] The present invention also relates to VHHs, conjugates, or pharmaceutical compositions as defined above for the treatment of pathologies affecting muscles, such as, but not limited to, muscle or neuromuscular diseases, such as myopathy, cardiomyopathy, muscular dystrophy (e.g., DMD, BMD, FSHD, Pompe disease, or familial hypertrophic cardiomyopathy), neuromuscular diseases (e.g., ALS, SMA, MS, CMT, or HD), or cancers of muscles (e.g., rhabdomyosarcoma or leiomyosarcoma).

[0282] The present invention also relates to VHHs, conjugates or pharmaceutical compositions as defined above, for imaging, diagnosis, prevention and / or treatment of muscle or neuromuscular diseases, such as myopathy, cardiomyopathy, muscular dystrophy (e.g., DMD, BMD, FSHD, Pompe disease or familial hypertrophic cardiomyopathy), neuromuscular diseases (e.g., ALS, SMA, MS, CMT or HD) or cancers of muscle (e.g., rhabdomyosarcoma or leiomyosarcoma).

[0283] In one particular embodiment, the invention also relates to VHHs, conjugates, or pharmaceutical compositions as defined above for the prevention and / or treatment of muscle or neuromuscular diseases, such as myopathy, cardiomyopathy, muscular dystrophy (e.g., DMD, BMD, FSHD, Pompe disease, or familial hypertrophic cardiomyopathy), and neuromuscular diseases (e.g., ALS, SMA, MS, CMT, or HD), wherein the VHH binds to TfRs on the surface of both muscle cells and CNS cells.

[0284] In another specific embodiment, the invention also relates to VHHs, conjugates, or pharmaceutical compositions as defined above for the prevention and / or treatment of muscular or neuromuscular diseases, such as myopathy, cardiomyopathy, muscular dystrophy (e.g., DMD, BMD, FSHD, Pompe disease, or familial hypertrophic cardiomyopathy), or neuromuscular diseases (e.g., ALS, SMA, MS, CMT, or HD), wherein the VHH, conjugate, or composition is administered intracerebral, intraventricular, or intrathecally.

[0285] In another specific embodiment, the invention also relates to VHHs, conjugates, or pharmaceutical compositions as defined above for the prevention and / or treatment of muscle or neuromuscular diseases, such as myopathy, cardiomyopathy, muscular dystrophy (e.g., DMD, BMD, FSHD, Pompe disease, or familial hypertrophic cardiomyopathy), neuromuscular diseases (e.g., ALS, SMA, MS, CMT, or HD), wherein the VHH, conjugate, or composition is administered intracerebrally, intraventricularly, or intrathecally, and wherein the VHH binds to TfRs on the surface of both muscle cells and CNS cells.

[0286] This invention also relates to VHHs, conjugates, or pharmaceutical compositions as defined above, wherein the conjugated agent is or comprises a virus or virus-like particle, such as a recombinant virus. In fact, this invention can be used to enhance the delivery of recombinant (e.g., replication-defective or attenuated) viruses (e.g., adenovirus, adeno-associated virus, lentivirus, retrovirus, etc.) or virus-like particles used in gene therapy to any TfR-rich muscle tissue. Conjugation with a virus or VLP can be, for example, by conjugation with the capsid protein of said virus.

[0287] The present invention also relates to a method of preventing or treating any of the aforementioned conditions or diseases by administering the VHH, conjugate, or composition of the present invention to a subject in need.

[0288] The present invention also relates to the use of the VHH, conjugate, or composition of the present invention in the preparation of a medicament for treating any of the above-mentioned conditions or diseases.

[0289] The following examples are for illustrative purposes only and are not intended to limit the scope of the claims provided herein.

[0290] Example

[0291] Example 1: Determination of the binding characteristics of VHH with hTfR, mTfR and rhTfR

[0292] The binding properties of VHH with affinity to TfR from different species (i.e., human (h), mouse (m), and rhesus monkey (rh)) were tested using flow cytometry, and the epigenetic affinity (K) was determined. d app All experiments were performed in 96-well plates using 2 x 10⁻⁶ wells. 5Cells were placed per well and shaken at 4°C. During the 30-min interval, CHO cell lines expressing TfR fused with EGFP or CHO WT cells were saturated with 2% PBS / BSA to avoid nonspecific binding, and then incubated with gradually increasing concentrations of purified VHH for 1 hr. After one wash in 4% PBS / BSA, cells were incubated with anti-6His-tagged antibody (mouse) for 1 hr, washed twice with 2% PBS / BSA, and incubated with an Alexa647-conjugated anti-mouse secondary antibody for 1 hr. After two final washes in 2% PBS / BSA, cells were fixed by incubation with 2% PBS / PFA for 15 min, washed once with PBS, and finally resuspended in PBS. Fluorescence levels were assessed using an Attune NxT flow cytometer (Thermo Fisher Scientific). Experimental data were nonlinearly fitted using GraphPad Prism® software to determine epigenetic properties. K d constant.

[0293] No nonspecific markers were observed under control conditions where cells were incubated with either VHH D12 or C5neg. All tested VHHs induced concentration-dependent changes in signaling, confirming binding to the receptor of interest. No markers were detected in CHO WT control cells (not shown) using any of the tested VHHs. Table 5 below summarizes the epigenetic K obtained for the tested VHHs. d Most VHHs exhibit cross-species reactivity and show apparent K values ​​ranging from 0.1 nM to 4 µM. d Combining all three human, mouse, and rhesus monkey TfRs highlights the immense interest and diversity of the resulting VHH library.

