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

Abstract

The present invention relates to Camelidae heavy chain only variable domain (VHH) molecules that bind to TfR, coupling compounds comprising such VHH molecules and their use for transporting molecules of pharmaceutical interest to cells and organs, for example under pathological conditions of CNS or PNS, including cancer.

Description

Technical Field

[0001] This invention relates to transferrin receptor (TfR) binding molecules and their uses. Specifically, this invention relates to camelid heavy chain variable domain (VHH) molecules that bind to TfRs at the surface of cell membranes, such as the blood-brain barrier (BBB). More specifically, this invention relates to conjugated compounds comprising such VHH molecules, and their use for, for example, the transport of pharmacologically interesting molecules to cells of the nervous system (including the central nervous system (CNS: brain, spinal cord, and retina) and the peripheral nervous system (PNS: nerves and ganglia)) or to tissues or organs (including muscle tissue) expressing TfR, and / or for the treatment of various diseases of the nervous system (including cancer and neuromuscular diseases). Background Technology

[0002] The nervous system consists of the central nervous system (CNS) and the peripheral nervous system (PNS). The CNS consists of the brain, spinal cord, and retina, which is an extension of the CNS. The PNS consists of nerves (cranial nerves and spinal nerves) and ganglia (cranial ganglia, dorsal root ganglia or DRG, autonomic ganglia) where the cell bodies of sensory neurons are located.

[0003] According to global industry analysts ( Global Industry Analysts According to data from [source missing], the global market for drugs treating central nervous system (CNS, brain and spinal cord) pathologies was approximately $100 billion in 2015, of which nearly $9 billion represented products generated by drug delivery technologies. Jain, 2008, Jain Pharma Biotech Report, Drug Delivery in CNS Disorders (Drug Delivery in CNS disorders) Therefore, neurology is one of the three major therapeutic areas today, alongside cardiovascular medicine and oncology. Although the number of people worldwide suffering from CNS disorders and pathologies is greater than the number suffering from cardiovascular disease or cancer, neurology remains an underdeveloped market. This can be explained by the fact that 98% of potential drugs for treating CNS pathologies do not cross the body's barrier (BBB). Pardridge, 2003, Mol. Interv., 3, 90-105 ).

[0004] In fact, the brain is protected by two major physiological barrier systems from potentially toxic substances: the blood-brain barrier (BBB) ​​and the blood-brain cerebrospinal fluid barrier (BCSFB). The BBB is considered the primary pathway for the uptake of plasma ligands. Its surface area is approximately 5000 times that of the BSFB. The total length of the constituent vessels of the BBB is approximately 600 km. 3 The cerebral cortex contains blood vessels equivalent to 1 km². The total surface area of ​​the BBB is estimated to be 20 m². 2 ( De Boer et al., 2007, Clin. Pharmacokinet., 46 (7), 553-576Therefore, the cerebral endothelium that makes up the BBB represents a large surface area for potential exchange between blood and nerve tissue. However, due to its unique properties, this cerebral endothelium is also a major obstacle to the use of drugs to treat CNS disorders.

[0005] In fact, the brain's blood-brain barrier (BBB) ​​is composed of brain capillary endothelial cells (BCECs), which exhibit unique properties not found in the pore-forming endothelial cells that make up the vascular systems of other organs. BCECs form tight junctions and are surrounded by the basement membrane, astrocyte terminales, pericytes, microglia, and neurons. Together, these cells form a highly selective barrier that controls molecular exchange between the blood and the brain, maintains brain homeostasis, and very effectively protects the brain from toxins and pathogens. A drawback is that the BBB is also impermeable to most molecules, including drugs and imaging agents. Generally, only a small number of small lipophilic molecules (approximately 450 to 600 Daltons) can cross the BBB (accounting for only 2% of all drug candidates). Most (if not all) higher molecular weight molecules, such as therapeutic peptides, proteins, and antibodies, which have shown promising results in in vitro and animal studies for treating CNS disorders, do not cross the BBB.

[0006] Therefore, the BBB is considered a major obstacle to be overcome in the development of new therapies for treating CNS disorders. Neuwelt et al., 2008, Lancet Neurol., 7, 84-96 One of the research priorities associated with the discovery of molecules used to treat, diagnose, or image CNS pathologies is to develop strategies that allow / enhance the passage of active substances across the BBB.

[0007] One approach to bypassing the brain's biological barrier (BBB) ​​is to administer drugs directly into the central nervous system (CNS), such as through injection into the ventricles, intracerebral, or intrathecal injection, or by disrupting the BBB. However, this highly invasive method has drawbacks (such as cost and short-term efficacy) and potential risks. Furthermore, many drugs, including those that do not cross the BBB (due to their large size and / or inadequate physicochemical properties), do not cross the plasma membranes of target cells in both the CNS and pneumoneurotic system. Therefore, strategies are needed to facilitate the delivery of such drugs to target cells in the nervous system.

[0008] Pharmacological strategies based on the addition of lipids or lipophilic groups to the active substance (transcellular lipophilic diffusion, TLD) or the use of liposomes have been envisioned. Zhou et al., 1992, J. Control. Release, 19, 459-486 However, the addition of lipid or lipophilic groups or the use of liposomes typically produces large and nonspecific complexes with an optimal limit greater than 450 Daltons, which are relatively ineffective for crossing the BBB. Levin, 1980, J. Med. Chem., 23, 682-684 ; Schackert et al., 1989, Selective Cancer Ther., 5, 73-79Among the strategies evaluated for delivering protein therapeutics to the brain, hijacking cellular mechanisms involved in the transport of natural nutrients and endogenous ligands across the BBB appears to be the safest and most efficient (Fang et al., 2017; Jones and Shusta, 2007; Pardridge et al., 1992). The transport of macromolecules across the BBB can be facilitated by receptor-mediated transcytosis (RMT), a physiological process involving the binding of ligands to receptors expressed by BCECs, internalization via endocytosis, intracellular transport, and dissociation from the receptor in sorting endosomes, followed by release outside the BBB lumen (Tuma and Hubbard, 2003; Xiao and Gan, 2013). In this regard, WO2010 / 046588 and WO2011 / 131896 disclose various peptides with high affinity for LDL receptors capable of transporting drugs or other molecules across the BBB.

[0009] Another receptor studied for transporting drugs across the BBB is the transferrin receptor (TfR), which participates in the transport of iron into the brain via its ligand transferrin (Tf) (Fishman et al., 1987). This receptor has been shown to be highly expressed in the brain endothelium (Jefferies et al., 1984; Pardridge et al., 1987), although it is also abundant in blood cells and the lungs (Chan and Gerhardt, 1992). Although the use of Tf as a transporter has been investigated (Chang et al., 2009; Jain et al., 2011; Yan et al., 2013), the transport mechanism of this molecule is saturable and competes with endogenous Tf. Anti-TfR monoclonal antibodies have been investigated as vectors for brain delivery, including OX26 antibodies targeting rat TfR (Moos and Morgan, 2001; Pardridge et al., 1991; Ulbrich et al., 2009), or 8D3 antibodies targeting mouse TfR (Pardridge, 2015; Zhang and Pardridge, 2005; Zhou et al., 2010) and R17-217 antibodies (Lee et al., 2000; Pardridge, 2015; Ulbrich et al., 2009) (see also WO2012075037, WO2013177062, WO201275037, WO2016077840, WO2016208695). However, these antibodies have drawbacks, including their lack of cross-species reactivity, and especially their non-binding to human TfR, which hinders preclinical or clinical studies. Furthermore, the ability of such antibodies to effectively transport drugs across the BBB remains controversial.

[0010] Besides the central nervous system (CNS), the peripheral nervous system (PNS) is located outside the brain and spinal cord, connecting the CNS to the limbs and organs. While most PNS exhibit low permeability between blood and nerve tissue, much like the blood-brain barrier in the CNS, the DRG exhibits high permeability due to its loose blood-nerve interface (Ahimsadasan et al., StatPearls 2022, PMID: 30335324). This vascular tissue provides a robust blood supply to the human DRG. Therefore, despite advances in this field, there remains a need for other effective methods and agents capable of improving drug delivery to the CNS or PNS. Summary of the Invention

[0011] This invention provides specific VHH molecules that advantageously target transferrin receptors (TfR) in tissues of the nervous system, including the central nervous system (CNS) and the peripheral nervous system (PNS), and also provides their use for the efficient delivery of various therapeutic agents to the CNS or PNS. This invention also discloses VHH molecules that bind to human, non-human primate, and / or rodent TfR and can deliver drugs across the BBB to the CNS via transcytosis. This invention demonstrates that the VHH molecules of this invention can migrate efficiently across the CNS and deliver conjugated drugs or imaging agents in vivo. Therefore, such VHHs represent valuable molecules for therapeutic or diagnostic methods.

[0012] More specifically, this invention provides conjugates comprising such VHH molecules, optimized for targeting the nervous system and mediating efficient functional delivery of drugs to CNS or PNS cells. This invention demonstrates that the conjugates of this invention can effectively accumulate in the CNS or PNS and deliver conjugated therapeutic agents within CNS or PNS tissues, thus making them suitable for the highly effective prevention and treatment of various CNS and PNS diseases.

[0013] One object of 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) at least one therapeutic compound or a carrier containing such therapeutic compound, The VHH molecule binds to TfR on the surface of nerve cells, and The VHH molecule comprises: - CDR1, comprising an amino acid sequence selected from SEQ ID NO: 1, 5, 9, 17, 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 - CDR2, comprising an amino acid sequence selected from SEQ ID NO: 2, 6, 73, 113, 115, 128, 160, 162, 164, 166, 169, 171, 176, 187, 191, 195, 199, 202, 206, 416, 419, 431, 608, 611, and 712, and - CDR3, which contains an amino acid sequence selected from SEQ ID NO: 3, 7, 11, 117, 119, 121, 123, 177, 180, 188, 192, 196, 200, 203, 207, 452, 455, 609, 612, 713-715, and 741-744. The conditions are: - When CDR1 contains SEQ ID NO: 1, 5, 9 or 17, then CDR2 is not selected from SEQ ID NO: 2, 6 and 73, and CDR3 is not selected from SEQ ID NO: 3, 7 and 11; - When CDR2 contains SEQ ID NO: 2, 6 or 73, then CDR1 is not selected from SEQ ID NO: 1, 5, 9 and 17, and CDR3 is not selected from SEQ ID NO: 3, 7 and 11; - When CDR3 contains SEQ ID NO: 3, 7 or 11, then CDR1 is not selected from SEQ ID NO: 1, 5, 9 and 17, and CDR2 is not selected from SEQ ID NO: 2, 6 and 73.

[0014] Preferably, the therapeutic compound is selected from peptides, polypeptides, proteins, antibodies, and nucleic acids, wherein the nucleic acid is preferably selected from mRNA, ribozymes, or oligonucleotides, and 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). Preferably, the nervous system cells are CNS cells, such as neurons, glial cells (e.g., astrocytes, oligodendrocytes, ependymal cells, or microglia), choroid plexus cells, pericytes, endothelial cells, or fibroblasts of the brain, spinal cord, or retina; or PNS cells, such as neurons, glial cells (e.g., Schwann cells, satellite cells, macrophages, endothelial cells, or fibroblasts), such as nerve cells of cranial ganglia, dorsal root ganglia, or autonomic ganglia; or cancer cells of the CNS or PNS system.

[0015] Another object of the present invention relates to a coupling compound, wherein the VHH molecule comprises: 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: 392, 2 and 3 ... 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 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.

[0016] Another object of the present invention relates to a coupling compound, wherein the VHH molecule comprises an amino acid sequence selected from any one of SEQ ID NO: 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.

[0017] Another object of the present invention relates to a coupling compound, wherein the VHH molecule comprises an amino acid sequence selected from any one of SEQ ID NO: 242-271, 274, 275, 675, 676 and 701.

[0018] Another object of the present invention relates to a coupling compound, wherein the VHH molecule comprises an amino acid sequence selected from any of SEQ ID NO: 285-299.

[0019] Another object of the present invention relates to a coupling compound, wherein the VHH molecule comprises an amino acid sequence selected from any of SEQ ID NO: 613-615.

[0020] Another object of the present invention relates to a coupling compound, wherein the VHH molecule further comprises a tag and / or a connector.

[0021] Another object of the present invention relates to a coupling compound wherein the VHH molecule is humanized and preferably selected from SEQ ID NO: 130-149, 152-154, 252-271 and 273-275, 752-765 and 773-786.

[0022] Another object of the present invention relates to a coupling compound wherein the VHH molecule binds to human, non-human primate and / or rodent TfR, preferably TfR1.

[0023] Another object of the present invention relates to a coupling compound wherein the VHH molecule can cross the human blood-brain barrier (“BBB”).

[0024] Another object of the present invention relates to the coupling compound, wherein the VHH molecule has an affinity (K0.1 nM to 2500 nM) of the coupling compound. d Combined with TfR.

[0025] The coupling compounds of the present invention may also contain at least one additional compound, such as 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 or a serum albumin moiety. Preferably, the additional compound is an Fc fragment.

[0026] Another object of the present invention relates to coupling compounds, wherein the Fc fragment is an Fc heterodimer comprising an Fc modified with the sequence of SEQ ID NO: 664 on the peduncle arm and an Fc modified with the sequence of SEQ ID NO: 665 on the mortar arm.

[0027] The present invention also provides a pharmaceutical composition comprising a coupling compound as defined herein and a pharmaceutically acceptable excipient, support, or carrier.

[0028] The present invention also provides nucleic acids, vectors, and host cells encoding VHH or chimeric agents as defined above.

[0029] The present invention also provides a method for preparing VHH or chimeric agents, the method comprising culturing host cells as defined above under conditions that allow the expression of the nucleic acids.

[0030] The present invention also provides a method for preparing coupling compounds, the method comprising covalently or non-covalently coupling one or more VHHs as defined above to a molecule or a drug or a scaffold.

[0031] Another object of the present invention relates to a coupling compound as defined above, which is used as a drug.

[0032] Another object of the present invention relates to the use of the coupling compounds of the present invention as defined herein for enhancing the bioactivity of the substance of interest and / or for delivery in nerve tissue.

[0033] Another object of the present invention relates to a method for improving or enabling molecules to cross the BBB using coupling compounds as defined herein.

[0034] Another object of the present invention relates to a method for using coupling compounds as defined herein to improve or enable molecules to cross the plasma membrane of target cells in the nervous system.

[0035] The conjugates of the present invention or compositions containing such conjugates may be administered systemically, intravenously, intramuscularly, subcutaneously, intracerebrally, intraventricularly, or intrathecally.

[0036] This invention can be used in any mammal, particularly human subjects. It is suitable for the prevention or treatment of any neurological disorder, such as any CNS disease, including Alzheimer's disease, dementia, Parkinson's disease, stroke, Creutzfeldt-Jakob disease, multiple sclerosis, spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), epilepsy, migraine, encephalitis, lysosomal storage disorders of the brain, CNS pain, or CNS cancers such as brain cancer (e.g., glioblastoma). It is also suitable for the treatment of any peripheral neuropathy, such as peripheral neuropathy caused by traumatic injury, infection, metabolic problems, genetic causes and toxin exposure, autoimmune diseases, inflammation, or DRG cell disorders. It is also suitable for the treatment of any complications of PNS diseases, such as neuropathic pain. Attached Figure Description

[0037] Figure 1 : TfR expression at the BBB. Western blotting was performed on membrane fractions of brain microvessels (BMV) and brain microvascular endothelial cells (BMEC) from mice, rats, pigs, and non-human primates (NHP; rhesus monkeys). The amount of protein loaded is indicated below the image. nd: undigested; dig-: digested.

[0038] Figure 2 : Validation of CHO cell lines expressing human or mouse TfR. (A) Atlas of plasmid constructs used to generate the CHO-hTfR-EGFP cell line. (B) Representative confocal micrographs of CHO-hTfR-EGFP cells (green) incubated with Tf-Alexa647 (250 µg / ml, red) at 37°C for 1 h. Cell nuclei are labeled with 0.5 μg / mL Hoechst#33342 (blue). Co-labeling in the merged images is shown in yellow. (C) Western blots of cell membrane preparations of CHO cells expressing hTfR-EGFP and mTfR-EGFP compared to CHO WT, using rabbit anti-TfR antibody (1 / 1000) or mouse anti-GFP antibody (1 / 1000), followed by HRP-conjugated anti-rabbit or anti-mouse secondary antibody (1 / 10000).

[0039] Figure 3VHH A and VHH Z bind to and endocytose the cell surface of CHO cells expressing hTfR and mTfR. Representative confocal micrographs of CHO-hTfR-EGFP and CHO-mTfR-EGFP cells (green), cells incubated with 20 µg / ml VHH A (A, B) and control VHH Z (C, D) at 37°C for 1 hr. After PFA fixation, the cell membrane was permeabilized with or without Triton X-100 and detected using mouse anti-cMyc antibody (1 / 1000) and Alexa594-conjugated anti-mouse secondary antibody (1 / 800, red). Cell nuclei were labeled with 0.5 μg / mL Hoechst#33342 (blue). Co-labeling in the merged images is shown as yellow / orange.

[0040] Figure 4 : Epigenetic K of VHH in CHO cell lines expressing hTfR and mTfR d Assay. (A) CHO-hTfR-EGFP and CHO-mTfR-EGFP cells were incubated with various concentrations of VHH at 4°C for 1 hr and detected with mouse anti-6His antibody (1 / 1000) and Alexa647-conjugated anti-mouse secondary antibody (1 / 200 or 1 / 400). Measurements were performed using flow cytometry. The fluorescence intensity ratio at each spot was normalized to the corresponding EGFP signal (receptor expression) and arbitrary units were obtained. Data are presented as mean ± SEM of 3 independent experiments. (B) Characteristics of selected VHH: molecular weight (Da); theoretical pI; epigenetic K on human TfR. d (nM); Epigenetic K on mouse TfR d (nM). Data are presented as the mean ± SEM of 3 independent experiments. NB: Not bound.

[0041] Figure 5: Competition assay between VHH and Tf. (A) Principle of the competition assay. First, CHO-hTfR-EGFP cells were incubated with a series of diluted competitors at 4°C for 1 hour. Next, tracers at a concentration of EC90 were added and incubated at 4°C for 1 hour. The tracers were then revealed using an appropriate revelation system. Measurements were performed using flow cytometry. The fluorescence intensity ratio at each point was normalized with the corresponding EGFP signal (receptor expression) and arbitrary units were obtained. (B) CHO-hTfR-EGFP cells were incubated with competitors (Tf). Then tracers at a concentration of EC90 (VHH) were added and detected using mouse anti-cMyc antibody (1 / 50) and Alexa647-conjugated anti-mouse secondary antibody (1 / 200). (C) CHO-hTfR-EGFP cells were incubated with competitors (VHH). Then tracers at a concentration of EC90 (Tf-Alexa647) were added and detected directly. The data are presented as the mean ± SEM of three independent experiments.

[0042] Figure 6 VHH conjugation strategy. Using chemical conjugation or recombinant fusion, VHHs can be used to carrier all kinds of molecules, including but not limited to peptides, siRNAs, dyes, nanoparticles (NPs), liposomes, imaging agents, and antibodies. Furthermore, VHHs can be used to carrier molecules as monovalent (VHHs) or multivalent (VHHs). n ) Coupled objects.

[0043] Figure 7 The VHH A-Fc and VHH Z-Fc fusion proteins bind to and are endocytosed on the cell surface of CHO cells expressing hTfR and mTfR. Representative confocal micrographs of CHO-hTfR-EGFP and CHO-mTfR-EGFP cells (green) are shown. Cells were incubated with 50 nM VHH A-Fc (A, B) and control VHH Z-Fc (C, D) at 37°C for 1 hr. After PFA fixation, the cell membrane was permeabilized with or without Triton X-100 and detected using an Alexa594-conjugated anti-hFc antibody (1 / 1000, red). Cell nuclei were labeled with 0.5 μg / mL Hoechst#33342 (blue). Co-labeling in the merged images is shown as yellow / orange.

[0044] Figure 8 : Phenotypic K in CHO cell lines expressing hTfR and mTfR by VHH-Fc and Fc-VHH dAssay. (A) CHO-hTfR-EGFP and CHO-mTfR-EGFP cells were incubated with various concentrations of VHH-Fc or Fc-VHH at 4°C for 1 hr and detected with Alexa647-conjugated anti-hFc antibody (1 / 400). Measurements were performed using flow cytometry. The fluorescence intensity ratio at each spot was normalized to the corresponding EGFP signal (receptor expression) and arbitrary units were derived. Data are presented as mean ± SEM of 3 independent experiments. (B) Characteristics of selected VHH-Fc and Fc-VHH: molecular weight (Da); epigenetic K on human TfR. d (nM); Epigenetic K on mouse TfR d (nM). Data are presented as mean ± SEM of 3 independent experiments. NB: No binding for control VHH (VHHZ).

[0045] Figure 9 Uptake and transport of VHH A-Fc and VHH B-Fc fusion proteins in an in vitro BBB model. (A) Representative micrograph of a rat brain microvascular endothelial cell (rBMEC) monolayer used to detect the uptake of 500 nM VHH A-Fc and VHH B-Fc, co-incubated with 200 nM Tf-Alexa647 (red) on live cells for 2 hours, followed by PFA fixation and permeabilization of the cell membrane with Triton X-100, and detection using an Alexa488-conjugated anti-hFc antibody (1 / 50, green). Cell nuclei were labeled with 0.5 μg / mL Hoechst#33342 (blue). Co-labeled nuclei are shown in yellow in the merged image. (B) Schematic diagram of an in vitro BBB model, which is a co-culture system in which primary rBMECs are plated on a type IV collagen / fibronectin-coated filter membrane in the upper compartment (1), and primary astrocytes are in the lower compartment (2). (C, D) The VHH A-Fc, VHH B-Fc, and VHH Z-Fc fusion proteins are transported across the rBMEC monolayer from the intraluminal (upper) compartment to the extraluminal (lower) compartment. (C) 10 nM of VHH A-Fc, VHH B-Fc, and VHH Z-Fc are incubated in the intraluminal compartment for 24 hr, and their transport to the extraluminal compartment is assessed (referred to as 24hr). The insert containing the VHH-Fc solution is then transferred to another 96-well plate containing fresh transport buffer for another 48-hr transport period to the extraluminal compartment (referred to as +48hr). The kinetics of the experiments described in (D) and (C) are given (72hr transport is the sum of the 24hr and 48hr transport periods). The content of Fc fragments in the extracavitary compartment was quantified using an internal anti-Fc antibody ELISA assay. Absorbance units were converted to femtomolarity per insert (the surface area of ​​a 96-well plate insert is 0.143 cm²). 2Three independent experiments were performed on at least 12 inserts for each conjugate. Data are presented as mean ± SEM ( ). p ≤ 0.001).

[0046] Figure 10 Distribution of VHH-Fc fusion protein in WT C57Bl / 6 mice at 2 and 24 hr post-injection (pi). After plasma collection, VHH A-Fc, VHH A-Fc-Agly, and VHH Z-Fc fusions were injected into the tail vein at a dose of 5 mg / kg, and mice were perfused with saline at 2 or 24 hr pi. Intermediate plasma samples were also collected using retroorbital sampling at 15 min and 6 hr pi. The brain was processed to separate the brain parenchyma from the capillaries. The amount of VHH-Fc in each tissue was assessed using an internal anti-Fc antibody ELISA assay. Data are presented as mean ± SEM of VHH-Fc concentrations in plasma (A), parenchyma (B), and microvessels (C), or mean ± SEM of parenchyma to plasma ratio (D) and microvessel to plasma ratio (E). (4 < n < 12 per group at each time point;) p ≤ 0.05, p ≤ 0.01, p ≤ 0.001).

[0047] Figure 11 Epigenetic K in VHH A1 to A9 on CHO cell lines stably expressing hTfR and mTfR d Assay. (A) CHO-hTfR-EGFP and CHO-mTfR-EGFP cells were incubated with various concentrations of VHH at 4°C for 1 hr and detected with mouse anti-6His antibody (1 / 1000) and Alexa647-conjugated anti-mouse secondary antibody (1 / 400). Measurements were performed using flow cytometry. The fluorescence intensity ratio at each spot was normalized to the corresponding EGFP signal (receptor expression) and arbitrary units were obtained. Data are presented as mean ± SEM of three independent experiments. (B) Characterization of VHH: molecular weight (Da); theoretical pI; epigenetic K on human TfR. d (nM); Epigenetic K on mouse TfR d (nM). Data are presented as mean ± SEM from 3 independent experiments. NB: no binding, LB: low binding.

[0048] Figure 12 Epigenetic K in VHH A10 to A19 on CHO cell lines stably expressing hTfR and mTfR dAssay. (A) CHO-hTfR-EGFP and CHO-mTfR-EGFP cells were incubated with various concentrations of VHH at 4°C for 1 hr and detected with mouse anti-6His antibody (1 / 1000) and Alexa647-conjugated anti-mouse secondary antibody (1 / 400). Measurements were performed using flow cytometry. The fluorescence intensity ratio at each spot was normalized to the corresponding EGFP signal (receptor expression) and arbitrary units were obtained. Data are presented as mean ± SEM of three independent experiments. (B) Characterization of VHH: molecular weight (Da); theoretical pI; epigenetic K on human TfR. d (nM); Epigenetic K on mouse TfR d (nM). Data are presented as the mean ± SEM of 3 independent experiments. NB: Not bound.

[0049] Figure 13 Epigenetic K in CHO cell lines expressing hTfR and mTfR by the 13C3-HC-VHH fusion d Assays. (A) CHO-hTfR-EGFP and CHO-mTfR-EGFP cells were incubated with various concentrations of 13C3 fusions at 4°C for 1 hr and detected with Alexa647-conjugated anti-mouse antibody (1 / 400). Measurements were performed using flow cytometry. The fluorescence intensity ratio at each spot was normalized to the corresponding EGFP signal (receptor expression) and arbitrary units were derived. Data are presented as mean ± SEM of three independent experiments. (B) Characterization of the 13C3 fusions: molecular weight (Da); epigenetic K on human TfR. d (nM); Epigenetic K on mouse TfR d (nM). Data are presented as mean ± SEM from 3 independent experiments. NB: no binding, LB: low binding.

[0050] Figure 14Distribution of 13C3 monoclonal antibody and 13C3-HC-VHH fusion in WT C57Bl / 6 mice at 2 and 6 hr post-injection (pi). 13C3, 13C3-HC-VHH A, and 13C3-HC-VHH A1 were injected into the tail vein at a dose of 35 nmol / kg and mice were perfused with saline at 2 or 6 hr pi. The brain was processed to separate the brain parenchyma from the capillaries. The amounts of 13C3 and 13C3-HC-VHH A / A1 in each tissue / compartment were assessed using a qualified Meso Scale Discovery (MSD) direct coating (Abeta) immunoassay (%CV < 20%, recovery ± 30%). Data are presented as mean ± SEM concentrations of 13C3 and 13C3-HC-VHH A / A1 in the whole brain (A) and parenchyma (B) (1 < n < 4 per group per time point). p ≤ 0.05, p ≤ 0.01, p ≤ 0.001).

[0051] Figure 15 In vitro gene silencing activity of VHH-siGFPst1 bioconjugates. (A) VHH A-siGFPst1 and VHHB-siGFPst1 bioconjugates binding to hTfR. CHO-hTfR-EGFP cells were incubated with various concentrations of the specified compounds at 4°C for 1 hr. VHH was detected using mouse anti-6His primary antibody (1 / 1000) and Alexa647-conjugated anti-mouse secondary antibody (1 / 400). Measurements of cell surface signals associated with VHH were performed using flow cytometry. Results are expressed as the ratio of Alexa647-related fluorescence intensity of the test compound to background fluorescence intensity. (B) VHH A-siGFPst1 bioconjugates showed gene silencing efficiency. CHO-hTfR-EGFP cells were transfected with 25 nM of the specified compound for 72 h at 37°C using Dharmafect 1 (Dharmacon). The total fluorescence associated with EGFP protein was then quantified using flow cytometry and rationalized to the fluorescence of untreated (control) cells (set to 100%). p < 0.001. (C) The VHH A-siGFPst1 bioconjugate exhibited intrinsic gene silencing activity in the picomolar range after direct delivery to the cytoplasm. CHO-hTfR-EGFP cells were transfected with various concentrations of the VHH A-siGFPst1 bioconjugate for 120 hr at 37 °C using Dharmafect 1 (Dharmacon). Total fluorescence associated with EGFP protein was then quantified by flow cytometry and rationalized to the fluorescence of untreated (control) cells (set to 100%). Data were fitted using nonlinear regression (solid line) with GraphPad Prism® software to estimate IC50 (the concentration that allows for a 50% reduction in GFP protein levels) and maximum effect (bottom plateau). (D) The VHH A-siGFPst1 bioconjugate induced specific and efficient TfR-mediated gene silencing. CHO-hTfR-EGFP cells were incubated with 1 µM of the specified compound at 37 °C for 120 hr. The data is processed and analyzed as described in (B). p < 0.001 compared to untreated cells. (E) The hTfR-mediated binding and uptake of the VHH A-siGFPst1 bioconjugate allows for cytoplasmic delivery and subsequent gene silencing at nanomolar concentrations. CHO-hTfR-EGFP cells were incubated with various concentrations of the VHH A-siGFPst1 bioconjugate at 37°C for 120 hr. Total fluorescence associated with EGFP protein was then quantified by flow cytometry and rationalized to the fluorescence of untreated (control) cells (set to 100%). Data were processed and analyzed as described in (C). (F) The gene silencing effect of the VHH A-siGFPst1 bioconjugate was inhibited by co-incubation with excess free TfR-binding VHH A and B, but not by co-incubation with unrelated VHH Z. CHO-hTfR-EGFP cells were incubated at 37°C with 30 nM VHH A-siGFPst1 alone or in the presence of 100-fold excess of free VHH A, B, or Z for 120 hr. Data were processed and analyzed as described in (C). (G) Exposure to the VHH A-siGFPst1 bioconjugate during a short 6-hour pulse was sufficient to induce efficient gene silencing. CHO-hTfR-EGFP cells were incubated with various concentrations of VHH A-siGFPst1 bioconjugate during a short 6-hour pulse, followed by chasing in ligand-free medium for up to 120 hr. Data were processed and analyzed as described in (B). (H) The gene silencing effect of the VHH B-siGFPst1 bioconjugate was similar to that observed for VHH A-siGFPst1. CHO-hTfR-EGFP cells were incubated with 30 nM of VHH A-siGFPst1 or VHH B-siGFPst1 (based on the saturation concentration of IC50 obtained using VHH A-siGFPst1) at 37°C for 120 hr. Data were processed and analyzed as described in (C).

[0052] Figure 16PET imaging of VHH A-68Ga bioconjugates in a subcutaneous mouse model of glioblastoma. (A) VHH A-NODAGA and VHH A-68Ga bioconjugates bind hTfR as efficiently as unconjugated VHH A compounds. CHO-hTfR-EGFP cells were incubated at 4°C for 1 hr with various concentrations of the designated compounds. VHH was detected using mouse anti-6His primary antibody (1 / 1000) and Alexa647-conjugated anti-mouse secondary antibody (1 / 400). Cell surface signals associated with VHH were measured using flow cytometry. Results are expressed as the ratio of Alexa647-related fluorescence intensity of the test compound to background fluorescence intensity. (B) PET imaging of mice administered VHH A-68Ga on day 28 after implantation of U87-MG cells (2 hr post-injection). Glioblastomas are indicated by circles in the sagittal view.

[0053] Figure 17 Competition assay between VHH and phage-VHH. CHO-hTfR-EGFP cells were incubated at 4°C with gradually increasing concentrations of VHH (competitor) for 1 hour. Phage-VHH (tracer) at EC80 concentration was then added and incubated for another 1 hour. Phage-VHH was detected using anti-M13-A647 antibody (1 / 200). Fluorescence levels were assessed using flow cytometry. The fluorescence intensity ratio at each spot was normalized to the corresponding EGFP signal (receptor expression) and assigned to arbitrary units. Data are presented as mean ± SEM of independent experiments. (A) Competition between VHH and phage-H1 identified after phage display selection on rhTfR. (B) Competition between VHH and phage-C5 identified after phage display selection on rhTfR. (C) Competition between VHH and phage-B6 identified after phage display selection on the apical domain of TfR. (D) Competition between VHH and phage-C5 identified after phage display selection on the apical domain of TfR.

[0054] Figure 18: Competition assay between VHH and Tf. (A) CHO-hTfR-EGFP cells were incubated at 4°C with gradually increasing concentrations of Tf (competitor) for 1 hr. Then, VHH (tracer) at EC80 concentration or Tf-A647 as a positive control was added, and the cells were incubated for another 1 hr. VHH was detected using mouse anti-6His antibody (1 / 1000) and Alexa647-conjugated anti-mouse secondary antibody (1 / 400). Fluorescence levels were assessed using flow cytometry. The fluorescence intensity ratio at each spot was normalized to the corresponding EGFP signal (receptor expression) and arbitrary units were obtained. The left Y-axis corresponds to VHH binding, and the right Y-axis corresponds to Tf-A647 binding. Data are presented as mean ± SEM of independent experiments. (B) CHO-hTfR-EGFP cells were incubated at 4°C with gradually increasing concentrations of VHH (competitor) or Tf as a positive control for 1 hr. Then, Tf-A647 (tracer) at a concentration of EC80 was added, and the mixture was incubated for another 1 hour. Fluorescence levels were assessed using flow cytometry. The fluorescence intensity ratio at each spot was normalized to the corresponding EGFP signal (receptor expression), and arbitrary units were obtained. Data are presented as mean ± SEM of independent experiments.

[0055] Figure 19 In WT C57Bl / 6 mice, the binding potential and in vivo distribution of the 13C3 monoclonal antibody and the 13C3-HC-VHH homodimer fusion complex with mTfR1 were assessed at 6 and 18 hours post-injection (pi). The binding of 13C3, 13C3-HC-C5h20, 13C3-HC-C5h9, 13C3-HC-C5h16, and 13C3-HC-C5h24 to mTfR (Bmax, Kmax, Kmax) were evaluated in a cell-based assay (A). d app Combination potential: Normalized Bmax / K d appRatio x 100). 35 nanomoles / kg of 13C3 and 13C3-HC-C5h20 / C5h9 / C5h16 / C5h24 were injected into the tail vein, and mice were perfused with saline after injection. The brain was processed to separate the brain parenchyma and extracellular fluid (ECF) from the capillaries. The amounts of 13C3 and 13C3-HC-C5h20 / C5h9 / C5h16 / C5h24 in each tissue / compartment were assessed using a qualified Meso Scale Discovery (MSD) direct coating (Abeta) immunoassay (%CV < 20%, recovery ± 30%). Data were presented as mean ± SEM concentrations of 13C3 and 13C3-HC-C5h20 / C5h9 / C5h16 / C5h24 in plasma (B), whole brain (C), parenchyma (E), and ECF (F), and as brain / plasma concentration percentages (D) (n=4 per group per time point). p ≤ 0.05, p ≤ 0.01, p ≤ 0.001).

[0056] Figure 20 In WT C57Bl / 6 mice, at 6, 18, and 42 hours post-injection (pi) (VHH) 1k -hFc LALA The binding potential and in vivo distribution of the heterodimer fusion complex with mTfR1 were assessed. In a cell-based assay (A), the (C5) group was evaluated. neg ) 1k -hFc LALA (C5) h18 ) 1k -hFc LALA and (C5) h19 ) 1k -hFc LALA Binding potential with mTfR: Normalized Bmax / K d app Ratio x100. (C5) neg ) 1k -hFc LALA (C5) h18 ) 1k -hFc LALA and (C5) h19 ) 1k -hFc LALA Mice were administered a subcutaneous injection of 70 nanomoles / kg, followed by saline perfusion. The brain was treated to separate the brain parenchyma and extracellular fluid (ECF) from the capillaries. The concentration of C5+ in each tissue / compartment was assessed using an anti-hFc antibody ELISA assay. neg ) 1k-hFc LALA (C5) h18 ) 1k -hFc LALA and (C5) h19 ) 1k -hFc LALA The amount (%CV < 20%, recovery ± 30%) was measured. Data are presented as hFc in plasma (B), whole brain (C), parenchyma (D), ECF (E), and liver (F). LALA and (C5) h18 / h19 ) 1k -hFc LALA Average concentration ± SEM (n=4 for each time point in each group); p ≤ 0.05, p ≤ 0.01, p ≤ 0.001).

[0057] Figure 21 In C57Bl / 6 or B-hTfR mice, the VHH-neurotensin (NT8-13) fusion was induced by hypothermia following a single IV injection of 500 nmol / kg. The effects of VHH-neurotensin (NT8-13) fusion on C57Bl / 6 or B-hTfR mice were evaluated in a cell-based assay. neg -NT(8-13), B8 h1 -NT(8-13), B8 V1 -NT(8-13), B8 v31 -NT(8-13), C5 h18 -NT(8-13), C5 v30 The apparent affinity of C12b-NT(8-13) and C12b-NT(8-13) for mTfR(A) or hTfR(B). The C5... neg -NT(8-13), B8 h1 -NT(8-13), B8 V1 -NT(8-13), B8 v31 -NT(8-13), C5 h18 -NT(8-13), C5 v30 -NT(8-13) and C12b-NT(8-13) were administered intravenously at 500 nanomoles / kg to C57Bl / 6 mice (C) or B-hTfR mice (D). Mice body temperature was assessed every 15 min post-injection until 90 min, then every 30 min until 180 min (n=4 to 3 per time point per group). Results are expressed as mean + / - SEM. The VHH-NT(8-13) fusion variant has a p ≤ 0.05 compared to the C5neg-NT(8-13) fusion variant. p ≤ 0.01, p ≤ 0.001. The area under the curve (AUC) was determined by comparison with the mouse temperature baseline (≈ 38°C). Differences between groups were tested using one-way ANOVA and Tukey's post-hoc test (Graph Pad 9.4.1).