[0294] Table 5:

[0295] NB: Not bound; ND: Not measured

[0296] Surface plasmon resonance (SPR) assays were also used to evaluate some VHH binding properties. Extracellular 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 a mouse IgG1 Fc fragment, and recombinant proteins were produced and purified internally. The interaction between VHHs and the receptors was tested using a Biacore T200 (GE Healthcare). The receptors were either directly immobilized on an HC 1500M or CM5 sensor chip (Xantec), or immobilized on previously immobilized anti-mouse IgG. VHHs were injected into the flow cell using single-cycle or multi-cycle kinetics. Table 6 below summarizes the tested VHH binding properties. These results further illustrate the cross-species reactivity of VHHs and the diversity of binding parameters.

[0297] Table 6:

[0298] ND: Not measured

[0299] Example 2: Oligonucleotide sequence and its coupling with TfR-binding VHH

[0300] The in vitro and in vivo experiments described in the examples provided herein were performed using the chemically modified siRNA or gapmer ASO sequences listed in Table 7 below. The RNAi-active siRNA sequences used in the examples below target mice and rats (siSOD1m) or humans and non-human primates (NHPs, including olive baboons, rhesus macaques, and cynomolgus monkeys) as described in WO2019217459 (which are adapted from siSOD1m to match human and NHP sources). SOD1 The ubiquitous superoxide dismutase 1 (SOD1) mRNA (siSOD-1) is used in the following examples. The single-stranded gapmer, rBase H-active antisense oligonucleotide (ASO) used targets the ubiquitously expressed long non-coding RNA (lncRNA) MALAT-1, which is preferentially enriched in nucleoli, where it regulates post-transcriptional RNA processing, and is modified by introducing the following sequence into SEQ ID NO: 405 in Table 7 below: mc "(i.e., 2'-O-methoxyethyl-5-methyl-cytidine; 2'MOE meC) replaces 2'-MOE-C (i.e., 2'-O-methoxyethyl-cytidine) adapted from Tripathi et al."

[0301] Table 7:

[0302] siSOD1m: A double-stranded siRNA targeting superoxide dismutase 1 (SOD1) mRNA in mice and rats; siSOD1h: Targets human and non-human primates (NHPs), including olive baboons ( Papio anubis ), macaques ( Macaca mulatta Crab-eating macaques ( Macaca fascicularis The siRNA duplex of superoxide dismutase 1 mRNA; hMALAT1 -ASO: Antisense oligonucleotide targeting the gapmer of human MALAT-1 long noncoding RNA (ASO); lowercase indicates targeting the 2'- of adenosine, cytidine, guanosine, or uridine nucleotides, respectively. O -Methyl (2'-OMe) sugar modification; italic capitalization indicates 2'-deoxy-2'-fluoro (2'F) sugar modification of adenosine, cytidine, guanosine or uridine nucleotides, respectively; Indicates the linkage between phosphate thioester (PS) nucleosides; VP indicates vinyl phosphonate; L indicates a linker (coupling arm); dN indicates a deoxynucleotide; lowercase italics indicate modification of the 2'-O-methoxyethyl (2'-MOE) sugar of adenosine, guanosine, or thymidine nucleotides; m c It represents 2'-O-methoxyethyl-5-methylcytidine (2'MOE meC).

[0303] The oligonucleotides, purchased from Horizon Discovery Biosciences Ltd. or GeneLink Inc., have coupling arms, such as a hexylamino linker, introduced into the 3'-terminus of the sense strand of the siRNA duplex or the 5'-terminus of the gapmer ASO. The overall coupling strategy involves pooling synthesis, such as the strategy described in WO 2020 / 144233, consisting of the following parallel modifications: i) modification of the VHH or VHH-hFc heterodimer to introduce, for example, an azido linker with site specificity; and ii) modification of the oligonucleotide to introduce, for example, a constrained alkyne group complementary to the azido functional group. In the final step, preferably using a copper-free click reaction, the functionalized azido-VHH or VHH-hFc-azido and alkyne-oligonucleotide precursors are interlinked to produce VHH-oligonucleotide or heterodimer VHH-hFc-oligonucleotide conjugates with stable linkers (see [link to relevant documentation]). Figure 14 The inventors employ alternative methods to produce VHH-oligonucleotide conjugates having other types of joints (e.g., cleavable disulfide joints or longer PEG joints) or another coupling chemistry (e.g., thiol-maleimide chemistry) (see [link]). Figure 13 ).

[0304] Example 3: Evaluation of the binding of VHH-oligonucleotide conjugates to mTfR, rhTfR and hTfR in living cells