[0058] Figure 22 Heterodimer (VHH) in B-hTfR mice 1k The -Fc-NT(8-13) fusion complex exhibited binding affinity for hTfR, in vivo brain uptake, and hypothermia induction following a single IV injection of 20 nanomolars / kg. (C5neg) was evaluated in a cell-based assay (A). 1k -hFc LALA -NT(8-13), (C5) h18 ) 1k -hFc LALA -NT(8-13), (C10a) 1k -hFc LALA -NT(8-13) and (H1) 1k -hFc LALA -NT(8-13) apparent affinity for hTfR. (C5neg) 1k -hFc LALA -NT(8-13), (C5) h18 ) 1k -hFc LALA -NT(8-13), (C10a) 1k -hFc LALA -NT(8-13) and (H1) 1k -hFc LALA -NT(8-13) was administered intravenously to B-hTfR mice at 20 nanomoles / kg. 24 hours post-injection, blood was recovered via cardiac puncture and the mice were perfused with saline solution. The brain was processed to separate the brain parenchyma and extracellular fluid (ECF) from the capillaries. The volume of blood in each tissue / compartment (VHH) was assessed using an anti-hFc antibody ELISA assay. 1k -hFc LALA -NT (8-13) levels. Data were presented as plasma (B), whole brain (C), ECF (D), and parenchyma (E) in the VHH. 1k -hFc LALA -NT(8-13) concentration mean ± SEM (n=4 for each time point in each group); p ≤ 0.05, p ≤ 0.01, p ≤ 0.001). This is for the study of (C5h18). 1k -hFcLALA -NT(8-13) has the ability to rapidly cross the BBB, connecting the fusion with the negative control fusion (C5neg). 1k -hFc LALA -NT(8-13) were injected together into B-hTfR mice at a dose of 20 nanomoles / kg. The body temperature of the mice was assessed every 15 min until 90 min after injection, and then every 30 min until 240 min after injection (n=4 per group at each time point). The results are expressed as mean + / - SEM. (C5h18) 1k -hFc LALA -NT(8-13) fusion compared to (C5neg) 1k -hFc LALA -NT(8-13) p ≤ 0.05, p≤ 0.01, p ≤ 0.001 (F).

[0059] Figure 23 : Coupling Strategy. An example of a coupling strategy for producing VHH-oligonucleotide (A) or heterodimeric VHH-hFc-oligonucleotide (B) conjugates with stable linkers. This general coupling strategy involves a pooling synthesis having the following parallel modifications: (i) modification of VHH (A) or heterodimeric VHH-hFc (B) to introduce, for example, an azido linker with site specificity; and (ii) modification of the oligonucleotide to introduce, for example, a constrained alkyne complementary to the azido functional group. In the final step, the functionalized azido-VHH (A) or VHH-hFc-azido (B) and the alkyne-oligonucleotide precursor are preferably linked together using a copper-free click reaction to produce VHH-oligonucleotide or VHH-hMc-oligonucleotide conjugates with stable linkers.

[0060] Figure 24 : Overall structure of VHH-oligonucleotides. (A) Overall structure of VHH-oligonucleotide conjugates 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-extending entity, wherein the oligonucleotide and VHH are linked to the 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).

[0061] Figure 25Epigenetic Ki on cells expressing mouse and human TfR. VHH-oligonucleotide conjugates (C5-siSOD1m-5'VP, C5-hFc-siSOD1m-5'VP, C5-siSOD1h, B8-siSOD1h, or B8-MALAT1-ASO1) and free VHH (C5 or B8) compete with fluorescent reference TfR-binding VHH for concentration-dependent binding and epigenetic binding affinity values ​​(Ki / app) of mouse TfR (A) expressed by mouse Neuro-2A cells or human TfR (B) expressed by MCF-7 cells.

[0062] Figure 26 In vitro silencing activity. Potential for downregulation of TfR-binding VHH-oligonucleotide conjugates on mRNA or long coding RNA lncRNA after free uptake in mouse Neuro-2A or human LN229 cell lines. Figure 26 A shows the results in mouse cells from Neuro-2a cells obtained using TfR-binding VHH-siRNA or VHH-hFc-siRNA conjugates (VHH-siSOD1m-5'VP and VHH-hFc-siSOD1m-5'VP, where VHH is C5) compared to unconjugated siSOD1m-5'VP. SOD1 mRNA levels were downregulated in a concentration-dependent manner. Figure 26 B shows the use of targeted mice. SOD1 Various examples of TfR-binding VHH-siRNA conjugates of mRNA (i.e., VHH-siSOD1m, VHH-thiol-Mal-siSOD1m, or VHH-ΔHis-siSOD1m, where VHH is or contains C5, B8, B8h1, C5V1, C5V13, C5h18, C5h19, or C5V7) were obtained from mouse cells in Neuro-2a cells. SOD1 Downregulation of mRNA levels, and such Figure 25 The figure shows the epigenetic binding affinity (Ki / app) of the test conjugate to TfR evaluated on mouse Neuro-2A cells. Figure 26 C shows the use of targeted mice MALAT-1 VHH- of long non-coding RNA MALAT1 -ASO1 conjugate (VHH- MALAT1 -ASO1, where VHH is obtained from B8) MALAT-1 Downregulation of lncRNA levels. Figure 26 D shows human LN229 cells obtained using TfR-binding VHH-siRNA (VHH-siSOD1h, where VHH is B8). SOD1 mRNA levels were downregulated in a concentration-dependent manner.

[0063] Figure 27 Distribution of TfR in brain microvessels and parenchymal cells. Immunohistochemistry was used to investigate the distribution of TfR in brain microvessels (co-stained with anti-collagen IV antibody) and parenchymal cells, including neurons (co-stained with anti-NeuN antibody).

[0064] Figure 28 CNS knockdown effect of VHH-siRNA conjugate. In wild-type C57Bl / 6 mice, local administration of VHH-siRNA conjugate (where VHH is C5) to the ICV resulted in... SOD1 CNS-specific knockdown of mRNA.

[0065] Figure 29 CNS knockdown effect and siRNA biodistribution. The figure illustrates the biodistribution of siSOD1m-5'VP in the CNS brain and spinal cord, assessed by in situ hybridization, in wild-type C57Bl / 6 mice after local ICV administration of PBS or VHH-siRNA conjugate (where VHH is C5), and in rodents. SOD1 mRNA levels. Figure 29 A illustrates a general sampling and bioanalysis method for the left and right hemispheres and spinal cord. Figure 29 Figures B and 29E show the overall brain (29B, sagittal section) and spinal cord (29E, coronal section) distribution of the siSOD1m-5'VP antisense strand (miRNAScope®, left panel), compared with that in mice. SOD1 Widespread downregulation of mRNA levels is associated with RNAScope® (right figure). Figure 29 CD, 29F, and 29G show magnified views of different regions of the brain (29C, D, G, sagittal section) and spinal cord (29F, coronal section), as well as using neuron-specific... MAP2 , DAT (Dopaminergic neurons) or ChAT (cholinergic interneurons) mRNA probes, astrocyte-specific GJA1 mRNA probes, oligodendrocyte-specific... OLIG2 mRNA probes or microglia-specific TMEM119 Co-staining of mRNA probes.

[0066] Figure 30 Optimization of CNS functional delivery of VHH-siRNA conjugates. In wild-type C57Bl / 6 mice, after repeated IV or SC administration of VHH-siRNA (15 mg / kg siRNA molar equivalent, QDx4) or VHH-hFc-siSOD1m-5'VP (1.5 mg / kg siRNA molar equivalent, Q2Dx3) conjugates, the mice showed improved CNS functional delivery. SOD1CNS-specific knockdown of mRNA.

[0067] Figure 31 CNS knockdown effect and siRNA biodistribution following systemic administration of VHH-hFc-siRNA conjugate. In wild-type C57Bl / 6 mice, after repeated IV administration of PBS at a total dose of 4.5 mg / kg (1.5 mg / kg siRNA molar equivalent, Q2Dx3), the CNS knockdown effect and siRNA biodistribution were assessed using RT-qPCR on the right hemisphere and spinal cord, or in situ hybridization on the left hemisphere and part of the lumbar spinal cord. SOD1 TfR-dependent CNS knockdown of mRNA, and biodistribution of siSOD1m-5'VP assessed using in situ hybridization in the left hemisphere and part of the lumbar spinal cord. Figure 31 C and 31I show the overall brain (31C, sagittal section) and spinal cord (31I, coronal section) distribution of the siSOD1m-5'VP antisense strand (miRNAScope®, left panel), compared with rodents. SOD1 Widespread downregulation of mRNA levels is associated with RNAScope® (right figure). Figure 31 DL shows magnified views of different regions of the brain (31D-H, J, K, sagittal section) and spinal cord (31I, 31L, coronal section), as well as using... MAP2 , DAT Neuron-specific co-staining of dopaminergic neurons or ChAT (cholinergic interneurons) mRNA-specific probes.

[0068] Figure 32 Differential biodistribution. In wild-type C57Bl / 6 mice, differential siSOD1m-5'VP biodistribution in microvessels of the brain and spinal cord compared to that in the parenchyma was observed after repeated IV administration of PBS or TfR-binding VHH-hFc-siSOD1m-5'VP conjugate containing a stable linker or nuclease-sensitive linker (dTdT) between the siRNA 3'SS and VHH-hFc portions.

[0069] Figure 33 CNS knockdown effect of a single subcutaneous administration of the VHH-hFc-siRNA conjugate in mice. Following a single subcutaneous administration of the TfR-binding VHH-hFc-siSOD1m-5'VP conjugate (where VHH is C5), murine knockdown was observed in the CNS tissues and brain regions of wild-type C57Bl / 6 mice. SOD1Dose-dependent downregulation of mRNA levels, estimated ED50 value and maximum downregulation effect (Max KD) Figure 33 AC). The potential of TfR-binding VHH-hFc-siSOD1 conjugates containing different VHH variants (where VHH is B8h1, C5, or C5V13) to mediate knockdown of murine SOD1 mRNA in CNS tissues was compared after a single subcutaneous (SC) administration of an intermediate dose of 1.5 mg / kg (siRNA molar equivalent) in wild-type C57Bl / 6 mice. Figure 33 D).

[0070] Figure 34 The potential of the VHHTfR-NT(8-13) fusion complex at 500 nmol / kg to induce hypothermia in B-hTfR mice. VHHTfR-NT(8-13) was administered intravenously (iv, bolus) to B-hTfR mice at a dose of 500 nmol / kg. Mouse body temperature was measured before injection and every 15 to 60 min post-injection, and then every 30 to 180 min post-injection. Mouse body temperature after VHHTfR-NT(8-13) injection (A). Area under the curve (AUC) relative to basal body temperature (38°C) (B). Mouse body temperature at maximum hypothermia induced by VHHTfR-NT(8-13) (C) and total duration of hypothermia (D). Results are presented as mean + / - SEM. VHHTfR-NT compared to C5neg-NT p < 0.05 p < 0.01, p < 0.001. Differences between groups were tested using GraphPad Prism version 10.1 with one-way ANOVA and Dunnett's post-hoc test.

[0071] Figure 35 The correlation between hypothermia parameters induced by VHHTfR-NT(8-13) in B-hTfR mice and hTfR binding parameters. Hypothermia parameters (AUC, maximum hypothermia, duration of hypothermia) and TfR binding parameters (K... on K off and K D Pearson correlation matrix between (A and B). Positive correlation is shown in blue, and negative correlation in red. p-values ​​of the Pearson correlation matrix. Significant correlations are shown in bold (B). hTfR K D A graph illustrating the correlation between the maximum low-temperature AUC and the low-temperature value. For each VHHTfR-NT, the maximum low-temperature value is indicated by a color gradient (C) from red (no low temperature) to blue (very high low temperature). K is obtained through linear regression. offA graph illustrating the correlation between the maximum low temperature and the maximum low temperature.

[0072] Figure 36 In B-hTfR mice, a heterodimer (C12b-L) exhibits a monovalent TfR binding mode. AST -HC) 1A’ Brain uptake of the -13C3 fusion. A modified mutant monoclonal antibody, 13C3, was introduced into the CH3 heavy chain. A’B’ and (C12b-L AST -HC) 1A’ The overall structure of the -13C3 fusion protein (A) was evaluated using cell-based assays. AST -HC) 1A’ -13C3's apparent affinity for hTfR (B). The 13C3... A’B’ and (C12b-L AST -HC) 1A’ -13C3 was injected into the tail vein at a dose of 70 nanomoles / kg. Blood samples were collected at 2, 6, 18, 24, 30, and 72 hours post-injection, and mice were perfused with saline at 6, 18, or 72 hours post-injection. The brain was processed to separate the brain parenchyma and ECF from the capillaries. 13C3 concentrations in each tissue / compartment were assessed using a qualified MesoScale Discovery (MSD) direct coating (Abeta) immunoassay. A’B’ and (C12b-L AST -HC) 1A’ -13C3 levels (%CV < 20%, recovery ± 30%). Injection dose / ml and percentage of AUC in plasma (C), injection dose / g and percentage of AUC in the whole brain (D, E), and posterior ventricle, parenchyma, ECF, and microvessels (F, G). Data are presented as mean ± SEM, n = 4 to 8 per group per time point. Differences between groups were tested using GraphPad Prism version 10.1 with one-way ANOVA and Dunnett's post-hoc test. 13C3 A’B’ Compared to (C12b-L) AST -HC) 1A’ -13C3 p ≤0.05, p ≤ 0.01, p ≤ 0.001.

[0073] Figure 37The CNS knockdown effect of VHH-siRNA conjugates after topical administration in B-hTfR1 mice. In B-hTfR1 mice, the TfR-binding VHH-siSOD1 conjugate (where VHH is B8) was evaluated after topical ICV administration. h1 Or B8 V32 ) Mediates rodent CNS tissues SOD1 The potential for mRNA knockdown was explored and compared with that of lipophilic C16 (palmitic acid)-siSOD1 conjugates or non-binding C5neg-siSOD1 conjugates.

[0074] Figure 38 The CNS knockdown effect of the heterodimeric VHH-hFc-siRNA conjugate following systemic administration in B-hTfR mice. In B-hTfR1 mice, the TfR-binding VHH-siSOD1 conjugate (where VHH is C5) was evaluated after a single subcutaneous (SC) administration of 4.5 mg / kg (siRNA molar equivalent). V30 B8 V31 B8 V32 C5 h9 C5 V5 Or C5 h18 ) Mediates rodent CNS tissues SOD1 The potential for mRNA knockdown was explored. Surface plasmon resonance was used to assess the binding affinity of conjugates to human TfR for each test.

[0075] Figure 39 The CNS knockdown effect of the human / NHP cross-reactive heterodimer VHH-hFc-siRNA conjugate on B-hTfR mice after systemic administration. In B-hTfR1 mice, the TfR-binding VHH-hFc-siSOD1 conjugate (where VHH is B8) was evaluated after multiple IV boluses (Q2D x3) at 1.5 mg / kg (siRNA molar equivalent). V40 B8 V32 B8 V31 Or B8V 31h5 ) Mediates rodent CNS tissues SOD1 The potential for mRNA knockdown was explored and compared with that of non-binding C5neg-hFc-siSOD1. Surface plasmon resonance was used to assess the binding affinity of the conjugates for human and rhesus / cynomolgus monkey TfR for each assay.

[0076] Figure 40Following systemic administration of the VHH-hFc-siRNA conjugate, siRNA biodistribution and target knockdown were observed in the dorsal root ganglia. In B-hTfR1 mice, 14 days after multiple IV boluses (Q2D x3) of the TfR-binding VHH-hFc-siSOD1 conjugate (where VHH is B8V32), the distribution of the siSOD1m-5'VP antisense strand in the dorsal root ganglia was assessed using in situ hybridization (miRNAScope®, left panel) and in rodents. SOD1 mRNA knockdown (RNAScope®, right image); SOD1 mRNA is shown in red; using neuron-specific... MAP2 mRNA probes are co-stained green. Detailed Implementation

[0077] This invention provides novel TfR binders that can be used to transport molecules, such as therapeutic agents, imaging agents, or diagnostic agents, across the BBB and the plasma membrane of any TfR-expressing cell in the central nervous system (CNS) or peripheral nervous system (PNS) (collectively, the nervous system). More specifically, this invention discloses modified VHH molecules that bind TfR, conjugates containing such modified VHH molecules, and their use for the prevention or treatment of CNS or PNS disorders.

[0078] Most therapeutic oligonucleotides on the market or in preclinical and clinical development for CNS disorders contain single-stranded ASOs with high phosphate-thioester (PS) content, thus providing relative lipophilicity (Goto et al., 2022). Strategies proposed to allow functional delivery of siRNA to CNS tissues via topical application are also few. These strategies include using palmitic acid (C16) as a lipophilic handle to attach to the siRNA (Brown et al., 2022), or using a bivalent siRNA scaffold containing an extended 3'AS single-stranded overhang with high PS content (Alterman et al., 2019). Therefore, all these strategies rely on relative lipophilicity to support nonspecific binding to the cell membrane and increase tissue retention in the CNS after topical application, ultimately allowing for significant cellular uptake, mRNA target binding, and induction of its pharmacological effects. In contrast, the VHH-containing conjugates of the present invention advantageously rely on targeting TfR to induce active receptor-mediated uptake and intracellular delivery of the therapeutic payload.

[0079] Interestingly, the inventors have demonstrated that TfR-binding VHH-siRNA conjugates, upon local CNS administration, have the potential for broad distribution and efficient functional delivery in CNS tissues, with little or no effect in excretory organs. Maximum knockdown effect and ED50 have been found to exceed those of lipophilic palmitic acid (C16)-siRNA conjugates. Therefore, the VHH of this invention has the potential to address CNS disorders at a lower effective dose than delivery strategies that rely on nonspecific tissue retention.

[0080] The inventors also showed that, compared with the non-carrier-based control 13C3 A’B Compared to antibodies, the TfR-binding conjugates of the present invention, such as (C12b-L... AST -HC) 1A’ -13C3 showed a significant brain uptake advantage (three-fold) after systemic administration, thus demonstrating that the TfR-targeting VHH of the present invention can be used to effectively deliver therapeutic antibodies to the brain.

[0081] The inventors have also demonstrated that the VHH-siRNA and VHH-ASO conjugates of the present invention induce concentration-dependent inhibition of the binding and uptake of the reference compound in cells expressing mouse or human TfR, with an apparent K i Values ​​and apparent values ​​of free VHH (e.g., C5 or B8) K i The values ​​were similar, thus demonstrating the efficient binding of VHH-siRNA and VHH-ASO conjugates to TfR.

[0082] Furthermore, the inventors have demonstrated that the functional delivery of oligonucleotides is observed not only in the central nervous system (CNS) but also in the peripheral nervous system (PNS), including neurons in the dorsal root ganglion (DRG). Therefore, the VHH of the present invention also has the potential to address PNS disorders.