[0305] The binding of VHH-oligonucleotide conjugates to mTfR, rhTfR, and hTfR was evaluated using a competitive assay on live cells expressing receptors of interest: mouse neuroblastoma Neuro-2a cells (N2a), CHO cells genetically engineered by the inventors to stably express rhesus monkey TfR fused to the C-terminus of eGFP protein (CHO-rhTfR-GFP cells), or human breast ductal carcinoma MCF-7 cells. MCF-7 and N2a cells were grown at 37°C and 5% CO2 in Dulbecco modified Eagle medium (DMEM) GlutaMAX supplemented with 10% v / v FBS. CHO-rhTfR-GFP cells were grown at 37°C and 5% CO2 in Ham F12 GlutaMAX supplemented with 10% v / v FBS. Cells were seeded at a density of 50,000 cells / well in 96-well plates two days prior to the experiment. On the day of the experiment, free VHH or VHH-oligonucleotide conjugates, diluted at various concentrations, were incubated at different concentrations 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 at 37°C for 3 hours with a subsaturated concentration of the fluorescent reference compound C5-Alexa680 (100 nM in N2a and CHO-rhTfR-GFP cell lines, and 10 nM in MCF-7 cells). After treatment, the cells were washed with D-PBS and detached by incubating with trypsin / EDTA at 37°C for 5 minutes, followed by the addition of cold medium (4°C) to inhibit trypsin activity. Resuspended cells were transferred to 96-well (V-bottom) plates containing 1% fetal bovine serum (FBS) / 0.02% sodium azide in phosphate-buffered saline (PBS) and centrifuged at 2000 rpm for 5 min at 4 °C. After removing the supernatant, 5 mM EDTA in D-PBS and 4% paraformaldehyde (PFA) (v / v 1:1) were added to the wells, and cells were fixed at room temperature for 15 min. PFA was removed by centrifugation (5 min, 3000 rpm, 4 °C), and cells were resuspended in 5 mM EDTA in D-PBS. Cells were then processed using an Attune-equipped plate. TM Attune for NxT v3.1.2 TMThermo Fisher Scientific NxT flow cytometer was used to quantify A680-related fluorescence signals in cells. Experimental data (three parallel experiments) were then analyzed using GraphPad Prism. ® Perform nonlinear fitting to determine the appearance K i Suppression constant. Figure 1 The figure shows a representative inhibition curve, and the table shows the mean ± SD of at least two independent experiments.

[0306] In cells expressing human, rhesus monkey, and mouse TfR, all tested VHH-oligonucleotide conjugates (e.g., B8-siSOD1m, C5-siSOD1m, B8-siSOD1h, C5-siSOD1h, B8-MALAT1-ASO) induced concentration-dependent inhibition of binding to and uptake of the reference compound. K i The values ​​were similar to or lower than those of free VHH, confirming efficient and cross-species binding with TfR. Figure 1 ).

[0307] Example 4: In vitro functional delivery and gene silencing potential of TfR-binding VHH-oligonucleotide conjugates after free uptake in human and mouse cell lines

[0308] Human cell lines MCF-7 (mammary ductal carcinoma cells), Igrov-1 (ovarian cancer cells), and MIA-PaCa-2 (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 and 5% CO2. For the free uptake assay, cells were seeded at a density of 2000 cells / well in 96-well plates. After 24 hours, the culture 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. The cells were then washed with D-PBS and harvested in lysis buffer from the SuperScript™ IV CellsDirect™ cDNA Synthesis Kit (Invitrogen) (for human cell lines) or the Nucleospin RNA XS Kit (Macherey-Nagel) (for N2a cells) as recommended by the manufacturer. For experiments using the Nucleospin RNA XS kit, cell lysates were stored at -20°C prior to 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 the TaqMan™ Rapid Universal PCR Master Mixture (2X) AmpErase™ UNG-Free 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, normalized to the expression of the reference RpL13 or GAPDH gene, based on raw quantitative cycle (Cq) values ​​(Bustin et al.), and expressed as mRNA levels relative to untreated cells. Experimental data (triple replicates) are presented as mean ± standard deviation (SD) and analyzed using GraphPad Prism. ® The software uses a three-parameter logarithmic (inhibitor) and response nonlinear regression analysis to determine the IC50. Figure 2 (human cell lines) and Figure 3 (Mouse N2a cells) show representative inhibition curves and estimated IC50 values.

[0309] Although free siSOD1h or siSOD1h coupled with unbound C5neg VHH... hSOD1mRNA levels were not affected, but TfR-binding VHH-siSOD1h conjugates (e.g., C5-siSOD1h, C5V8-siSOD1h, or B8-siSOD1h, which contain the following VHH molecules: C5, C5V8, or B8, respectively) showed potent and concentration-dependent downregulation of hSOD1 mRNA in various human cell lines. Figure 2 A and Table 1). Free VHH- uptake on MCF-7 cells. MALAT1 Similar potent knockdown effects were observed after using ASO conjugates. Figure 2 B). Similar specific and potent knockdown effects were also observed in mouse N2a cell lines after free uptake using various VHH-siSOD1m conjugates (i.e., conjugates containing the following VHH molecules: 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 (which contain VHH molecules, such as B8 as described in Table 1). Figure 3 (A and 3B).

[0310] These results demonstrate that the VHH-oligonucleotide conjugates of the present invention bind efficiently to human or mouse TfRs on the cell surface, followed by TfR-mediated endocytosis, endosome escape, and cytoplasmic or nuclear delivery of active oligonucleotides, thereby allowing for efficient regulation of target gene expression using different oligonucleotide modalities.

[0311] Example 5: In vivo functional delivery and mRNA knockdown in muscle tissue of TfR-binding VHH-siRNA conjugate in wild-type mice

[0312] The study evaluated the mediating effect of VHH-siSOD1m conjugate on muscle tissue in wild-type C57Bl / 6 mice after systemic administration. SOD1 The potential for mRNA knockdown. Mice (4-8 per group) were administered the drug via intravenous (IV bolus) or subcutaneous (SC) injection of PBS vector (control), unconjugated siSOD1m, or a specified VHH-siSOD1m conjugate, as shown in the image. Figure 4-7Dosage was administered as shown. At the designated time post-injection, tissue samples were removed, 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 homogenate 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 in an Agilent fragment analyzer system. Relative RNA expression levels were quantified by RT-qPCR using the TaqMan™ Rapid Universal PCR Master Mixture (2X) AmpErase™ UNG-Free 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 reference RpL13 gene, based on raw quantitative cycle (Cq) values ​​(Bustin et al.). Results are expressed as mean ± standard error of mean (SEM) and presented as mRNA levels relative to PBS-injected control animals. When administered at a low dose of 3 mg / kg (siRNA molar equivalent) via IV or SC in wild-type mice, the TfR-binding VHH-siSOD1m conjugate showed target activity in the gastrocnemius muscle. SOD1 mRNA levels were downregulated by approximately 70% to 80%, and in the myocardium, downregulated by approximately 60%. Figure 4 No or little effect was observed in the liver or lungs. Figure 4 Furthermore, no effect was observed in any tissue using the non-binding C5neg-siSOD1m conjugate. Figure 5 The results presented in this embodiment demonstrate tissue selectivity and confirm the TfR-dependent functional delivery of these TfR-binding conjugates in vivo at low therapeutic doses. Dose-response studies using one of these conjugates showed that the knockdown effect was 80% at the second-maximum dose in skeletal muscle at a dose of 3 mg / kg and in cardiac muscle at a concentration of 15 mg / kg, while unconjugated siRNA at concentrations up to 15 mg / kg had no effect. Figure 6The 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 assessments of these conjugates indicated that a single administration of a low dose of 3 mg / kg of the TfR-binding VHH-siRNA conjugate was sufficient to induce a potent and durable knockdown effect lasting for more than one month. Figure 7 This indicates that early TfR-mediated uptake in muscle tissue allows for efficient intracellular delivery of VHH-siRNA conjugates, and that active siRNA is continuously released from the endolysosomal compartment into the cytosol, with the target mRNA molecule loaded by the RNA-induced silencing complex (RISC) and continuously degraded.

[0313] Example 6: Generation and evaluation of the in vitro and in vivo functional delivery and mRNA knockdown potential of TfR-binding VHH-hFc-siRNA conjugates

[0314] To introduce a half-life-extending or stabilizing portion with improved pharmacokinetic characteristics, TfR-binding VHHs can be conjugated to oligonucleotides via an antibody or antibody fragment scaffold. This strategy is illustrated here using VHH-hFc-siRNA conjugates, which are generated using the same overall conjugation strategy described for direct VHH-siRNA conjugates. Furthermore, the VHH-hFc-siRNA conjugates also comprise a human IgG1-derived Fc dimer (hFc) having two arms referred to as a "groove" and a "mortar." In this embodiment, various TfR-binding VHH-hFc-siRNA heterodimer conjugates were generated using the "groove" technique. Each of these VHH-hFc-siRNAs contains a selected VHH at the N-terminus of the Fc dimer "groove" arm and a transglutaminase (TGase)-specifically recognized tag sequence (i.e., a Q-tag) inserted into the C-terminus of the Fc dimer "mortar" arm. The VHH-hFc-siRNA heterodimer is site-specifically modified on the Q-tag to introduce an azide linker. The resulting heterodimer VHH-hFc-Q-tag-azido intermediate is coupled to alkyne-siRNA using a copper-free click reaction to generate a VHH-hFc-siRNA conjugate with a stable linker. Figure 14 More specifically, the VHH-hFc-siSOD1m and VHH-hFc-siSOD1h conjugates were generated using the same siSOD1m and siSOD1h sequences as described in the preceding examples, and their biological properties in different systems were evaluated. The VHH used to generate the VHH-hFc-siRNA conjugates were C5 and B8 variants (e.g., ...). Figure 8 , 9(as shown in SEQ ID NO: 664 and SEQ ID NO: 665 on the "groove" arm). These conjugates are generated using a modified IgG1-derived Fc dimer having the sequence of SEQ ID NO: 664 on the "groove" arm and the sequence of SEQ ID NO: 665 on the "mortar" arm. The linker between VHH and hFc used in the "groove" arm is GGGGSGGGGS (SEQ ID NO: 630). For example, for the generation of C5-Fc conjugates, the complete amino acid sequence for the "groove" arm is shown in SEQ ID NO: 666, and the complete amino acid sequence for the "mortar" arm is shown in SEQ ID NO: 667. These sequences of the "groove" arm and the "mortar" arm are encoded by SEQ ID NO: 668 or 669, respectively.

[0315] First, the VHH-hFc-siSOD1 conjugate exhibits cross-species characteristics and, compared to free VHH, shows similar or slightly higher binding rates to mTfR and hTfR. Figure 8 Second, on human breast cancer MCF-7 cells ( Figure 9 A) or mouse neuroblastoma Neuro-2a cells ( Figure 9 B) Using the same in vitro free uptake assay as described in the previous embodiments, the VHH-hFc-siSOD1 conjugate exhibited specificity, potency, and concentration dependence. SOD1 Third, it was demonstrated that the VHH-hFc-siSOD1m conjugate mediated murine mRNA downregulation in muscle tissue after intravenous (IV bolus) administration in wild-type C57Bl / 6 mice. SOD1The ability to knock down mRNA was assessed. Mice (n=4–8 per group) were administered a single intravenous (IV bolus) injection of 0.5 or 1.5 mg / kg (siRNA molar equivalent) of a PBS carrier (control), a non-binding (C5neg) conjugate, or a TfR-binding (C5) VHH-hFc-siSOD1m conjugate. Fourteen days post-administration, tissue samples were 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 a fragment analyzer RNA kit in an Agilent fragment analyzer system. Relative RNA expression levels were quantified by RT-qPCR using the TaqMan™ Rapid Universal PCR Master Mixture (2X) AmpErase™ UNG-free 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 reference RpL13 gene, based on raw quantitative cycle (Cq) values ​​(Bustin et al.). Results are expressed as mean ± standard error of mean (SEM) and presented as mRNA levels relative to PBS-injected control animals.