[0083] Surprisingly, the inventors have demonstrated for the first time that, following local CNS administration, such as intracerebral, intraventricular (ICV), or intrathecal (IT) administration, the conjugates of the present invention can be functionally delivered to muscle tissue, even at doses similar to those used for muscle delivery via systemic administration. Therefore, the conjugates according to the present invention can address both muscular 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 present invention can be used to simultaneously target the neuronal components (particularly nervous system cells) and muscle components (particularly muscle cells) of 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 present invention are functionally delivered to both nervous system and muscle tissues after local CNS (IT or ICV) administration, and can simultaneously bind to TfRs on the surface of both nervous system cells and muscle cells after local CNS administration. Therefore, the conjugates according to the invention can be used not only by systemic administration but also by local CNS (IT or ICV) administration to address and treat neuromuscular disorders selected from spinal muscular atrophy, amyotrophic lateral sclerosis, peroneal muscular atrophy, multiple sclerosis and Huntington's disease.

[0084] Finally, the inventors have demonstrated that significant KD can be obtained in CNS tissues following systemic (IV or SC) administration of the TfR-binding VHH-hFc-siRNA conjugate in hTfR-expressing mice. Furthermore, several VHH variants and conjugates exhibit 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.

[0085] TfR is involved in the incorporation of iron transported by its transferrin ligands and in the regulation of cell growth (Neckers and Trepel, 1986). Two types of transferrin receptors exist: the TfR1 receptor 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 the 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 to a transferrin molecule. 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).

[0086] 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 and disease stage or prognosis. Therefore, targeting TfR with drugs may be suitable for cancer treatment, as well as for the plasma membrane of any TfR-expressing cell that crosses the BBB and the central nervous system (CNS) or peripheral nervous system (PNS). 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. The inventors also demonstrated that these VHH molecules retain TfR binding capacity, migrate across in vitro BBB models, and exhibit brain-targeting properties in vivo when fused with human IgG1 Fc regions or drugs (e.g., antibodies, siRNA) or imaging agents. They further demonstrated that these VHH molecules retain in vivo TfR binding capacity and efficient delivery when fused with oligonucleotides such as siRNA or ASO or NODAGA scaffolds. The VHH molecules exhibit appropriate levels of affinity and specificity upon TfR binding for suitable endocytosis.

[0087] Therefore, the present invention provides novel TfR-binding molecules, which represent valuable agents for drug targeting. Thus, one object of the present invention relates to VHH molecules, wherein said VHH molecules bind to human, non-human primate, and / or rodent (e.g., rat or mouse) TfR. Preferably, said VHH can cross the plasma membrane of human BBB and any TfR-expressing cells in the central nervous system (CNS) or peripheral nervous system (PNS), or bind to TfR-expressing tissues such as cancer. The present invention also relates to conjugates comprising such VHHs, their preparation, compositions comprising them, and their uses.

[0088] definition

[0089] 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. As used herein, the term "treatment" refers to any action aimed at improving a patient's health, such as treating, stopping, preventing, and blocking disease. The nervous system consists of the central nervous system (CNS) and the peripheral nervous system (PNS). The CNS comprises the brain, spinal cord, and retina, which is an extension of the CNS. The PNS comprises nerves (cranial and spinal nerves) and ganglia (cranial ganglia, dorsal root ganglia or DRGs, autonomic ganglia) where the cell bodies of sensory neurons reside. The PNS is located outside the brain and spinal cord and connects the CNS to the limbs and organs. The disease being treated can be any disease, particularly any CNS disease affecting the structure or function of the brain, spinal cord, or retina. The CNS diseases mentioned can be Alzheimer's disease, Parkinson's disease, stroke, Creutzfeldt-Jakob disease, dementia, multiple sclerosis, spinal muscular atrophy (SMA), amyotrophic lateral sclerosis, epilepsy, migraine, encephalitis, lysosomal storage disorders of the brain, CNS pain, or CNS tumors such as brain tumors such as glioblastoma. The treated diseases can also be any PNS disease, such as peripheral neuropathy caused by traumatic injury, infection, metabolic problems, genetic causes and toxin exposure, autoimmune diseases, inflammation, or DRG cell disorders. More specifically, PNS diseases can be selected from peripheral neuropathy, such as diabetic neuropathy, cancer and chemotherapy-induced peripheral neuropathy (CIPN), HIV-induced neuropathy, leprosy (HD), infection-induced neuropathy such as Lyme disease, traumatic nerve injury (such as carpal tunnel syndrome and sciatica); hereditary sensorimotor peripheral neuropathy, such as peroneal muscular atrophy (CMT), Friedreich ataxia (FA), giant axonal neuropathy (GAN); autoimmune and inflammatory induced peripheral neuropathy, such as Guillain-Barré syndrome, lupus, rheumatoid arthritis, chronic inflammatory demyelinating polyneuropathy (CIDP), multifocal motor neuropathy, Sjögren's syndrome; DRG cell disorders, such as sensory polyneuropathy (e.g., dorsal root ganglion lesions or sensory neuron diseases), sensory polygangliopathy, sensory polyradiculopathy, central sensory axonopathy, sensory polyradiculopathy; any complication of PNS disease, such as neuropathic pain; and PNS cancer. It refers to both curative and / or preventative treatment of the disease. In the context of cancer, this specifically includes alleviating symptoms, reducing inflammation, inhibiting cancer cell growth, and / or reducing tumor size.For example, in the case of cancer, the response to treatment includes relief of cachexia, increased survival time, prolonged time before tumor progression, reduced tumor mass, reduced 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 the criteria established by the American Institute for Cancer Research 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).

[0090] The term "nervous system cells" as used herein refers to any cell present in the nervous system. Typically, these nervous system cells are CNS cells, such as any neurons, glial cells (including astrocytes, oligodendrocytes, ependymal cells, or microglia), choroid plexus cells, pericytes, endothelial cells, or fibroblasts in the brain, spinal cord, or retina; or PNS cells, such as any neurons, glial cells (including Schwann cells), satellite cells, macrophages, endothelial cells, or fibroblasts, such as nerve cells in cranial ganglia, dorsal root ganglia, or autonomic ganglia; or cancer cells in the CNS or PNS system.

[0091] 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 state. For example, in treating cancers, lesions, or disorders of the nervous system, such as the brain or other tissues, or pathologies, lesions, or disorders of the CNS or PNS, 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 the disease or disorder, but will provide treatment for it, 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.

[0092] It should be understood that a person's "therapeutic effective dose" will specifically depend on a variety of factors, including the activity / efficacy of the active substance, its timing of administration, route of administration, toxicity, 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. The "VHH molecule" used herein corresponds to the variable region of a naturally occurring, heavy-chain-only Camelidae antibody that does not contain a light chain. VHHs have a very small molecular weight, approximately 12-15 kDa. They contain a single-chain molecule that can bind its homologous antigen using a single structural domain. The antigen-binding surface of a VHH is typically more convex (or protruding) than that of a conventional antibody, which is typically 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 which 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 traditional human antibody VH, several amino acids are replaced in the FR2 region and complementarity-determining region (CDR) of VHH. For example, highly conserved hydrophobic amino acids in the FR2 region (such as Val42, Gly49, Leu50 and / or Trp52) are usually replaced by hydrophilic amino acids (Phe42, Glu49, Arg50, Gly52), making the overall structure more hydrophilic and contributing to high stability, solubility and anti-aggregation properties.

[0093] The VHH molecule according to the present invention is a polypeptide containing an antigen-binding domain (or consisting of or substantially consisting of a heavy chain antibody (HcAb) only).

[0094] As used herein, the term “and / or” should be regarded 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 regarded as specifically disclosing (i) A, (ii) B, and (iii) each of A and B as if each were described separately.

[0095] 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.

[0096] VHH molecules

[0097] 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.

[0098] The VHH molecule of the present invention typically comprises or is composed of the following formula: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FRn represents the framework region and CDRn represents the complementarity-determining region.

[0099] 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 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.

[0100] The "identity %" between amino acid (or nucleic acid) sequences can be determined using techniques known per se in the art. Typically, the identity % between two nucleic acid or amino acid sequences is determined using a computer program such as 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 % 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 or 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).

[0101] 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, 671-674, 710 or 711.

[0102] 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.

[0103] 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.

[0104] 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 or 713-715, 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.

[0105] 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.

[0106] 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, wherein the CDR3 domain comprises a subset 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 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.

[0107] In another specific embodiment, the VHH molecule of the present invention comprises: - CDR1, said CDR1 comprising an amino acid sequence selected from SEQ ID NO: 1, 5, 9, 17, 125, 175, 179, 182, 184, 186, 190, 194, 198, 201, 205, 392, 410, 413, 426, 434, 437, 607, 610, 671-674, 710, and 711, 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 its entire length; and - CDR2, wherein CDR2 comprises an amino acid sequence selected from SEQ ID NO: 2, 6, 73, 113, 115, 128, 160, 162, 164, 166, 169, 171, 176, 187, 191, 195, 199, 202, 206, 416, 419, 431, 608, 611, and 712 or a variant thereof, the 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 the sequences thereof throughout its length; and - CDR3, wherein CDR3 comprises an amino acid sequence selected from SEQ ID NO: 3, 7, 11, 117, 119, 121, 123, 177, 180, 188, 192, 196, 200, 203, 207, 452, 455, 609, 612, 713-715, and 741-744 or a variant thereof, the 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 the sequences in its entire length. The VHH has TfR binding capability. The conditions are: - When CDR1 contains SEQ ID NO: 1, 5, 9 or 17, then CDR2 is not selected from SEQ ID NO: 2, 6 and 73, and CDR3 is not selected from SEQ ID NO: 3, 7 and 11; - When CDR2 contains SEQ ID NO: 2, 6 or 73, then CDR1 is not selected from SEQ ID NO: 1, 5, 9 and 17, and CDR3 is not selected from SEQ ID NO: 3, 7 and 11; - When CDR3 contains SEQ ID NO: 3, 7 or 11, then CDR1 is not selected from SEQ ID NO: 1, 5, 9 and 17, and CDR2 is not selected from SEQ ID NO: 2, 6 and 73.

[0108] 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.

[0109] In another preferred embodiment, the VHH molecule of the present invention comprises: - CDR1, wherein CDR1 comprises an amino acid sequence selected from the following: SEQ ID NO: 1, 5, 9, 17, 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 - CDR2, wherein CDR2 comprises an amino acid sequence selected from the following: SEQ ID NO: 2, 6, 73, 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, wherein CDR3 comprises an amino acid sequence selected from the following: SEQ ID NO: 3, 7, 11, 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. The conditions are: - When CDR1 contains SEQ ID NO: 1, 5, 9 or 17, then CDR2 is not selected from SEQ ID NO: 2, 6 and 73, and CDR3 is not selected from SEQ ID NO: 3, 7 and 11; - When CDR2 contains SEQ ID NO: 2, 6 or 73, then CDR1 is not selected from SEQ ID NO: 1, 5, 9 and 17, and CDR3 is not selected from SEQ ID NO: 3, 7 and 11; - When CDR3 contains SEQ ID NO: 3, 7 or 11, then CDR1 is not selected from SEQ ID NO: 1, 5, 9 and 17, and CDR2 is not selected from SEQ ID NO: 2, 6 and 73.

[0110] 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; - 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.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] The preferred VHH molecule of the present invention comprises the FR domain as defined below.

[0118] 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.

[0119] In one specific implementation, E in position 1 can be replaced by Q.

[0120] In one specific implementation, V in position 5 can be replaced by Q.

[0121] In one specific implementation, E in position 6 can be replaced by Q.

[0122] In one specific implementation, G in position 10 can be replaced with K or A.

[0123] In one specific implementation, L in position 11 can be replaced by V or E.

[0124] In one specific implementation, P in position 14 can be replaced by A.

[0125] In one specific implementation, G in position 16 can be replaced by D.

[0126] In another specific implementation, K in position 19 can be replaced by R.

[0127] In one specific implementation, A in position 23 can be replaced by V or T.

[0128] In another specific embodiment, S in position 25 can be replaced by D.

[0129] 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.

[0130] 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.

[0131] 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 VQLVESGGRLVQAGGSRLLCCTAS (SEQ ID NO: 620).

[0132] 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.

[0133] In one specific implementation, M in position 1 can be replaced by I or V.

[0134] In one specific implementation, R in position 2 can be replaced by G, H, or S.

[0135] In one specific implementation, Y in position 4 can be replaced by F or V.

[0136] In one specific implementation, R in position 5 can be replaced by G.

[0137] In one specific implementation, Q in position 6 can be replaced by R or E.

[0138] In one specific implementation, A in position 7 can be replaced by R.

[0139] In one specific implementation, G in position 9 can be replaced by I or E.

[0140] In one specific implementation, Q at position 11 can be replaced by E, G, I, or D.

[0141] In one specific implementation, R in position 12 can be replaced by L.

[0142] In one specific implementation, E in position 13 can be replaced by N or H.

[0143] In one specific implementation, L in position 14 can be replaced by F, W, S, or Q.

[0144] In one specific implementation, V in position 15 can be replaced by Q or I.

[0145] In one specific implementation, A in position 16 can be replaced by M or S.

[0146] In one specific implementation, T in position 17 can be replaced by G or S.

[0147] 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.

[0148] 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).

[0149] 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.

[0150] 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).

[0151] 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.

[0152] In one specific implementation, Y in position 1 can be replaced by N.

[0153] In one specific implementation, Y in position 2 can be replaced by A.

[0154] In one specific implementation, A in position 3 can be replaced by P or I.

[0155] In one specific implementation, D in position 4 can be replaced by S or N.

[0156] In one specific implementation, A in position 17 can be replaced by S.

[0157] In one specific implementation, K in position 29 can be replaced by R.

[0158] In one specific implementation, P in position 30 can be replaced with A.

[0159] 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.

[0160] 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); NYPDSMKGRFTISRDNAKNTVYLQINSLKSEDTAVYYC (SEQ ID NO: 49); or a variant thereof.

[0161] 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).

[0162] 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. 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. A specific illustrative example of the FR4 sequence is SEQ ID NO: 50.

[0163] 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).

[0164] Specific examples of TfR-binding VHH molecules of the present invention are those comprising molecules selected from SEQ ID NO: 4 (VHH A), 8 (VHHB), 12 (VHH C), 16 (VHH D), 18 (VHH A1), 20 (VHH A2), 22 (VHH A3), 24 (VHH A4), 26 (VHHA5), 28 (VHH A6), 30 (VHH A7), 32 (VHH A8), 34 (VHH A9), 68 (VHH A10), 70 (VHH A11), 72 (VHH A12), 74 (VHH A13), 76 (VHH A14), 78 (VHH A15), 80 (VHH A16), 82 (VHH A17), 84 (VHHA18), 86 (VHH A19), 87 (VHH A20), 88 (VHH A19), 80 (VHH A16), 82 (VHH A17), 84 (VHHA18), 86 (VHH A19), 87 (VHH A20), 88 (VHH A19), 80 (VHH A10 ...1), 82 (VHH A12), 84 (VHHA18), 86 (VHH A19), 87 (VHH A20), 88 (VHH A19), 80 (VHH A11), 82 (VHH A12), A21), 89 (VHH A22), 90 (VHH A23), 91 (VHH A24) and 92 (VHH A25), 114, 116, 118, 120, 122, 124, 126, 127, 129-149, 152-159, 161, 163, 165, 167, 168, 170, 172-174, 178, 181, 183, 185, 189, 193, 197, 204, 208, 393, 411, 414, 417, 420, 422, 42 4, 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).

[0165] Other 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 (as detailed in Table 1 below when x is 0). In one particular embodiment, the VHH of the present invention is humanized.

[0166] For humanization, one or more FR and / or CDR domains can be modified by substitution of one or more amino acids. In this regard, in a particular embodiment, the VHH is humanized by selected modification (e.g., amino acid substitution) of the FR1 domain. The FR1 domain typically consists of a sequence of 25 amino acid residues. Typical humanization sites in FR1 are 19R or 23A or both (e.g., by reference to any of SEQ ID NOs: 35-39, 331, 332, 400 or any variant thereof as described 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.

[0167] 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.

[0168] 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 described herein).

[0169] 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.

[0170] 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.

[0171] Another specific example of the humanized FR2 includes SEQ ID NO: 343, in which Q11 and R12 are modified to 11G and 12L, respectively.

[0172] 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.

[0173] Another specific instance of the humanized FR2 includes SEQ ID NO: 345, where Q11 is modified to 11G.

[0174] Another specific instance of the humanized FR2 includes SEQ ID NO: 346, where Y4 is modified to 4V.

[0175] 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.

[0176] Another specific example of the humanized FR2 includes SEQ ID NO: 348, in which Q11 and R12 are modified to 11G and 12L, respectively.

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] In another particular embodiment, the VHH is humanized by selected modifications of the FR3 domain. Typical humanization sites in FR3 are selected from 6V, 17A or S, 20T, 21L, 25M, 26N, 29R, 30A and any combination thereof (by reference, for example, any of SEQ ID NO: 45-49, 366-379 or any variant thereof as defined herein).

[0182] Therefore, a specific example of such humanized FR3 includes SEQ ID NO: 46, in which one or more or all of M6, T17, A20, V21, I25, D26 and K29 ​​are modified to 6V, 17A, 20T, 21L, 25M, 26N and 29R respectively.

[0183] Another specific example of the humanized FR3 includes SEQ ID NO: 366, in which V21 and K29 ​​are modified to 21L and 29R, respectively.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 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.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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.

[0200] 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.

[0201] 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.

[0202] In another specific implementation, FR1 and / or FR2 and / or FR3 are humanized.

[0203] Specific examples of the humanized TfR-binding VHH molecule of the present invention are molecules comprising or composed of amino acid sequences selected from any of SEQ ID NO: 236-241, 252-271, 273-275, 752-765, or 773-786 (see Table 1). In another specific embodiment, the VHH molecule may also comprise one or more tags suitable for, for example, purification, conjugation, detection, etc. In the context of the present invention, the term "tag" includes any peptide sequence attached to the polypeptide VHH molecule of the present 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.

[0204] 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).

[0205] Typically, the one or more tags are located at the C-end of the VHH.

[0206] In another particular embodiment, the VHH molecule may further include one or more connectors.

[0207] 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).

[0208] 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).

[0209] 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).

[0210] 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.

[0211] 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).

[0212] 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 VHHs 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 comprise the following tag sequence at the C-terminus: AAAEQKLISEEDLNGAAHHHHHHGS (SEQ ID NO: 51), where the single underscore is the myc tag, the double underscore is the His6 tag (the remaining residues are linkers such as the Ala linker AAA, or residues generated by cloning).

[0213] 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.

[0214] 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, 130-149, 152-154 or 752-765 or composed of said amino acid sequences (see Table 1).

[0215] 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).

[0216] 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.

[0217] 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.

[0218] 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 consists of LQRs.

[0219] 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.

[0220] 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.

[0221] 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 a recombinant cell or membrane preparation 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.