[0316] Although no or almost no rodents were observed in any tissue analyzed after treatment with the non-binding conjugate (C5neg-hFc-siSOD1m). SOD1 Downregulation of mRNA levels, but treatment with the TfR-binding conjugate (C5-hFc-siSOD1m) induced potent muscle tissue selectivity and specificity, with knockdown exceeding 70% in the gastrocnemius, diaphragm, or myocardium at a dose of 1.5 mg / kg, and similar or slightly lower effects at a dose of 0.5 mg / kg, while no effect was observed in the liver or lungs. Figure 10A). Notably, 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 is 4-fold and 8-fold lower than the ED50 values ​​obtained after a single SC administration of the direct C5-siSOD1m conjugate, respectively. Figure 6 The results showed improvement with the introduction of the extended half-life Fc backbone. Similarly, another conjugate containing an additional 5'-VP on the antisense strand of siSOD1m (C5-hFc-siSOD1m-5'VP) was administered as a single SC in wild-type mice at various doses. High potency (over 80%) and muscle-selective KD were observed, with ED50 ranging from 0.1 to 0.6 mg / kg (siRNA molar equivalents). The results presented in this example confirm the tissue selectivity and TfR-dependent functional delivery designed for these TfR-binding VHH-hFc-siRNA conjugates when administered as a single dose via IV or SC to wild-type mice at low therapeutic doses.

[0317] Example 7: In hTfR1 + / + In vivo functional delivery of TfR-binding VHH-siRNA conjugates and mRNA knockdown in muscle tissue in mice

[0318] The VHH-siSOD1m conjugate was evaluated for its role in mediating the effects of constitutively human TfR1-expressing transgenic mice (B-hTfR1 mice, Biocytogen) in muscle tissue. SOD1 The ability to knock down mRNA was tested. PBS vector (control), unconjugated siSOD1m, or VHH-siSOD1m conjugate containing a cross-species rodent / NHP / human TfR1-binding VHH (C5) or a human TfR1-binding VHH (B6) were administered as a single subcutaneous (SC) injection at a dose of 3 mg / kg (siRNA molar equivalent). hTfR1 + / +In mice (3-4 mice per group). Seven days after administration, tissue samples were removed, 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 CryolysEvolution 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 in an Agilent fragment analyzer system. Relative RNA expression levels were quantified by RT-qPCR using the TaqMan™ Rapid Universal PCR Master Mixture (2X) AmpErase™ UNG-Free Kit (Applied Biosystems) and commercially available TaqMan™ probes for mouse SOD1 and RpL13 genes (Applied Biosystems). Based on raw quantitative cycle (Cq) values ​​(Bustin et al.), expression data were analyzed using the DDCq method, normalized to the expression of the reference RpL13 gene. Results are expressed as mean ± standard error of mean (SEM) and presented as mRNA levels relative to PBS-injected control animals.

[0319] Although no rodents were observed in any of the test samples analyzed after treatment with uncoupled siSOD1m. SOD1 Downregulation of mRNA levels, but a single subcutaneous injection of 3 mg / kg (siRNA molar equivalent) hTfR1 + / + In mice, both hTfR-binding conjugates induced similarly potent muscle tissue-selective effects, knocking down approximately 70-80% in the gastrocnemius and diaphragm, and approximately 40-50% in the myocardium, but had no effect in the liver or lung. Figure 11 The results presented in this embodiment demonstrate the cross-species rodent / human ability of the TfR-binding VHH-siRNA conjugate to undergo TfR-dependent binding, functional uptake, and target mRNA downregulation in vivo in mouse muscle tissue expressing human TfR at low therapeutic doses.

[0320] Example 8: Knockdown effect of VHH-siRNA conjugate on muscle after local ICV administration in B-hTfR mice

[0321] The VHH-siSOD1m conjugate was evaluated in B-hTfR mice (transgenic hTfR-ECD). KI / KI In mice, administration of Biocytogen to the mid-lateral ventricle (ICV) mediates the effect of cytotoxicity in muscle tissue. SOD1 The potential for mRNA knockdown. Currently, oligonucleotides acting on muscle tissue after local CNS administration (e.g., intrathecal or lateral ventricle) for neuromuscular disorders have not been developed. This study demonstrates for the first time that conjugates according to the invention are functionally delivered to muscle tissue after local CNS administration, and that conjugates according to the invention can target muscle cells via two administration routes: systemic and local CNS administration. B-hTfR mice (2 males and 2 females per group) were administered 1, 10, or 100 µg (siRNA molar equivalent) of PBS carrier (control), non-binding C5neg-siSOD1m-5'VP, or the TfR-binding VHH-siSOD1m-5'VP conjugate (B8h1-siSOD1m-5'VP, K) with high or medium binding affinity to hTfR. D = 0.6 nM; B8V32-siSOD1m-5'VP, K DA single ICV injection (54.8 nM) was administered to the right ventricle (5-10 µL, 0.75 µL / min). Seven days post-injection, tissue samples including brain regions (from the ipsilateral hemisphere), spinal cord, liver, and kidneys were 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 homogenate using the rNeasy 96 QIAcube HT kit (QIAGEN) in the QIAcube HT system. RNA samples were analyzed and quantified using the Fragment Analyzer RNA Kit in an Agilent fragment analyzer system. Relative RNA expression levels were quantified by RT-qPCR using the TaqMan™ Rapid Universal PCR Master Mixture (2X) without AmpErase™ UNG kit (Applied Biosystems) and commercially available TaqMan™ probes for mouse SOD1, RpL13, and RpL30 genes (Applied Biosystems). Expression data were analyzed using the ΔΔCq method (multiplex qPCR) normalized to the expression of reference RpL13 and RpL30 genes, based on raw quantitative cycle (Cq) values ​​(Bustin et al.). Results are expressed as mean ± standard error of mean (SEM) and presented as mRNA levels relative to PBS-injected control animals. Both TfR-binding conjugates showed potent knockdown in muscle tissue, and the medium-affinity conjugate B8V32-siSOD1-5'VP had no effect in excretory organs, while the high-affinity B8h1-siSOD1-5'VP conjugate had only mild to moderate effects in the kidneys and liver. Figure 15 At a dose of 100 µg / animal (corresponding to approximately 3 mg / kg (siRNA molar equivalent)), the maximum effect was approximately -80% in all tested muscle tissues. Using the non-bound C5neg-siSOD1-5'VP conjugate, only a moderate effect was observed at the highest tested dose of 100 µg, confirming that the TfR-bound VHH-siSOD1 conjugate is involved in TfR-mediated uptake and functional delivery in muscle tissue.