[0222] As shown in the experimental section, VHH molecules bind to TfR both in vitro and in vivo. They exhibit sufficient affinity for K... dThe molecular weight is from about 0.01 nM to about 4 µM, more preferably from about 0.1 nM to about 2500 nM, from about 0.1 nM to about 1000 nM, from about 1 nM to about 2500 nM, from about 1 nM to about 1000 nM, from about 10 nM to about 1000 nM, from about 0.1 nM to about 500 nM, from about 0.1 nM to about 250 nM, from about 0.1 nM to about 100 nM, from about 1 nM to about 100 nM, or from about 10 nM to about 100 nM.

[0223] In the context of this invention, the term K d This 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 k on 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.

[0224] In another specific embodiment, the VHH molecule of the present invention is a cross-species molecule with a Kc of about 0.1 nM to about 2000 nM. d Combining human and non-human primate TfR.

[0225] In one particular embodiment, the VHH molecule of the present invention, after systemic administration (e.g., intravenous or subcutaneous), is in a K+ concentration of about 1 nM to about 2500 nM. d Combine with TfR.

[0226] In another specific embodiment, the VHH molecule of the present invention is administered topically (e.g., intrathecally or intraventricularly) at a concentration of about 0.1 nM to about 250 nM of K. d Combine with TfR.

[0227] In another embodiment, the conjugate according to the invention comprising a VHH-siRNA variant, such as the VHH-siSOD1 variant, binds to human and non-human primate TfRs (more preferably as evaluated using SPR) in the range of 150 nM to 1 µM, preferably 150 to 400 nM. Preferably, the non-human primate TfR is a rhesus monkey or cynomolgus monkey TfR.

[0228] In another specific embodiment, the conjugate according to the invention, for example, a conjugate comprising a VHH-NT variant, binds to human and non-human primate TfRs in the range of about 1 nM to about 1800 nM. In yet another embodiment, the conjugate comprising the VHH-NT variant exhibits a wide range of hTfR affinity, K D / K Deq In the range of approximately 1 nM to approximately 350 nM. As shown in the experimental data, the VHH-NT variant exhibits a higher affinity (K0.05) compared to the same variant without NT. D The gain is approximately 2-3 times, meaning that the supported molecule can influence the TfR binding properties of the final conjugate. Interestingly, when using the Pearson correlation matrix, various TfR binding parameters (e.g., k) measured by SPR are compared... on K off and K D When compared with all in vivo parameters (i.e., hypothermia AUC, maximum hypothermia temperature, and hypothermia duration) measured in NT-induced cryoassays for each VHH-NT variant, the inventors found that the affinity (KAUC) measured by SPR was significantly higher than that measured by SPR. D There is a significant negative correlation between K and the maximum low temperature, indicating that K D With a maximum affinity of 350 nM, the lower the affinity for hTfR, the higher the temperature, resulting in a higher potential for BBB crossing.

[0229] In another embodiment of the invention, the dissociation rate (K) measured by SPR is... off There is a significant negative correlation between hTfR and the maximum low temperature, which implies a rapid dissociation rate (between 6E-03 and 1E-01 s). -1 Within the range, it is beneficial for the efficient BBB crossing of NT neuropeptides.

[0230] In another embodiment of the invention, the key parameter leading to efficient BBB crossing of the VHH-NT variant and enabling it to be delivered to the CNS in sufficient quantities to activate NTSR expressed at the surface of CNS parenchymal cells is a K0 between 6 and 350 nM. D and 6E-03 to 1E-01 s -1 K between offPreferably, the VHH-NT variant is selected from the C5 and B8 variants listed in Table 8.

[0231] In one specific embodiment of the invention, the VHH molecule is a humanized or non-humanized C5 or a variant thereof, comprising or consisting of any one of SEQ ID NOs: 213, 216-271, 274, 275, 675, 676, and 701 (as listed in Table 1). In another specific embodiment, the VHH molecule is a C5 or a variant thereof, which is a cross-species VHH molecule that binds to human, non-human primate (NHP), and rodent TfR. In one specific embodiment, the C5 or a variant thereof includes a tag sequence at the N- and / or C-terminus. Specific examples of such variants include or consist of 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. In another specific embodiment, the VHH molecule is a humanized variant of C5 that includes or consists 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-terminal ends. Specific examples of such variants include 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 which contains or consists of SEQ ID NO: 265 or 143 or consists of SEQ ID NO: 274 or 15 ...

[0232] 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.

[0233] In another specific embodiment, the VHH molecule of the present invention is B8 or a variant thereof, which cross-reacts with human and non-human primate species.

[0234] 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.

[0235] In another specific embodiment, the VHH molecule of the present invention is B8 or a variant thereof, comprising any one of SEQ ID NO: 677, 678, 681, 682, 702-709, 693-700, 745-786, preferably any one of SEQ ID NO: 677, 678, 681, 682, 752-765 or 773-786, or composed of thereof.

[0236] In another specific embodiment, the VHH molecule is a humanized variant of B8, comprising SEQ ID NO: 273, 152 (e.g., B8h1 variant), 752-765, or 773-786 (e.g., B8h1 variant). V31h1-5 h 9 Or B8 V32h1 (variants of h6, h9-14) or composed of them.

[0237] 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.

[0238] 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 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.

[0239] 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.

[0240] 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.

[0241] Furthermore, the binding of the VHH of this 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 to TfR in vitro and to be transported across the BBB to the CNS in vivo, exhibiting transcytosis. Therefore, such VHHs represent potent agents for drug delivery or targeting.

[0242] 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).

[0243] Coupled

[0244] Another object of the present invention relates to conjugates (which may also be interchangeably referred to herein as "conjugate compounds" or "chimeric agents") comprising one or more VHH molecules as defined above, conjugated with at least one additional compound, particularly with at least one additional molecule, agent, compound, or scaffold of interest. Such additional compound may be a different VHH or a molecule that is not a VHH. The at least one additional molecule, agent, or compound of interest may be any molecule, agent, or compound, such as a half-life-extending moiety, stabilizing group, or scaffold, therapeutic (i.e., active) compound, drug or pharmaceutical product, diagnostic agent, imaging molecule, tracer, etc., or a carrier containing such a therapeutic, diagnostic, or imaging compound. In one particular aspect, the chimeric agent (i.e., conjugate) may comprise two additional compounds, i.e., i) a half-life-extending moiety, stabilizing group, or scaffold, and ii) a therapeutic, diagnostic, or imaging compound, or a carrier containing them. The therapeutic compound is, for example, selected from peptides, polypeptides, proteins, antibodies, nucleic acids, and any fragments thereof. In one particular embodiment, the therapeutic compound is a nucleic acid molecule, such as mRNA, ribozyme, or oligonucleotide, 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, bridging nucleic acid (BNA), virus, diagnostic agent, tracer, etc.

[0245] Examples of conjugate 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 drugs, anti-inflammatory drugs, or adjuvants); peptides, polypeptides, or proteins (e.g., enzymes, hormones, neurotrophic factors, neuropeptides, cytokines, apolipoproteins, growth factors, antigens, antibodies or portions of antibodies, adjuvants, etc.); nucleic acids (e.g., RNA or DNA of human, viral, animal, eukaryotic, prokaryotic, plant, or synthetic origin, 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, "molecules, pharmaceuticals, or compounds of interest" can be any pharmaceutical (active) ingredient, whether chemical, biochemical, natural, or synthetic. Typically, the description of "small chemical molecules, pharmaceuticals, or compounds" refers to molecules of pharmacological interest with a maximum molecular weight of 1,000 Daltons, usually between 300 and 700 Daltons.

[0246] The carrier can be selected from, for example, viruses, virus-like particles (VLPs), cell-derived vesicles (CDVs), exosomes, lipid carriers, and polymer carriers, preferably lipid nanoparticles (LNPs), micelles, or liposomes.

[0247] The conjugated compound is typically a drug (e.g., a small drug, nucleic acid, or peptide, such as an antibody or fragment thereof) or an imaging agent suitable for treating or detecting neurological, infectious, or cancerous pathologies, preferably CNS pathologies, such as brain, spinal cord, or retinal pathologies like Alzheimer's disease, Parkinson's disease, stroke, Creutzfeldt-Jakob disease, dementia, multiple sclerosis, spinal muscular atrophy (SMA), amyotrophic lateral sclerosis, epilepsy, migraine, encephalitis, lysosomal storage disorders of the brain, CNS pain, or CNS cancers such as brain cancer (e.g., glioblastoma); or PNS pathologies, such as pathologies of nerve cells in cranial ganglia, dorsal root ganglia, or autonomic ganglia, such as peripheral neuropathy, such as diabetic neuropathy, cancer and chemotherapy-induced peripheral neuropathy (CIPN), HIV-induced neuropathy, leprosy (HD). Infection-induced neuropathy such as Lyme disease, traumatic nerve injury (e.g., carpal tunnel syndrome and sciatica); hereditary sensorimotor peripheral neuropathy such as peroneal muscular atrophy (CMT), Friedreich ataxia (FA), and giant axonal neuropathy (GAN); autoimmune and inflammatory-induced peripheral neuropathy such as Guillain-Barré syndrome, lupus, rheumatoid arthritis, chronic inflammatory demyelinating polyneuropathy (CIDP), multifocal motor neuropathy, and Sjögren's syndrome; DRG cell disorders such as sensory polyneuropathy (e.g., dorsal root ganglion lesions or sensory neuron diseases), sensory polygangliopathy, sensory polyradiculopathy, central sensory axonopathy, and sensory polyradiculopathy; any complication of PNS disease, such as neuropathic pain; and PNS cancer.

[0248] 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) at least one therapeutic compound or a carrier containing such therapeutic compound, The VHH molecule binds to TfR on the surface of nerve cells, and The VHH molecule described herein comprises: 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 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 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.

[0249] 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) at least one therapeutic compound or a carrier containing such therapeutic compound, The VHH molecule binds to both TfR on the surface of nerve cells and muscle cells, and The VHH molecule described herein comprises: 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 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 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, for the prevention or treatment of neuromuscular diseases such as spinal muscular atrophy, amyotrophic lateral sclerosis, peroneal muscular atrophy, multiple sclerosis or Huntington's disease, wherein the conjugate is preferably administered intracerebrally, intraventricularly, or intrathecally.

[0250] In addition to or as a substitute for the compound of interest, the conjugate may also contain a half-life-extending moiety or a stabilizing group to increase the plasma half-life of the VHH or the 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 moiety or a stabilizing group, (iii) the compound of interest, typically a therapeutic, diagnostic, or imaging compound, and optionally (iv) a carrier.

[0251] 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.

[0252] 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.

[0253] In another specific embodiment, the conjugate according to the invention comprises a half-life extension portion or a stabilizing group, which is a Fc fragment of human IgG1 or IgG4, preferably IgG1. Such a conjugate has the general formula VHH-hFc, such as VHH-hFc-siRNA or VHH-hFc-ASO.

[0254] 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.

[0255] In another embodiment, the conjugate according to the invention comprises a half-life extension portion or stabilizing group, which is a homodimer or heterodimer of an Fc fragment modified with IgG1 or IgG4, having a weakened or eliminated effector function and / or an extended half-life.

[0256] 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.

[0257] 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.

[0258] 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.

[0259] 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.

[0260] 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.

[0261] 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.

[0262] 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.

[0263] 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.

[0264] 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, to couple with at least one oligonucleotide, i.e., one or more oligonucleotides (e.g., 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.

[0265] 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 XXIII and...). Figure 23 (as shown in B and 24C).

[0266] In one particular embodiment, the conjugate according to the invention comprises a VHH-Fc heterodimer, wherein Fc comprises the following mutations: T366W on the "pestle" arm of the heterodimer, and / or T366S, L368A and Y407V on the "mortar" arm of the heterodimer.

[0267] 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.

[0268] 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. For example, in Example XXII and... Figure 20 and 22 Specific examples of such VHH-Fc heterodimers containing SEQ ID NO: 664 and 665 are described in the document.

[0269] All of the above-described mutations are defined according to the standard Kabat system used for numbering amino acid residues in antibodies. In the coupling compounds of the present 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 present invention is used for the delivery of at least one active pharmaceutical ingredient to a CNS or PNS 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 carried out using genetic engineering.

[0270] 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., CNS or PNS cells). 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 the side chain of one of the amino acids that make up the sequence (Majumdar and Siahaan, 2012). The compound of interest can be conjugated directly to the VHH or indirectly via a linker or spacer. Covalent chemical conjugation, with or without a linker, includes classical bioconjugation techniques, such as those using 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, acid anhydrides, thiols or carboxyl groups, 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. Illustrative strategies for coupling the VHH of the present invention to molecules or scaffolds are disclosed in [the relevant section]. Figure 6 middle.

[0271] In one particular implementation, 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 a VHH fused to the molecule is prepared and expressed in any suitable expression system to produce the conjugate.

[0272] The overall structure of the VHH-oligonucleotide conjugate of the present invention is shown in Figure 24 The illustrative strategy for coupling the VHH of the present invention with a molecule or scaffold is disclosed in [the document / concept]. Figure 23 middle.

[0273] In another specific embodiment, the coupling is performed using a thiol / maleimide chemistry technique. For this reaction to occur, VHH has a peptide sequence containing an additional cysteine ​​fused to its C-terminus. Specifically, the additional peptide tag is typically GGGGSCHHHHHH (SEQ ID NO: 399), where the single underscore is a Gly linker used as a spacer, and the double underscore is a 6His tag used for purification purposes.

[0274] Since VHH contains only cysteine ​​residues involved in disulfide bonding, the additional cysteine ​​introduced in the tag is the only cysteine ​​chemically reactive to maleimide. This allows for the specific coupling of VHH with maleimide-derived molecules of interest. The reaction proceeds in two steps. First, it is necessary to smoothly reduce VHH-GGGGSCHHHH, as the additional cysteine ​​in the tag can partially participate in disulfide bonding during production (forming a disulfide bond with another VHH-GGGGSCHHHHHH or a free cysteine). 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, forming a VHH-molecule conjugate linked in a covalent and stable manner. These two steps can be performed sequentially or together in place.

[0275] 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.

[0276] 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.

[0277] 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.

[0278] 3) Due to complementary reactive groups, the functionalized VHH and the molecule are coupled.

[0279] Such coupling strategies represent another object of the present invention. Specifically, one object of the present invention is a method for coupling two molecules using a Q-tag as defined above via a Tgase coupling reaction. Another object of the present invention is a VHH comprising a Q-tag. Yet another object of the present invention is a VHH molecule comprising a linker, such as a Gly linker, and a Q-tag.

[0280] The preferred VHH of this invention has the following structure: VHH-connector-His m -Connector-LQR, where: VHH is any VHH molecule; the linker is any molecular linker, such as an Ala or Gly linker (preferably the two linkers are different); m is an integer from 0 to 8, preferably m is 6 or 8.

[0281] 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.

[0282] 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 one specific 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. For example, such an oligonucleotide is an ASO or siRNA capable of regulating (silencing, inhibiting, or activating) the expression of a target gene.

[0283] 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%.

[0284] 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 nerve 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.

[0285] In one particular embodiment, the present invention relates to a coupling compound comprising: (i) One or more VHH molecules that bind to TfR on the surface of nerve 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.

[0286] In one particular embodiment, the present invention relates to a coupling compound comprising: (i) One or more VHH molecules that bind to TfR on the surface of nerve cells and contain: - CDR1, wherein CDR1 comprises an amino acid sequence selected from the following: SEQ ID NO: 1, 5, 9, 17, 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, wherein the variants have 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 the sequences thereof throughout their entire length; and - CDR2, wherein CDR2 comprises an amino acid sequence selected from the following: SEQ ID NO: 2, 6, 73, 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, wherein the variants have 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 the sequences thereof throughout their entire length; and - CDR3, wherein CDR3 comprises an amino acid sequence selected from the following: SEQ ID NO: 3, 7, 11, 117, 119, 121, 123, 177, 180, 188, 192, 196, 200, 203, 207, 452, 455, 609, 612, 713-715, 741-744 and variants thereof, wherein the variants have 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 the sequences in their entire length. The conditions are: - When CDR1 contains SEQ ID NO: 1, 5, 9 or 17, then CDR2 is not selected from SEQ ID NO: 2, 6 and 73, and CDR3 is not selected from SEQ ID NO: 3, 7 and 11; - When CDR2 contains SEQ ID NO: 2, 6 or 73, then CDR1 is not selected from SEQ ID NO: 1, 5, 9 and 17, and CDR3 is not selected from SEQ ID NO: 3, 7 and 11; - When CDR3 contains SEQ ID NO: 3, 7 or 11, then CDR1 is not selected from SEQ ID NO: 1, 5, 9 and 17, and CDR2 is not selected from SEQ ID NO: 2, 6 and 73.

[0287] (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.

[0288] In another specific embodiment, the present invention relates to a coupling compound comprising: (i) One or more VHH molecules that bind to TfR on the surface of nerve cells and contain: - CDR1, wherein CDR1 comprises an amino acid sequence selected from the following: SEQ ID NO: 1, 5, 9, 17, 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 - CDR2, wherein CDR2 comprises an amino acid sequence selected from the following: SEQ ID NO: 2, 6, 73, 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, wherein CDR3 comprises an amino acid sequence selected from the following: SEQ ID NO: 3, 7, 11, 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. The conditions are: - When CDR1 contains SEQ ID NO: 1, 5, 9 or 17, then CDR2 is not selected from SEQ ID NO: 2, 6 and 73, and CDR3 is not selected from SEQ ID NO: 3, 7 and 11; - When CDR2 contains SEQ ID NO: 2, 6 or 73, then CDR1 is not selected from SEQ ID NO: 1, 5, 9 and 17, and CDR3 is not selected from SEQ ID NO: 3, 7 and 11; - When CDR3 contains SEQ ID NO: 3, 7 or 11, then CDR1 is not selected from SEQ ID NO: 1, 5, 9 and 17, and CDR2 is not selected from SEQ ID NO: 2, 6 and 73.

[0289] (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.

[0290] In another specific embodiment, the present invention relates to a coupling compound comprising: (i) One or more VHH molecules that bind to TfR on the surface of nerve 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.

[0291] 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 nerve 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.

[0292] 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 nervous system 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.

[0293] 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.

[0294] 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 nervous system cells, and comprising or consisting of any one of SEQ ID NO: 12, 168, 170, 172-174, 178, 181, 183, 185, 215, 285-293; 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.

[0295] 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 on the surface of nervous system 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.

[0296] 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, 163-164, 164-165, 164 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.

[0297] 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.

[0298] 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.

[0299] 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.

[0300] In another embodiment, the present invention relates to a coupling compound comprising a VHH covalently linked to a dye.

[0301] 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 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.

[0302] 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 a heavy chain or light chain, or both, or to the C- or N-terminus of an Fc fragment. In one particular aspect, the VHH molecule is conjugated to the N-terminus of an Fc. In another particular 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.

[0303] 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.

[0304] In the chimeric agents of the present invention, when several VHHs are present, they may have similar or different binding specificities.

[0305] Nucleic acids, vectors, and host cells

[0306] 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.

[0307] Specific examples of such nucleic acid sequences include sequences containing any of SEQ ID NO: 301-329, 52-64 and 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.