[0322] These results confirm that the TfR-binding VHH-siRNA conjugate, when administered locally via the CNS at a dose similar to that used for muscle delivery via systemic administration, has the potential for potent functional delivery in muscle tissue. Therefore, the VHH of this invention has the potential to address both muscular and neuromuscular disorders not only through systemic administration but also through CNS administration.

[0323] Example 9: Knockdown effect of systemic administration of heterodimeric VHH-hFc-siRNA conjugate on muscle in B-hTfR mice

[0324] B-hTfR mice (n=4 per group) were administered a single subcutaneous (SC) dose of a TfR-binding VHH-hFc-siSOD1m-5'VP conjugate containing different VHH variants (C5V30, B8V31, B8V32, C5h9, C5V5, and C5h18 variants, as examples here), at a dose of 4.5 mg / kg (siRNA molar equivalent), and compared with PBS-injected mice (n=8). As assessed using SPR, all tested conjugates bound to human TfR with an affinity in the range of 150–400 nM, except for the E8- and C5-hFc-siSOD1m-5'VP conjugates, which did so due to their low dissociation rates. k off It exhibited sub-nanomolar binding affinity. Two weeks after administration, tissue samples, including muscle tissue (gastrocnemius, diaphragm, and myocardium), liver, and kidneys, were rapidly frozen in 10 volumes of NucleoProtect RNA stabilization reagent (Macherey-Nagel) and stored at -20°C until processing for use. SOD1qPCR quantification of 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 the tissue homogenate 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 in an Agilent fragment analyzer system. Relative RNA expression levels were quantified by RT-qPCR using the TaqMan™ Rapid Universal PCR Master Mixture (2X) AmpErase™ UNG-Free Kit (Applied Biosystems) and commercially available TaqMan™ probes for mouse SOD1, RpL13, and RpL30 genes (Applied Biosystems). Expression data were analyzed using the ΔΔCq method (multiplex qPCR) normalized to the expression of reference RpL13 and RpL30 genes, based on raw quantitative cycle (Cq) values ​​(Bustin et al.). Results are expressed as mean ± standard error of mean (SEM) and presented as mRNA levels relative to control animals injected with PBS. Results showed that KD was potent in all tested muscle tissues and similar across all tested VHH variants, with approximately 90% KD in the gastrocnemius muscle, approximately 80% in the diaphragm and myocardium, and no effect in excretory organs. Figure 16 ).

[0325] In another experiment, B-hTfR mice (n=4 males per group) were administered three IV doses of a TfR-binding VHH-hFc-siSOD1m-5'VP conjugate containing different VHH variants (B8V40, B8V31, B8V32, and B8V31h5 variants, as examples here) at a dose of 1.5 mg / kg (siRNA molar equivalent) and compared with mice injected with PBS (n=8). The TfR-binding conjugates evaluated here were chosen because they exhibited similar binding affinities (based on SPR) to human and non-human primate TfR (rhesus macaque or cynomolgus monkey TfR extracellular domains) in the range of 150 nM to 1 µM. K D Selected samples were chosen based on differences less than 2-fold. Two weeks after the last administration, tissue samples, including muscle tissue (gastrocnemius, diaphragm, and myocardium), liver, and kidneys, were rapidly frozen in 10 volumes of NucleoProtect RNA stabilization reagent (Macherey-Nagel) and stored at -20°C until processed as described in the preceding examples for use. SOD1qPCR quantification of mRNA was performed. Results are expressed as mean ± standard error of mean (SEM) and presented as mRNA levels relative to control animals injected with PBS. As previously observed after a single SC administration of 4.5 mpk (siRNA molar equivalent), all tested TfR-binding conjugates showed robust >80% KD in all tested muscle tissues and had no effect in excretory organs. Figure 17 ).

[0326] The results shown here demonstrate that the conjugates of this invention exhibit strong functional delivery potential in the muscle tissue of mice expressing hTfR at low doses. Furthermore, several VHH variants and conjugates showed very similar binding affinity (less than 2-fold difference) between human and non-human primate (rhesus / cynomolgus monkey) TfR, allowing for easier translation from preclinical settings in non-human primates to clinical studies in humans.

[0327] Example 10: In vivo functional delivery of TfR-binding VHH-siRNA conjugate and mRNA knockdown in muscle tissue in non-human primates

[0328] Evaluation was conducted on targeted human and non-human primates (NHPs). SOD1 The VHH-siSOD1h conjugate of the gene in the olive baboon ( Papio Anubis Muscle tissue mediation SOD1mRNA knockdown capability. PBS vector (control) or TfR-binding VHH-siSOD1h conjugate was administered intravenously (IV infusion, 30 min) at a single dose of 5 mg / kg (siRNA molar equivalent) to male olive baboons (10–17 months old, 2.6–4.5 kg). At the designated time after administration, approximately 100 mg of gastrocnemius, quadriceps, and tibialis anterior muscle samples were removed under general anesthesia, 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 the QIAcube HT system. RNA samples were analyzed and quantified using the Fragment Analyzer RNA Kit in an Agilent fragment analyzer system. Relative RNA expression levels were quantified by RT-qPCR using the TaqMan™ Rapid Universal PCR Master Mixture (2X) AmpErase™ UNG-free 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 reference RpL13 gene, based on raw quantitative cycle (Cq) values ​​(Bustin et al.). Results are expressed as mean ± standard error of mean (SEM) and presented as mRNA levels relative to PBS-injected control animals.

[0329] Compared to the control group injected with PBS, a single IV administration of the TfR-bound VHH-siSOD1h conjugate induced the target in all tested muscle tissues. SOD1 The knockdown of mRNA levels was potent and persistent, with 60% of cases showing a duration of downregulation lasting more than three months. Figure 13 ).

[0330] The results presented in this embodiment confirm the ability of TfR-binding VHH-siRNA conjugates to undergo TfR-dependent binding, functional uptake, and target mRNA downregulation in vivo in non-human primate muscle tissue at low therapeutic doses in rodents / NHPs / humans.

[0331] sequence list

[0332] Table 1: VHH molecule of the present invention and negative control C5neg A list of amino acid sequences of D12, the corresponding CDR1-3, and their identifiers designated herein as SEQ ID NO or SEQ.

[0333]

[0334] Table 2: List of nucleotide sequences encoding the VHH molecule

[0335] Table 3: List of FR (framework region) sequences of VHH molecules

[0336] Table 4: Examples of Tag Sequences and Connectors

[0337] References

[0338] Ait Benichou et al., Gene Ther. 2022 Jan 25. pii: 10.1038 / s41434-022-00316-7. doi: 10.1038 / s41434-022-00316-7.

[0339] Barrientos T. et al., (2015), “Metabolic catastrophe in Mice Lacking Transferrin Receptor in Muscle”, EBioMedicine 2(11): 1705-1717.

[0340] Bizot et al., Drugs. 2020 Jul 21. pii: 10.1007 / s40265-020-01363-3. doi:10.1007 / s40265-020-01363-3.

[0341] Bustin et al. Clin Chem. 2009 Apr;55(4):611-22.

[0342] 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.

[0343] Jefferies et al., Transferrin receptor on endothelium of brain capillaries, Nature 1984 Nov 8-14; 312(5990):162-3.

[0344] Debacker, AJ et al., (2020), “Delivery of Oligonucleotides to the Liver with GalNAc: From Research to Registered Therapeutic Drug”, Mol Ther 28(8): 1759-1771.

[0345] Johnsen KB et al., Targeting the transferrin receptor for brain drug delivery, Prog Neurobiol. 2019 Oct;181:101665.

[0346] Majumdar S. and Siahaan TJ., “Peptide-mediated targeted drug delivery” Peptide-mediated targeted drug delivery ), Med Res Rev. 2012 May;32(3):637-58.

[0347] 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.

[0348] Needleman, SB and Wunsch, CD, (1970), Journal of Molecular Biology, 48, 443-453.

[0349] Pardridge et al., Human blood-brain barrier transferrin receptor, Metabolism 1987 Sep;36(9):892-5.

[0350] Sambrook J, Russell D, (2001), Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor.

[0351] Tai, W., Current Aspects of siRNA Bioconjugate for In Vitro and In Vivo Delivery, Molecules, 2019 Jun13;24(12).

[0352] Tripathi et al., “The nuclear-retained noncoding RNA MALAT1 regulates alternativesplicing by modulating SR splicing factor phosphorylation”, Mol Cell. 2010 Sep24;39(6):925-38.

[0353] Xu W. et al., (2015), “Lethal Cardiomyopathy in Mice Lacking Transferrin Receptor in the Heart”, Cell Rep 13(3): 533-545.