[0308] The present invention also relates to a vector containing such nucleic acid, optionally under the control of a regulatory sequence (e.g., promoter, terminator, 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. Control sequences known to those skilled in the art are 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. The present invention also relates to the use of the nucleic acid or vector according to the present invention for transforming, transfecting, or transducing host cells or for preparing compositions (including pharmaceutical compositions) for transforming, transfecting, or transducing host cells.

[0309] The present invention also provides a host cell comprising one or more nucleic acids of the present invention and / or one or more vectors of the present 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.

[0310] use

[0311] The VHH molecule of the present invention can bind to TfR, thereby targeting / delivering the molecule to cells or organs 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 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.

[0312] 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.

[0313] The present invention also relates to the use of VHH as defined above as a carrier for the transport of compounds to / through cells or organs expressing TfR.

[0314] The present invention also relates to the use of VHH as defined above for the preparation of medicaments capable of crossing the plasma membrane of any TfR-expressing cell in the BBB and the central nervous system (CNS) or peripheral nervous system (PNS).

[0315] The present invention also relates to a method for enabling or improving molecular passage through the BBB, the method comprising coupling the molecule to the VHH of the present invention. The present invention further relates to a method for enabling or improving molecular passage through the plasma membrane of target cells in neural tissue using coupling compounds as defined above.

[0316] As explained above, the VHH of the present invention can be used to transport or deliver any compound, such as small drugs, proteins, polypeptides, peptides, amino acids, lipids, nucleic acids, viruses, liposomes, exosomes, etc.

[0317] 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.

[0318] 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.

[0319] 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).

[0320] 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 carries at least one substance of interest.

[0321] The term "pharmaceutically acceptable salt" refers to a non-toxic salt, 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, benzenesulfonates, benzoates, bicarbonates, bisulfates, tartrates, borates, hydrobromates, bromides, butyrates, camphorsulfonates, carbonates, hydrochlorides, chlorides, citrates, clavulanates, dihydrochlorides, diphosphates, ethylenediaminetetraacetate, calcium ethylenediaminetetraacetate, ethanedisulfonates, etolates, ethanesulfonates, fumarates, gluconate, gluconate, glutamates, glycolylarsanylate, hexafluorophosphates, hexyl isophthalate, hybamin, hydroxynaphthylcarbamate, iodides, and isothiocyanates. 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.

[0322] 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.

[0323] The compositions or conjugates of the present invention may be administered by any suitable route, and in a non-limiting manner by 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 oral), 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; topical 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.

[0324] 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.

[0325] 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, particularly those affecting the nervous system, infectious diseases, or cancer. The VHH of the present invention has the ability to target cells expressing TfR, particularly cells exhibiting significant expression of the receptor, such as cancer cells, neural or non-neural tissues, and / or across cell membranes, particularly the cell membranes of physiological barriers of the nervous system, and more particularly the blood-tumor barrier (BTB) cell membranes of cancerous neural tissues. TfR is enriched in organs such as bone marrow, placenta, and in the gastrointestinal tract. TfR is also highly expressed in brain endothelial cells but not in vascular endothelial cells of other tissues. Expression of TfR at the plasma membrane of purified brain microvessels and cultured endothelial cells from rats, mice, pigs, and non-human primates has been demonstrated.

[0326] In this respect, the present invention relates to the use of the drug conjugates or compositions described above for the prevention or treatment of CNS or PNS pathologies or disorders, brain tumors or other cancer cells, and bacterial, viral, parasitic or fungal infectious pathologies of the brain or other tissues.

[0327] The present invention also relates to VHH, conjugates or compositions as described above, for the diagnosis, imaging or treatment of CNS or PNS pathologies or disorders, brain tumors or other cancer cells, and bacterial, viral, parasitic or fungal infectious pathologies of the brain or other tissues of the CNS or PNS.

[0328] The present invention also relates to VHH, conjugates or compositions as described above, for the treatment, imaging and / or diagnosis of CNS or PNS tumors such as brain tumors or other types of cancer.

[0329] This invention relates to VHHs, conjugates, or compositions as defined above, for the prevention, treatment, imaging, and / or diagnosis of neurodegenerative diseases, such as, but not limited to, Alzheimer's disease, Parkinson's disease, stroke, Creutzfeldt-Jakob disease, dementia, multiple sclerosis, amyotrophic lateral sclerosis, and brain cancers such as glioblastoma.

[0330] The present invention also relates to VHHs, conjugates or compositions as defined above, for the prevention, treatment, imaging and / or diagnosis of neurological diseases, such as, but not limited to, epilepsy, migraine, encephalitis, CNS pain, etc.

[0331] The present invention also relates to VHHs, conjugates or compositions as defined above, for the prevention, treatment, imaging and / or diagnosis of rare diseases, such as, but not limited to, lysosomal storage diseases, Fabry disease, Fabry disease, ganglioside storage diseases GM1 and GM2, Gaucher disease, various mucopolysaccharidosis diseases, etc.

[0332] The present invention also relates to VHHs, conjugates or compositions as defined above, for the prevention, treatment, imaging and / or diagnosis of neuropsychiatric disorders, such as, but not limited to, depression, autism, anxiety disorders, schizophrenia, etc.

[0333] The present invention also relates to VHHs, conjugates or compositions as defined above, for the prevention, treatment, imaging and / or diagnosis of cancer, such as, but not limited to, glioblastoma, pancreatic cancer, ovarian cancer, hepatocellular carcinoma and the like.

[0334] The present invention also relates to VHHs, conjugates or compositions as defined above for the prevention, treatment, imaging and / or diagnosis of acquired peripheral neuropathy, such as, but not limited to, diabetic neuropathy, cancer and chemotherapy-induced peripheral neuropathy (CIPN), HIV-induced neuropathy, leprosy (HD), Lyme disease, traumatic nerve injury (e.g., carpal tunnel syndrome and sciatica).

[0335] The present invention also relates to VHHs, conjugates or compositions as defined above, for the prevention, treatment, imaging and / or diagnosis of hereditary sensorimotor peripheral neuropathy, such as, but not limited to, peroneal muscular dystrophy (CMT), Friedreich ataxia (FA), giant axonal neuropathy (GAN), etc.

[0336] The present invention also relates to VHHs, conjugates or compositions as defined above, for the prevention, treatment, imaging and / or diagnosis of autoimmune and inflammatory-induced peripheral neuropathy, such as, but not limited to, Guillain-Barré syndrome, lupus, rheumatoid arthritis, chronic inflammatory demyelinating polyneuropathy (CIDP), multifocal motor neuropathy, Sjögren's syndrome, etc.

[0337] The present invention also relates to VHHs, conjugates or compositions as defined above, for the prevention, treatment, imaging and / or diagnosis of DRG cell diseases, such as, but not limited to, sensory polyneuropathy (dorsal root ganglion disease or sensory neuron disease), sensory polygangliopathy, sensory polyradiculopathy, central sensory axonal disease (also known as posterior column disease), sensory polyradiculopathy, etc.

[0338] This invention also relates to VHHs, conjugates, or compositions as defined above for the prevention, treatment, imaging, and / or diagnosis of neuromuscular disorders, such as spinal muscular atrophy, amyotrophic lateral sclerosis (ALS), peroneal muscular atrophy, multiple sclerosis, and Huntington's disease. In one particular embodiment, the invention relates to VHHs, conjugates, or compositions as defined above for the prevention or treatment of neuromuscular disorders, such as spinal muscular atrophy, ALS, peroneal muscular atrophy, multiple sclerosis, and Huntington's disease, wherein the VHH binds to TfRs on the surface of both CNS cells and muscle cells.

[0339] In another specific embodiment, the present invention relates to VHHs, conjugates, or compositions as defined above for the prevention or treatment of neuromuscular disorders such as spinal muscular atrophy, amyotrophic lateral sclerosis, peroneal muscular atrophy, multiple sclerosis, and Huntington's disease, wherein the VHHs, conjugates, or compositions are administered intracerebrally, intraventricularly, or intrathecally.

[0340] In another specific embodiment, the present invention relates to VHHs, conjugates, or compositions as defined above for the prevention or treatment of neuromuscular disorders, such as spinal muscular atrophy, amyotrophic lateral sclerosis, peroneal muscular atrophy, multiple sclerosis, and Huntington's disease, wherein the VHH, conjugate, or composition is administered intracerebral, intraventricular, or intrathecally, and wherein the VHH binds to TfRs on the surface of both CNS cells and muscle cells.

[0341] This invention also relates to VHHs, conjugates, or compositions as defined above for the prevention or treatment of neuropathic pain. The invention further relates to VHHs, conjugates, or 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 increase 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 brain or cancer or any TfR-rich tissue. Conjugation with a virus or VLP can be, for example, by conjugation with the capsid protein of said virus.

[0342] 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.

[0343] 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.

[0344] Other aspects and advantages of the invention will become apparent from the following embodiments.

[0345] Example

[0346] Example 1: Verification of TfR expression at the BBB

[0347] We analyzed the cell membrane expression profiles of TfR in brain endothelial cells from various species. Membrane extracts were prepared from digested or undigested primary cultures of brain microvessels (BMV) and brain microvascular endothelial cells (BMEC) from rats, mice, pigs, and non-human primates (NHPs; rhesus monkeys) using the ProteoExtract Subcellular Proteomics Extraction Kit (Calbiochem, La Jolla, CA, USA). Figure 1 Membrane extracts were quantified using the BioRad DC protein assay (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions. Membrane proteins were separated on 4–12% polyacrylamide gels by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (ThermoFisher Scientific). Proteins were detected with a primary antibody against TfR (Genetex GTX102596; 1 / 1000) followed by a 1 / 10000 dilution of HRP-conjugated donkey anti-rabbit IgG secondary antibody (Jackson ImmunoResearch). Finally, proteins were detected by chemiluminescence. Figure 1 As shown, TfR is expressed in digested and undigested brain microvessels from rats, mice, pigs, and non-human primates. TfR is also expressed in brain endothelial cells from mice, rats, and pigs (note that only 1 µg of membrane protein was loaded on SDS-PAGE for brain microvascular endothelial cells, while 10 µg or 5 µg was loaded for brain microvessels). TfR expression is enhanced in digested NHP brain microvessels. These data demonstrate that TfR represents an effective target for designing molecules for in vivo application.

[0348] Example II: Construction of CHO cell lines stably expressing human and mouse TfR

[0349] A prerequisite for identifying and characterizing TfR-binding VHHs is the establishment of stable cell lines in eukaryotic cells (Chinese hamster ovary cells, CHO) that constitutively and rapidly express hTfR and mTfR. These cell lines are then used to i) identify and characterize agents that bind to receptors expressed in their native conformation on the cell surface; and ii) test whether such receptors can internalize these agents via endocytosis. To construct these cell lines, cDNA encoding hTfR was cloned using sequence information available in a database (accession number: NM_003234.3). Primers required for cDNA amplification by RT-PCR (see SEQ ID NO: 65 and 66, shown below) were selected, containing at their ends the restriction sites (EcoRI and SalI) required for cloning into the pEGFP-C1 expression vector (Clontech) (bold). Figure 2 -A).

[0350]

[0351] Total RNA obtained from human brain was used for RT-PCR amplification of a cDNA fragment encoding hTfR. After amplification, the PCR product was digested with EcoRI-SalI restriction enzyme and ligated into a pEGFP-C1 expression vector (Clontech) digested with the same restriction enzyme. Upon transfection into eukaryotic cells, this vector was able to express hTfR fused to EGFP at its N-terminus (i.e., the end of its intracellular domain) under the control of the CMV promoter. The construct was validated by complete sequencing of both strands after transformation into competent *E. coli* DH5α bacteria, obtaining isolated colonies, and preparing plasmid DNA. The plasmid encoding mTfR fused to EGFP was purchased from GeneCopoeia (plasmid reference: EX-Mm05845-M29). Transient transfection was performed in CHO-K1 cells and used to select stable transfectants through limiting dilution and resistance to the antibiotic (G418). These cell lines were amplified while maintaining selection pressure. Figure 2 In -B, confocal microscopy images taken after immunocytochemistry of Alexa647-conjugated transferrin (Tf-Alexa647) on fixed (PFA) cell lines confirmed the colocalization between EGFP (green) and Tf-Alexa647 (red), thus confirming good receptor expression and functional binding. Membrane expression of the receptor of expected size was examined by Western blotting on cell membranes extracted using the ProteoExtract subcellular proteomics extraction kit. Antibodies were targeted at either GFP or TfR. Proteins corresponding to the combined size (170 kDa) of EGFP and h / mTfR were identified using anti-GFP and anti-TfR antibodies. Figure 2-C). The CHO K1 wild-type (WT) cell line was used as a negative control, and the protein was not detected by the antibody. These data confirm the expression of the functional receptor on the cell surface of the CHO cell line.

[0352] Example III: (A) Generation of VHH in conjunction with TfR large alpaca ( Lama glama Four subcutaneous immunizations were performed using membrane preparations from CHO stable cell lines expressing the human and mouse receptors of interest. VHH libraries were constructed as previously described (Alvarez-Rueda et al., 2007; Behar et al., 2009). Briefly, the mRNA encoding VHH was amplified from total RNA of peripheral blood mononuclear cells isolated via a ficoll gradient by RT-PCR and cloned into pHEN1 phages. Repeated selection was performed to isolate phages capable of presenting VHHs exhibiting strong affinity for TfR expressed at the cell surface. A total of over 2000 clones were screened for their TfR binding ability, and approximately 450 clones were sequenced. VHHs with improved binding (with mouse, non-human primate, and / or human cell lines), cell penetration, and transport properties were obtained.

[0353] The first identified descriptive VHHs were VHH A (C5), VHH B (B8), VHH C (B6), and VHH D (H3) (see also the sequence listing). These VHHs did not bind to control CHO cell lines. Furthermore, TfR-binding VHHs with appropriate, improved binding properties were generated through site-directed mutagenesis. More specifically, site-directed mutagenesis was performed to introduce a single alanine substitution in the complementarity-determining regions (CDRs) 1, 2, and 3 of VHH A, producing VHH A1 through A9 (C5). V1 Up to C5 V9 VHH A1 and A2 were mutated in CDR1, VHH A3 and A4 in CDR2, and VHH A5 to A9 in CDR3. Furthermore, single-site mutagenesis was performed by replacing some CDR amino acids with structurally similar amino acids. VHH A10-A19 (C5) were obtained. V10 Up to C5 V19 VHH A10 and A11 are mutated in CDR1, VHH A12 to A14 are mutated in CDR2, and VHH A15 to A19 are mutated in CDR3. Furthermore, humanized TfR-binding VHHs were generated to improve in vivo efficacy by, for example, avoiding immunogenicity, and were named VHH A20-A25 (C5). h1 Up to C5 h6Additionally, tagged VHH molecules were generated to facilitate purification and / or coupling. The amino acid sequence of each of these VHHs is provided in the sequence listing.

[0354] (B) Generation of other VHHs in conjunction with TfR

[0355] Several strategies were further implemented to generate VHHs with different binding properties to TfR. To further improve the binding properties of C5, site-directed mutagenesis was performed in complementarity-determining regions (CDRs) 1, 2, and 3, resulting in variant C5. V20 Up to C5 V29 By introducing two alanine residues into CDR3, a negative control for C5 was also generated, namely C5. neg .

[0356] Furthermore, humanized C5 VHH was developed to improve in vivo efficacy, for example, by avoiding immunogenicity. Therefore, different humanization schemes were applied in framework regions (FRs) 1, 2, 3, and 4, resulting in variant C5. h8 Up to C5 h26 They exhibited a humanization rate between 89% and 96% compared to C5. The humanization model was also applied to VHH B8 and C5. V13 and C5 V16 This generated VHH B8 h1 C5 V13h20 and C5 V16h20 .

[0357] To generate B8 and B6 variants, as well as a novel C5 variant, yeast visualization combined with deep mutational scanning (DMS) was performed by CRO (Deeptope, Paris) to provide residue-level resolution at locations in the VHH interface, crucial for TfR binding. Because the DMS maps highlighted interesting sites modified to affect VHH binding, a large number of B8 variants (B8...) were generated through site-directed mutagenesis in either the CDR or FR. V1 To B8 V40 ), C5 variant (C5) V30 Up to C5 V32 ) and B6 variant (B6) V1 Up to B6 V4 ).

[0358] The TfR receptor comprises several extracellular domains. One strategy developed here is to perform phage display selection and screening on the TfR apical domain using the aforementioned VHH library. Over 700 clones were screened for their ability to bind to the TfR apical domain using an ELISA assay, and approximately 150 clones were sequenced. Sequence analysis then led to the isolation of 10 VHHs of interest that showed specific binding to the TfR apical domain. The illustrative VHHs are B7, H7, C12b, E4, E2, C3, F2a, E9, A9a, and B4a. Notably, VHHs B7, H7, C12b, and E4 showed similar CDR3 sequences compared to B6, suggesting they should bind to the same epitope.

[0359] Finally, a last strategy for enriching the diversity of VHHs in this invention was phage display selection on CHO cell lines stably expressing rhesus monkey TfR (rhTfR). Screening was then performed on CHO cell lines stably expressing either hTfR or rhTfR and on a CHO cell line not expressing TfR (CHO TRVb). Flow cytometry experiments were used to screen for the ability of over 180 clones to bind both rhTfR and hTfR, and approximately 30 clones were sequenced. This strategy allowed for the identification of specific VHHs that bind both hTfR and rhTfR, namely E8, C10a, and H1. The amino acid sequences of all generated VHHs are provided in the accompanying sequence listing and Table 1 below.

[0360] Example IV: Binding and endocytosis of purified VHH of the present invention

[0361] To confirm the ability of the selected VHH molecule to bind to TfR and be endocytosed, immunocytochemical experiments were performed. These involved incubating VHH in live CHO cell lines expressing TfR fused with EGFP, detecting the molecule using a mouse anti-cMyc primary antibody (ThermoFisher), followed by detection using an Alexa594-conjugated donkey anti-mouse secondary antibody (Jackson ImmunoResearch), and observing the results using confocal microscopy. Results obtained with VHH A are shown as an example. Figure 3 As shown, VHH and CHO-hTfR-EGFP ( Figure 3 -B) and CHO-mTfR-EGFP ( Figure 3 -A) Cell line binding and incorporation via endocytosis to accumulate in cells, as shown by triton permeation, unlike the control VHH (VHH Z). Figure 3 -C,D).

[0362] Example V: Determination of the binding affinity of VHH to human and mouse TfR (first-time identification)

[0363] The binding properties of the newly identified VHH with affinity for TfR were tested using flow cytometry, and the epigenetic affinity (K0.05) was determined. d app All experiments were performed in 96-well plates using 2-3 x 10⁻⁶ wells. 5 Cells / well were shaken at 4°C. CHO cell lines or CHO WT cells expressing TfR fused with EGFP were saturated with PBS / BSA 2% solution for 30 min to avoid nonspecific binding, then incubated with purified VHH at concentrations ranging from 50 µM to 1 pM for 1 hr. After one wash in PBS / BSA 2%, cells were incubated with anti-6His-tagged antibody (mouse) for 1 hr, washed twice with PBS / BSA 2%, and incubated with an Alexa647-conjugated anti-mouse secondary antibody for 45 min. After the final two washes in PBS / BSA 2%, cells were fixed (or not fixed) by incubation with PBS / PFA 2% for 15 min, washed once with PBS, and finally resuspended in PBS. Fluorescence levels were assessed using a MACSQuant flow cytometer (Miltenyi) or an Attune NxT flow cytometer (Thermo Fisher Scientific). No nonspecific labeling was observed under control conditions where cells were incubated with control VHH (VHH Z). All tested VHHs induced concentration-dependent signal migration, confirming binding to the receptor of interest. Figure 4 -A). No markers (not shown) were detected in CHO WT control cells using VHH in all tests. VHHK was calculated using GraphPad Prism software. d app ( Figure 4 -B). For all VHH, K d app All are within the same range, from 7.5 nM (VHH B) to 56 nM (VHH D) for mTfR and from 1.7 nM (VHH B) to 2.7 nM (VHH A) for hTfR.

[0364] Example VI: Competition assay between purified VHH with affinity for TfR and natural ligands

[0365] To evaluate the ability of the selected VHH to competitively bind to the receptor against transferrin (Tf) (the natural ligand of TfR), a competition assay was performed using flow cytometry. First, serially diluted competitors were incubated for 1 hour at 4°C on CHO cells expressing the receptor of interest fused with EGFP. Next, EC90 tracer was added and incubated for another 1 hour, followed by detection using an appropriate revealing system. Figure 5 TfR-binding VHH is used as a tracer ( Figure 5-B) and competitors ( Figure 5 -C). Under all conditions, there was no competition between VHH and the ligand Tf, indicating that VHH binds to TfR at a different epitope than Tf.

[0366] Example VII: (A) Determination of the binding affinity of VHH A1-A19 to human and mouse TfR VHH A1-A19 (C5) was tested using flow cytometry. V1 Up to C5 V19 The binding properties of TfR were determined, and the apparent affinity (K) was also determined. d app All experiments were performed in 96-well plates using 2 x 10⁻⁶ wells. 5 Cells / well were shaken at 4°C. CHO cell lines or CHO WT cells stably expressing hTfR or mTfR fused with EGFP were saturated with PBS / BSA 2% solution for 30 min to avoid nonspecific binding, then incubated with purified VHH at concentrations ranging from 50 µM to 5 pM for 1 hr. After one wash in PBS / BSA 2%, cells were incubated with anti-6His-tagged antibody (mouse) for 1 hr, washed twice with PBS / BSA 2%, and incubated with an Alexa647-conjugated anti-mouse secondary antibody for 45 min. After the final two washes in PBS / BSA 2%, cells were fixed by incubation with PBS / PFA 2% for 15 min, washed once with PBS, and finally resuspended in PBS and stored at 4°C. Fluorescence levels were assessed using an Attune NxT flow cytometer (Thermo Fisher Scientific). VHH A1-A19 induced concentration-dependent signal translocation in both cell lines (except for VHH A12), confirming their efficient binding to the receptor of interest. Figure 11 ;12). Although VHH A, VHH A1 to A4 and VHH A10 to A15 all showed similar B in cell lines expressing hTfR and mTfR. max (Curve plateau), but VHH A6 to A9 and VHH A16 to A19 showed mild to severe B in both cell lines. max Decreased, and with slight to strong curve migration. Compared to other VHH Ax, only VHH A12 showed lower B-values ​​in hTfR-expressing cell lines. max Strong curve migration was observed. No markers (not shown) were detected in CHO WT control cells using all tests of VHH. VHH K was calculated using GraphPad Prism software. d app ( Figure 11-B; 12-B). Regarding binding to human TfR, VHH A, A1 to A4, A6, A9 to A11, and A13 to A17 all showed a similarity of approximately 3-4 nM. d app Conversely, VHH A5, A8, A18, and 19 showed slightly lower affinities of 9.2 to 25 nM, while VHH A7 and A12 showed sharply decreasing affinities of 255 nM and 363 nM, respectively. Regarding binding to mouse TfR, VHH A and A9 showed similar affinity of approximately 50 nM. d app Except for VHH A5, A8 and A18, which showed significantly reduced affinity of 604 nM, 427 nM and 416 nM respectively, all other VHH Ax showed slightly lower affinity of 131 to 259 nM.

[0367] (B) Determination of the binding characteristics of VHH with hTfR, mTfR and rhTfR

[0368] The binding properties of VHH with affinity for 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. 5 Cells / well were collected and shaken at 4°C. CHO cell lines expressing hTfR, mTfR, or rhTfR fused with EGFP were saturated with PBS / BSA 2% solution for 30 min to avoid nonspecific binding, and then incubated with purified VHH at gradually increasing concentrations for 1 hr. After one wash in PBS / BSA 2%, cells were incubated with anti-6His-tagged antibody (mouse) for 1 hr, washed twice with PBS / BSA 2%, and incubated with an Alexa647-conjugated anti-mouse secondary antibody for 1 hr. After two final washes in PBS / BSA 2%, cells were fixed by incubation with PBS / PFA 2% 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 fitted using nonlinear fitting with GraphPad Prism® software to determine epigenetic properties. K d Constant. When cells were compared with controls VHH D12 or C5... neg No nonspecific markers were found under the incubation control conditions, confirming the C5 negative control. On hTfR, all tested VHHs induced concentration-dependent signal migration, confirming binding to the receptor of interest. Table 5 below summarizes the epigenetic K values ​​obtained for the tested VHHs. dMost 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 TfR studies highlights the immense interest and diversity of the resulting VHH library.

[0369] Table 5:

[0370] NB: Not bound; ND: Not measured

[0371] 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 antibodies. 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.

[0372] Table 6:

[0373] ND: Not measured

[0374] Example VIII: Binding and internalization of purified VHH-Fc fusion molecules with affinity for TfR, and affinity determination.

[0375] The anti-TfR VHH molecule of this invention was fused with an IgG Fc fragment. To generate the fusion protein, a DNA fragment encoding VHH (untagged) was amplified by PCR and cloned into the pINFUSE-IgG1-Fc2 vector (InvivoGen) to encode a human IgG1-Fc fragment containing VHH in its N-ter or C-ter. The fusion protein was prepared using the Expi293 expression system according to the manufacturer's instructions (Life Technologies). 72 hours after transfection, the supernatant was recovered and purified using a Protein A GraviTrap column (GE Healthcare). The purified fusion protein was quantified using an in-cell anti-Fc antibody ELISA. Immunocytochemical experiments were performed on CHO cell lines expressing TfR fused with EGFP, involving incubation of the VHH-Fc fusion protein on live cells, detection using an Alexa594-conjugated anti-hFc antibody (Jackson ImmunoResearch), and imaging with confocal microscopy to confirm the fusion protein's ability to bind to the target receptor of interest. The results confirmed that the conjugate of the present invention can bind and be endocytosed by cells. Figure 7 No binding of the control VHH-Fc conjugate (VHH Z-Fc) was observed on cells, demonstrating the specificity of the interaction.

[0376] The binding properties of VHH-Fc and Fc-VHH fusion proteins with affinity for TfR were tested in flow cytometry experiments, and the epigenetic affinity (K0) was determined. d app All experiments were performed in 96-well plates using 2-3 x 10⁻⁶ wells. 5 Cells / well were shaken at 4°C. CHO cell lines or CHO WT cells expressing receptors of interest fused with EGFP were saturated with 2% PBS / BSA and then incubated for 1 hour with purified VHH-Fc or Fc-VHH at concentrations ranging from 350 nM to 0.03 pM. After washing, cells were incubated for 1 hour with an Alexa647-conjugated anti-hFc antibody (Jackson ImmunoResearch). Immediately after the last three washes and resuspending the cells in PBS, fluorescence was measured using a MACSQuant flow cytometer (Miltenyi), and the results were analyzed using MACSQuant software. All VHH-Fc and Fc-VHH fusion proteins induced concentration-dependent migration of the signal, confirming binding to the receptor of interest. VHH-Fc and Fc-VHH K were calculated using GraphPad Prism software. d app ( Figure 8 -B). Through coupling with the Fc fragment, almost all VHH's K... d appBoth have been greatly improved, for TfR binding VHH-Fc and Fc VHH, K d app In the range of 0.44 nM to 51 nM.

[0377] Example IX. Internalization and transport of VHH in an in vitro BBB model according to the present invention.

[0378] We established co-culture models using rat or mouse brain microvascular endothelial cells (BMECs) and rat or mouse astrocytes. This type of in vitro BBB model was used to assess the passive or active transport of many molecules (especially pharmacological reagents) across BMECs, thereby extrapolating their ability to reach CNS tissues in vivo. The various models developed to date (bovine, porcine, mouse, and human) possess ultrastructural characteristics specific to the brain endothelium, particularly tight junctions, the absence of fenestrations, low permeability to hydrophilic molecules, and high electrical resistance. Furthermore, these models showed a robust correlation between measurements obtained from in vitro and in vivo assessments of the properties of various molecules crossing the BBB. All data obtained to date indicate that these in vitro BBB models mimic the in vivo situation by reproducing some of the complexities of the cellular environment present in vivo, while retaining the advantages associated with cell culture experiments. For example, the in vitro rat BBB model effectively utilizes the co-culture of BMECs and astrocytes (Molino et al., 2014). Prior to cell culture, membrane inserts (Corning, Transwell 1.0 μm pore size, for 96-well or 12-well plates) were treated with type IV collagen and fibronectin on the top to achieve optimal adhesion of BMECs and create basal layer conditions. Primary cultures of mixed astrocytes were established from the cerebral cortex of neonatal rats. Briefly, the meninges were removed, and cortical fragments were mechanically and then enzymatically dissociated in trypsin solution. The dissociated cells were seeded into glial cell culture medium (GCM) containing DMEM supplemented with 10% fetal bovine serum and then frozen in liquid nitrogen for later use. Primary cultures of BMECs were prepared from 5–6-week-old Wistar rats. Briefly, cortical fragments were mechanically and then enzymatically dissociated in collagenase / dispersin solution. The digested tissue was separated by density-dependent centrifugation in 25% bovine serum albumin. Microvascular deposits were seeded into DMEM / F12 endothelial cell culture medium (ECM) containing 20% ​​bovine anemia platelet-derived serum and 2 ng / ml basic fibroblast growth factor (bFGF) in culture flasks pre-coated with type IV collagen and fibronectin. Five days prior to co-culture establishment, astrocytes were thawed and seeded into 12-well or 96-well plates (extraluminal compartments). BMECs were then distributed onto the upper surface of the filter membrane in the co-culture (intraluminal compartments). Under these conditions, the in vitro model differentiated within 3 days, expressing junction-related proteins, and maintained optimal differentiation for the following 3 days. The binding / uptake at the BBB of the present invention's VHH (VHH-Fc), conjugated to the human Fc fragment of IgG1 antibody, was verified in the above-described in vitro rat model. Figure 9 VHH A-Fc or VHH B-Fc and Tf-Alexa647 were incubated with live rBMEC monolayer at 37°C for 2 hours. Figure 9A). After incubation, the cell monolayer was thoroughly washed and fixed with 4% PFA. The cell monolayer was permeabilized with 0.1% Triton X-100 solution. VHH-Fc was detected by immunostaining with an antibody against the human Fc fragment. Colocalization between the fluorescence signals of VHH A-Fc or VHH B-Fc and Tf-A647 was then assessed using confocal microscopy. Figure 9 A). The results showed that after 2 hours of co-incubation, VHH A-Fc and VHH B-Fc were readily internalized and almost completely colocalized with Tf-Alexa647. This analysis of colocalization of different TfR ligands (VHH A-Fc, VHH B-Fc, and Tf-A647) confirms the specificity of the VHH of the present invention for its target receptor. To transport the VHH-Fc across the rBMEC monolayer to the extracavitary compartment, the VHH-Fc was incubated at 10 nM for 24 to 72 hours in the intracavitary compartment of the culture system. Figure 9 -C, D). Prior to the experiment, filter inserts containing rBMEC monolayers were placed in 96-well plates containing fresh transport buffer (75 µl in the intraluminal compartment and 250 µl in the extraluminal compartment). To assess the integrity of the BBB in vitro and the absence of endothelial cell toxicity, VHH-FC was incubated with fluorescein (LY) (a small fluorescent molecule that does not cross the BBB). After 24 hours of incubation, the inserts were transferred to another 96-well plate containing fresh transport buffer for another 48-hour period. At the end of transport, the accumulated LY in the extraluminal compartment was quantified by fluorescence spectrophotometry, and the results were expressed as endothelial surface permeability (or Pe) in units of 10⁻⁶. -3 cm / min. If the Pe value of LY is greater than 0.6 x 10 -3 A flow rate of cm / min indicates that the in vitro barrier is considered "permeable" or "open." Transendothelial resistance (TEER), measured with an ohmmeter and expressed in ohm·cm², can also be used to measure the in vitro integrity of the BBB during the cross-BBB test. The mass threshold was set to >400 ohm·cm². Experiments performed showed that VHH-Fc is non-toxic and has no adverse effect on the permeability properties of the BBB (not shown). The Fc fragment content of the VHH-Fc of the present invention was quantified using an internal anti-Fc antibody ELISA assay in the influent (T0), the intraluminal compartment at the end of the transport experiment (T72 hr, product recovery), and the extraluminal compartment (24 hr and +48 hr transport periods), with a sensitivity between 0.5 and 50 femtomoles. Absorbance units were converted to femtomoles per insert (the surface area of ​​a 96-well plate insert is 0.143 cm²). 2Our results showed that VHH B-Fc and VHH A-Fc conjugates exhibited significantly higher transport than VHH Z-Fc (negative control), approximately 10-fold at 24 hr and 5-fold at 72 hr. This transport reached apparent saturation between 24 and 72 hr, further suggesting the involvement of a specific and saturable receptor-mediated process. Figure 9 -D).

[0379] Example X: Pharmacokinetics and organ uptake of VHH-Fc conjugate in vivo

[0380] To evaluate the potential of the VHH-Fc conjugates of the present invention to target organs rich in receptors of interest in vivo, conjugates VHH A-Fc, VHH A-Fc-Agly, and VHH Z-Fc were injected into the tail vein at 5 mg / kg and mice were perfused with saline at different time points. Plasma and brain were collected. The brain was treated by capillary depletion to separate the brain parenchyma from the capillaries. The amount of VHH-Fc in plasma, brain parenchyma, and microvessels was measured using an internal anti-Fc antibody ELISA. The results were presented as concentration (nM) or organ-to-plasma ratio (nM). Figure 10 The TfR-binding conjugates VHH A-Fc and VHH A-Fc-Agly exhibited significant brain targeting at 2 hr pi, with concentrations in the brain parenchyma of VHH A-Fc and VHH A-Fc-Agly being 0.25 and 0.32 nM, respectively, compared to 0.07 nM for the control VHH Z-Fc. Figure 10 -B). When observing the parenchyma-to-plasma ratio, a significant advantage was confirmed, particularly at 24 hr pi, when VHH A-Fc-Agly was still measurable in the brain parenchyma, while only 8 nM was present in the plasma. Figure 10 -D). In microvessels, VHH A-Fc and VHH A-Fc-Agly accumulated significantly more than VHHZ-Fc at 2 hr pi, with concentrations 9-fold and 5-fold higher, respectively. Furthermore, at 24 hr pi, the concentration of VHH A-Fc in microvessels was still 3-fold higher than that of VHH Z-Fc ( Figure 10 -C). These results were confirmed when the microvessel to plasma ratio was observed. Figure 10 These results demonstrate that the VHH targeting TfR of the present invention can be used for efficient delivery of drugs (especially protein cargoes) or to improve their pharmacokinetic properties.

[0381] Example XI: Design and production of therapeutic antibodies fused with VHH

[0382] The anti-TfR VHH A, A1, A5, A6, A7, and A8 (untagged) of this invention were fused with a mouse IgG1 13C3 monoclonal antibody that exhibits high specific affinity for β-amyloid peptide in fibrillary form (WO2009 / 065054). To generate the 13C3-HC-VHH fusion protein, a DNA fragment encoding a selected VHH was synthesized and cloned into a 13C3 heavy chain (HC) vector to encode a 13C3-HC-VHH conjugate containing a selected VHH sequence fused to the C-ter amino acid residues of the antibody heavy chain. In another set of experiments, a DNA fragment encoding a selected VHH was cloned into a 13C3 light chain (LC) vector to encode a 13C3-LC conjugate containing a selected VHH sequence fused to the C-ter amino acid residues of the antibody light chain. Expi293 was used. TM The expression system was used to produce the fusion protein according to the manufacturer's instructions (Life Technologies). 72 hours post-transfection, the supernatant was recovered and purified using a HiTrap® Protein G high-performance column (GE Healthcare). The purified fusion protein was quantified using 280 nm absorbance measurement.

[0383] The amino acid sequence of the 13C3-HC-VHHA conjugate is provided as SEQ ID NO: 93:

[0384] Bold text represents the 13C3 variable heavy chain sequence; underlined text represents the 13C3 constant heavy chain sequence; bold and underlined text represents the Gly linker; double-underlined MA and C-ter AAA residues are generated by cloning and may be optionally removed. The remainder is VHH.

[0385] The amino acid sequence of the 13C3-LC-VHHA conjugate is provided as SEQ ID NO: 94:

[0386] Bold text represents the 13C3 variable κ light chain sequence; FGGGTK is the J region; LEIKR is the multiple cloning site; underlined text represents the 13C3 constant κ light chain sequence; bold and underlined text represents the Gly linker; double-underlined MA and C-ter AAA residues are generated by cloning and may be optionally removed. The remainder is VHH.

[0387] Binding affinity determination

[0388] The binding properties of the 13C3 conjugate of this invention to TfR were tested using flow cytometry, and the apparent affinity (K) was determined. d appAll experiments were performed under the same conditions as described in Example VII, with the 13C3 construct incubated at concentrations ranging from 15 µM to 7 pM and detected using an Alexa647-conjugated anti-mouse antibody. All 13C3-HC-VHH fusion proteins induced concentration-dependent signal translocation in both hTfR and mTfR-expressing cell lines, confirming receptor binding (…). Figure 13 All 13C3 fusion proteins exhibited the same hTfR binding properties, with the exception of the VHH A7 fusion, which showed slightly lower B binding. max All fusion proteins exhibited different binding properties to mTfR, with the 13C3-HC-VHH A1 fusion protein showing the best binding affinity to mTfR. max It is 2 times lower than 13C3-HC-VHH A, while the A5 to A8 13C3 fusions show very low B. max The Ki of the 13C3 fusion was calculated using GraphPad Prism software. d app ( Figure 13 -B). For all fusions, the affinity for hTfR is similar, K d app Approximately 10-20 nM. Although B max The VHH A, A1, and A6 13C3HC fusions showed similar affinities of 10 to 20 nM, while the 13C3-HC-VHH A8 and 13C3-LC-VHH A fusions showed lower affinities of 315 nM and 106 nM, respectively.