[0354] 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. A coupling compound comprising: (i) One or more VHH molecules of the formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and (ii) One or more oligonucleotides, The VHH molecule binds to TfR on the surface of muscle cells, and the VHH molecule comprises SEQ ID NO: 1, 2 and 3, or SEQ ID NO: 5, 6 and 7, or SEQ ID NO: 9, 10 and 11, or SEQ ID NO: 13, 14 and 15, SEQ ID NO: 17, 2 and 3, or SEQ ID NO: 19, 2 and 3, or SEQ ID NO: 1, 21 and 3, or SEQ ID NO: 1, 23 and 3, or SEQ ID NO: 1, 2 and 25, or SEQ ID NO: 1, 2 and 27, or SEQ ID NO: 1, 2 and 29, or SEQ ID NO: 1, 2 and 31, or SEQ ID NO: 1, 2 and 33, or SEQ ID NO: 67, 2 and 3, or SEQ ID NO: 69, 2 and 3, or SEQ ID NO: 1, 71 and 3, or SEQ ID NO: 1, 73 and 3, or SEQ ID NO: 1, 75 and 3, or SEQ ID NO: 1, 71 and 3, or SEQ ID NO: 1, 73 and 3, or SEQ ID NO: 1, 75 and 3, or SEQ ID NO: 1, 2, and 3. SEQ ID NO: 1, 2 and 77, or SEQ ID NO: 1, 2 and 79, or SEQ ID NO: 1, 2 and 81, or SEQ ID NO: 1, 2 and 83, or SEQ ID NO: 1, 2 and 85, or SEQ ID NO: 392, 2 and 3, or SEQ ID NO: 1, 113 and 3, or SEQ ID NO: 1, 115 and 3, or SEQ ID NO: 1, 2 and 117, or SEQ ID NO: 1, 2 and 119, or SEQ ID NO: 1, 2 and 121, or SEQ ID NO: 1, 2 and 123, or SEQ ID NO: 125, 2 and 3, or SEQ ID NO: 17, 73 and 3, or SEQ ID NO: 17, 128 and 3, or SEQ ID NO: 5, 160 and 7, or SEQ ID NO: 5, 162 and 7, or SEQ ID NO: 5, 164 and 7, or SEQ ID NO: 5, 166 and 7, or SEQ ID SEQ ID NO: 9, 169 and 11, or SEQ ID NO: 9, 171 and 11, or SEQ ID NO: 175, 176 and 177, or SEQ ID NO: 179, 176 and 180, or SEQ ID NO: 182, 176 and 177, or SEQ ID NO: 184, 176 and 177, or SEQ ID NO: 186, 187 and 188, or SEQ ID NO: 190, 191 and 192, or SEQ ID NO: 194, 195 and 196, or SEQ ID NO: 198, 199 and 200, or SEQ ID NO: 201, 202 and 203, or SEQ ID NO: 205, 206 and 207, or SEQ ID NO: 410, 6 and 7.Or SEQ ID NO: 413, 6 and 7, or SEQ ID NO: 5, 416 and 7, or SEQ ID NO: 5, 419 and 7, or SEQ ID NO: 426, 6 and 7, or SEQ ID NO: 5, 431 and 7, or SEQ ID NO: 434, 6 and 7, or SEQ ID NO: 437, 6 and 7, or SEQ ID NO: 5, 6 and 452, or SEQ ID NO: 5, 6 and 455, or SEQ ID NO: 607, 608 and 609, or SEQ ID NO: 610, 611 and 612, or SEQ ID NO: 671, 2 and 3, or SEQ ID NO: 672, 2 and 3, or SEQ ID NO: 673, 6 and 7, or SEQ ID NO: 674, 6 and 7, or SEQ ID NO: 1, 2 and 713, or SEQ ID NO: 5, 6 and 714, or SEQ ID SEQ ID NO: 674, 164 and 7, or SEQ ID NO: 710, 6 and 7, or SEQ ID NO: 5, 6 and 715, or SEQ ID NO: 674, 712 and 7, or SEQ ID NO: 711, 6 and 7, or SEQ ID NO: 673, 6 and 741, or SEQ ID NO: 673, 6 and 742, or SEQ ID NO: 673, 6 and 743, or SEQ ID NO: 673, 6 and 744, or SEQ ID NO: 673, 431 and 741, or SEQ ID NO: 673, 431 and 742, or SEQ ID NO: 673, 431 and 743, or SEQ ID NO: 673, 6 and 7, or SEQ ID NO: 674, 6 and 7.

2. The coupling compound according to claim 1, wherein the VHH molecule comprises an amino acid sequence selected from any one of SEQ ID NO: 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 coupling compound according to claim 1 or 2, wherein the VHH molecule comprises an amino acid sequence selected from any one of SEQ ID NO: 213, 216-271, 274, 275, 675, 676 and 701.

4. The coupling compound according to claim 1 or 2, wherein the VHH molecule comprises an amino acid sequence selected from any one of SEQ ID NO: 215 and 285-299.

5. The coupling compound according to claim 1 or 2, wherein the VHH molecule comprises an amino acid sequence selected from any of SEQ ID NO: 613-615.

6. The coupling compound according to any one of the preceding claims, wherein the VHH molecule further comprises a tag and / or a connector.

7. The coupling compound according to any one of the preceding claims, wherein the VHH molecule is humanized.

8. The coupling compound according to any one of the preceding claims, wherein the VHH molecule binds to human, non-human primate and / or rodent TfR1.

9. The conjugation compound according to any one of the preceding claims, wherein the oligonucleotide 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 bridging nucleic acid (BNA).

10. The coupling compound according to any one of the preceding claims, wherein the muscle cells are skeletal muscle cells, cardiomyocytes, or muscle cancer cells.

11. The coupling compound according to any one of the preceding claims, further comprising at least one additional compound, preferably a half-life extension portion or stabilizing group or scaffold such as an antibody or a fragment thereof (e.g., an Fc fragment), a VHH molecule, PEG, serum albumin, a serum albumin-binding moiety, more preferably an Fc fragment, wherein the Fc fragment is even more preferably an Fc heterodimer comprising an Fc modified with the sequence of SEQ ID NO: 664 on the club arm and an Fc modified with the sequence of SEQ ID NO: 665 on the mortis arm.

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

13. The coupling compound according to any one of claims 1 to 11 or the composition according to claim 12, for the treatment of muscle or neuromuscular diseases, preferably selected from myopathy, cardiomyopathy, muscular dystrophy (e.g., DMD, BMD, FSHD, Pompe disease or familial hypertrophic cardiomyopathy), neuromuscular diseases (e.g., ALS, SMA, MS, HD or CMT) and muscle cancers (e.g., rhabdomyosarcoma or leiomyosarcoma).

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

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

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