[0389] Example XII: Brain uptake of the 13C3-HC-VHH and 13C3-LC-VHH fusion in vivo

[0390] To evaluate the potential of the VHH conjugate to promote antibody brain uptake in this invention, 13C3-HC-VHH A and 13C3-HC-VHHA1 conjugates or uncarriered 13C3 were injected into the tail vein of C57Bl6 mice at a dose of 35 nanomoles / kg. Mice were perfused with saline solution at different time points. Brains were collected at 2 hr and 6 hr post-injection (pi). Half of the mouse brain was treated using a capillary depletion method to separate the brain parenchyma from the capillary network, the method comprising centrifuging the resuspended half-brain homogenate on 20% dextran solution (Sigma Aldrich) and recovering the parenchymal fraction. The other half of the mouse brain was directly processed (homogenized and lysed) for whole-brain quantification. The amount of 13C3-HC-VHH conjugate in the whole brain and brain parenchyma was measured using an in-house qualified Meso Scale Discovery (MSD) direct coating (Abeta) immunoassay. (CV < 20%, recovery ± 30%). Results are presented as concentrations (nM) ( Figure 14The results showed that, compared with the control uncarriered 13C3 antibody, the TfR-binding conjugates 13C3-HC-VHH A and 13C3-HC-VHH A1 exhibited significant brain uptake advantages at 2 and 6 hr p1 (i.e., compared with the control uncarriered 13C3 antibody). Figure 14 -A). At 6 hr pi, the whole-brain concentrations of 13C3-HC-VHH A and 13C3-HC-VHH A1 were 8 and 5 times that of the uncarriered 13C3 antibody, respectively. The cross-brain penetration of 13C3-HC-VHH A and 13C3-HC-VHH A1 was confirmed by the fact that at 6 hr pi, the concentrations measured in brain parenchyma with depleted microcapillary networks were 10 and 9 times that of the uncarriered 13C3, respectively. Figure 14 -B). Further brain uptake studies confirmed that 13C3-HC-VHH A and 13C3-LC-VHH A (light chain carrier version) exhibited BBB crossing at a dose of 70 nanomoles / kg, and their substantial accumulation at 4 hr pi was 6 times and 5 times that of the uncarriered 13C3 antibody, respectively.

[0391] Example XIII: Synthesis of VHH-siRNA Conjugate

[0392] A chemically modified anti-GFP siRNA (i.e., siGFPst1) containing high resistance to nucleases was conjugated to a tagged VHH A to generate the VHH A-siGFPst1 bioconjugate. Using the same conjugation strategy, siGFPst1 was conjugated to an unrelated VHH Z as a negative control, which had the same structure and size as the VHH A-siGFPst1 conjugate but lacked TfR targeting ability. The conjugation strategy involved a pooled synthesis with the following parallel modifications: (i) modification of VHH to introduce an azide linker with site specificity; and (ii) modification of siGFPst1 to introduce a constrained azide moiety complementary to the azide functional group. In the final step, the functionalized VHH-azid and the alkyne-siGFPst1 precursor were linked together using a copper-free click reaction.

[0393] Synthesis of VHH-azides

[0394] Site-specific coupling with VHH was achieved using a bacterial transglutaminase (BTG)-based ligation strategy. The BTG enzyme catalyzes the formation of an isopeptide bond between a glutamine residue inserted into a tag sequence (i.e., the Q-tag) specifically recognized by the BTG enzyme and an amino-functionalized substrate. The introduced amino-functionalized substrate is a heterobifunctional linker containing an amino moiety at one end of what we have identified as the substrate of the BTG enzyme, and an azide moiety at the other end for coupling with siGFPst1 via copper-free click chemistry.

[0395] BTG coupling scheme: 3-Azide-1-propylamine (20 equivalences / Gln) was dissolved in PBS (1X) and added to the internally generated Q-tagged VHH. BTG (Zedira, Darmstadt, Germany) was then introduced into the mixture (0.1 U / nmolGln), allowing it to react overnight at 37 °C. The crude mixture was purified by chromatography on a Protino Ni-ida 1000 packed column according to the manufacturer's instructions to separate the VHH-azid from excess starting material and potential byproducts. Absorbance was read at 280 nm to calculate the amount of purified VHH-azid construct, thereby calculating the coupling yield (in the range of 70–80%). The final VHH-azid was characterized by LCMS analysis to check its identity and purity.

[0396] Synthesis of alkyne-siGFPst1

[0397] siGFPst1, purchased from Dharmacon, has a 3' amine modification on the sense chain (N6-siGFPst1) to allow for further functionalization of the alkyne moiety required for click chemical coupling with VHH-azides.

[0398] siGFPst1 functionalization scheme

[0399] N6-siGFPst1 (1 equivalent) was dissolved in NaB (0.09 M; pH 8.5) coupling buffer to obtain a final concentration between 0.3 and 0.8 mM. DBCO-NHS (20 equivalents, DMSO) was then added to this solution. The reaction mixture was stirred at room temperature for 2 hours. Alkyne-siGFPst1 was purified by precipitation in cold anhydrous ethanol. The absorbance was read at 260 nm to calculate the amount of purified alkyne-siGFPst1 construct, thereby calculating the coupling yield (in the range of 40–50%). The final alkyne-siGFPst1 was characterized by analytical HPLC to check its identity and purity.

[0400] Synthesis of VHH-siGFPst1

[0401] The VHH-azide and alkyne-siGFPst1 precursor were finally coupled by a copper-free click chemistry reaction to obtain the final conjugate VHH-siGFPst1.

[0402] VHH-siGFPst coupling scheme: Alkyne-siGFPst1 (2 equivalents) was dissolved in PBS (1X) and added to VHH-azid (1 equivalent, in PBS (1X), final concentration in the range of 100 µM). The reaction mixture was allowed to stir overnight at room temperature. The final conjugates were first purified by gel filtration chromatography on a Superdex 75 column and then concentrated using an Amicon ultracentrifuge filter (10 K). The absorbance was read at 260 nm to calculate the amount of purified VHH-siGFPst1 construct, thereby calculating the conjugation yield (total yield in the range of 30%). The final VHH-siGFPst1 (VHH A-siGFPst1 and VHH Z-siGFPst1) were characterized by analytical SEC-HPLC and agarose gel electrophoresis to check their identity and purity.

[0403] Example XIV: In vitro gene silencing activity of VHH-siRNA bioconjugates

[0404] Specific cell targeting and productive intracellular delivery of therapeutic nucleic acids (especially siRNAs and oligonucleotides) remain a major challenge. Without assistance, the structural and physicochemical characteristics of these molecules (as multi-charged hydrophilic oligomers) prevent them from entering any subcellular compartment. The VHH of this invention is used for transmembrane transport of small interfering RNA (siRNA) into the cytosol.

[0405] First, as described in Example VII (Determination of Binding Affinity of VHH A1-A19), the apparent hTfR binding affinity (Kb) of VHH A-siGFPst1 and VHH B-siGFPst1 bioconjugates at concentrations ranging from 2 µM to 30 pM was evaluated by adding them to the same CHO-hTfR-GFP cells at 4 °C for 1 hour. d app The molecules bound to the cell surface were quantified by immunocytochemistry using anti-6His antibody, and the experimental data were fitted using nonlinear regression with GraphPad Prism® software. As previously shown with free VHH A and VHH B, the VHH A-siGFPst1 and VHH B-siGFPst1 bioconjugates exhibited concentration-dependent and saturable binding to the cell surface target hTfR. d app The values ​​are in the same low nanomolar range as the uncoupled VHH A and VHH B ( Figure 15 A). No significant binding was observed using the control VHH Z.

[0406] Secondly, the intrinsic silencing activity of the VHH-siGFPst1 bioconjugate was assessed in a live CHO cell line stably expressing TfR fused with EGFP (CHO-hTfR-EGFP cells) by transfecting 25 nM of the conjugate directly into the cytosol using Dharmafect 1 (Dharmacon). The total cellular amount of GFP was quantified by flow cytometry 72 hours post-transfection. The results showed that the VHH A-siGFPst1 conjugate induced a reduction in GFP protein levels of approximately 85%, within the same range as the unconjugated siGFPst1 or the control VHH Z-siGFPst1 conjugate. Figure 15 B). This confirms that conjugation of VHH A or Z does not prevent siRNA from RISC loading and exerting its silencing activity. In another series of experiments, the VHH A-siGFPst1 conjugate was transfected into CHO-hTfR-EGFP cells at concentrations ranging from 10 nM to 1 pM, and the total cellular amount of GFP was quantified by flow cytometry 120 hours post-transfection. This resulted in a concentration-dependent decrease in GFP protein levels, with an IC50 of 50.4 pM, and a maximum silencing efficiency of -90.2% under these conditions. Figure 15 C).

[0407] Third, the ability of VHH A to trigger hTfR-mediated endocytosis and subsequent pharmacological delivery to the cytosol of target cells upon conjugation with siGFPst1 was evaluated. Incubation of 1 µM VHH A-siGFPst1 or the control VHH Z-siGFPst1 bioconjugate at 37°C for 120 h on CHO-hTfR-GFP cells allowed for free uptake, delivery to the cytosol, and gene silencing at both the mRNA transcript and protein levels. This resulted in a significant reduction of approximately 70% in GFP protein levels when using the TfR-binding VHH A-siGFPst1 bioconjugate, while no silencing was observed when using the control VHH Z-siGFPst1 bioconjugate. Figure 15 D). Next, the VHH A-siGFPst1 bioconjugate was incubated on CHO-hTfR-GFP cells at concentrations ranging from 3 µM to 10 pM for 120 hr. This resulted in a concentration-dependent decrease in GFP protein levels, with an IC50 of 2.73 ± 0.23 nM, and a maximum silencing efficiency of -61.6 ± 2.9% under these conditions. Figure 15 E). This confirms that the cell surface binding of the VHH A-siGFPst1 bioconjugate to hTfR and subsequent endocytosis allows it to be delivered into the cytosol in pharmacological amounts, with an IC50 in the same nanomolar range as the hTfR binding affinity of the bioconjugate.

[0408] Fourth, the silencing effect of hTfR on the VHH A-siGFPst1 bioconjugate observed after free uptake in CHO-hTfR-GFP cells was confirmed in a competition assay. In this experiment, VHH A-siGFPst1 was incubated alone or in the presence of 100-fold excess of free VHH A, B, or Z at 37°C for 120 hours. The results showed that the approximately 60% reduction in GFP protein levels was almost completely negated in the presence of free VHH A or VHH B (GFP protein levels remained at 85% and 96% of the control levels, respectively). Importantly, no competition was observed when an excess of irrelevant VHH Z was used. Figure 15 F). This clearly confirms that the silencing effect of the VHH A-siGFPst1 bioconjugate is indeed due to hTfR-mediated cellular uptake and subsequent delivery in the cytoplasm.

[0409] Fifth, the TfR-mediated GFP silencing effect of the VHH A-siGFPst1 bioconjugate was evaluated using a pulse tracking procedure. CHO-hTfR-GFP cells were exposed to VHH A-siGFPst1 at concentrations ranging from 300 nM to 1 pM for a short period (6 hours) and then tracked in ligand-free medium until a total duration of 120 hr. This experiment allowed for assessment of the contribution of early cellular uptake to the silencing effect previously observed through continuous incubation over 120 hr. As observed with continuous incubation, the VHH A-siGFPst1 bioconjugate again induced a concentration-dependent decrease in GFP protein levels, with a similar IC50 of 1.24 nM and a maximum silencing efficiency of -54.2%. Figure 15 (G). This result indicates that the effects previously observed after 120 hours of continuous incubation are largely due to productive TfR-mediated uptake within the first 6 hours. This finding is particularly interesting because, in vivo, the plasma pharmacokinetic characteristics of such bioconjugates, when administered via intravenous or subcutaneous bolus, typically allow tissues to be exposed to therapeutic levels within just a few hours. Therefore, the TfR-targeting VHH described here represents a viable tool for in vivo targeting and efficient gene silencing.

[0410] Finally, the ability of VHH B to induce hTfR-mediated endocytosis and subsequent gene silencing was assessed by incubating 30 nM of the VHH B-siGFPst1 bioconjugate on CHO-hTfR-GFP cells for 120 hours. The results showed a reduction in GFP levels of approximately 60%, similar to levels obtained using the VHH A-siGFPst1 bioconjugate, confirming that these VHHs exhibit similar TfR targeting and intracellular delivery potential. Figure 15H). To our knowledge, receptor-mediated hepatocyte uptake via the desialylate glycoprotein receptor (ASGPR) using trigonal GalNAc as a targeting ligand is the only ligand / receptor system capable of inducing specific and efficient gene silencing at nanomolar concentrations. However, since ASGPR is expressed only in hepatocytes in vivo, the in vivo therapeutic application of this system for therapeutic nucleic acids is limited to liver targets. Therefore, this invention provides a novel ligand / receptor system for the targeted and intracytoplasmic delivery of nanomolar concentrations of therapeutic nucleic acids (e.g., siRNA) to extrahepatic organs and tissues expressing TfR.

[0411] Example XV: Synthesis of VHH-NODAGA Conjugate

[0412] VHH A design with Q-tag

[0413] In this embodiment, a DNA fragment encoding VHH A with an Ala adapter, His tag, Gly adapter and Q tag (AAA-His tag-GGG-LQR sequence) introduced at its C-terminus was synthesized and cloned into the pHEN1 vector.

[0414] BTG-based preparation of VHH A-azides: 3-Azide-1-propylamine (20 equivalences / Gln) was dissolved in PBS (1X) and added to internally produced VHH A with an LQR tag. BTG (Zedira, Darmstadt, Germany) was introduced into the mixture (0.1 U / nmol Gln). The reaction mixture was then allowed to react overnight at 37°C. The crude mixture was purified by chromatography on a ProtinoNi-ida 1000 packed column, according to the manufacturer's instructions, to separate the VHH A-azid from excess starting material and potential byproducts. Absorbance was read at 280 nm to calculate the amount of purified VHH A-azid construct, thereby calculating the coupling yield (in the range of 70-80%). The final VHH A-azid was characterized by LCMS analysis to check its identity and purity.

[0415] Click chemistry of coupling VHH A-azides with commercially available alkynes -NODAGA

[0416] VHH A-azide (1 equivalent) was allowed to react with heterobifunctional NODAGA-BCN (5 equivalents) (Chematech, Dijon, France) in PBS at room temperature. The reaction was monitored by LCMS. After the reaction was complete, the final conjugate was purified by chromatography on a Protino Ni-ida 1000 packed column according to the manufacturer's instructions to separate VHH A-azide from excess starting material and potential byproducts. The absorbance was read at 280 nm to calculate the amount of purified VHHA-NODAGA construct, thereby calculating the coupling yield (in the range of 50-60%). The final VHH A-NODAGA was characterized by LCMS analysis to check its identity and purity.

[0417] Example XVI: PET imaging of VHH-68Ga bioconjugate in a subcutaneous mouse model of glioblastoma

[0418] Glioblastoma is the most common primary malignant brain tumor, and the human primary glioblastoma cell line U87 is known to express high levels of TfR. To evaluate the glioblastoma targeting of VHH of the present invention, radiolabeled VHH A-NODAGA bioconjugate was intravenously administered to mice previously implanted with glioblastoma cells (xenograft model), and PET scan imaging was performed.

[0419] Radiolabeling and binding affinity verification of VHH A-NODAGA

[0420] First, VHH A-NODAGA was radiolabeled using 68Ga chloride. Gallium was obtained as 68Ga3+ using a commercially available TiO2-based 68Ge / 68Ga generator (Obninsk). The radiolabeling reaction was performed by reacting 60 μg of VHH A-NODAGA with 74–148 MBq (2–4 mCi) of 68Ga in 400 μL ammonium acetate buffer (1M, pH 6) at 25°C for 10 min. The obtained VHH A-68Ga had a radiochemical purity (RPC) >95%. Following radiolabeling, as described in Example VII (Determination of Binding Affinity of VHH A1-19), the apparent hTfR binding affinity (KbA) of VHH A-NODAGA and VHH A-68Ga bioconjugates at concentrations ranging from 2 µM to 30 pM was evaluated on the same CHO-hTfR-GFP cells at 4°C for 1 hr. d appThe VHHA bioconjugates bound to the cell surface were quantified by immunocytochemistry using anti-6His antibody, and the experimental data were fitted using nonlinear regression with GraphPad Prism® software. The VHHA-NODAGA and VHHA-68Ga bioconjugates exhibited concentration-dependent and saturable binding to the cell surface target receptor hTfR. d app The value is in the same low nanomolar range as the uncoupled VHH A ( Figure 16 A). No significant binding was observed using the control VHH Z. This confirms the coupling of VHH A with the NODAGA ligand and that the radiolabeling scheme does not alter its ability to specifically bind to hTfR.

[0421] PET scan imaging

[0422] Animal studies were conducted according to a protocol approved by the Aix-Marseille Ethic comity (Comity 14). Four-week-old female BALB / c nude mice were obtained from Charles River Inc. Mice (n=6) were subcutaneously implanted with 100 µL of U87-MG cells (2 × 10⁻⁶) in complete Corning medium containing 50% Matrigel between their shoulders. 6 On day 28 post-implantation (when the tumor reaches 300-700 mm)... 3 VHHA-68Ga was administered intravenously in animals at a single bolus dose of 5 ± 1 MBq. Following administration, biodistribution in glioblastoma xenografts and other tissues was assessed using PET imaging. Three mice underwent PET / CT scans within 2 hours, and another three mice underwent PET / CT scans 2 hours after injection (pi). PET and PET / CT studies were performed on a microPET / microCT rodent model scanner (nanoPET / CT®, Mediso). Anesthesia was induced with 5% isoflurane and maintained with 1.5%. To improve image quality, 20 million coincidence events were acquired per mouse for each static PET emission scan (energy window, 400–600 keV; time: one FOV 20 min). For bimodal PET / CT, CT images (35 kVp, exposure time 350 ns, medium zoom) were acquired, and anatomical registration and attenuation correction were applied to the corresponding PET scans. Imaging images of animals injected with VHH A-68Ga showed significant accumulation at the tumor site. Figure 16B, 1.46% ID / g) and a good tumor / muscle ratio (4.0). Therefore, the experiment showed that glioblastoma cancer was clearly and selectively imaged and labeled with VHHA-68Ga on day 28, consistent with the known high expression level of TfR.

[0423] Example XVII: Competition assay between purified VHH and phage-VHH

[0424] To assess whether the selected VHHs competed with each other for binding to the TfR receptor, a competition assay was performed using flow cytometry. All experiments were performed in 96-well plates using 2 x 10⁻⁶ cells / well. 5 Cells / well were shaken at 4°C. CHO cell lines expressing hTfR fused with EGFP were saturated with 2% PBS / BSA for 30 min to avoid nonspecific binding, then incubated with gradually increasing concentrations of VHH (competitor) for 1 hr. Then, EC80 phage-VHH (tracer) was added and incubated for another 1 hr. To detect phage-VHH, cells were washed twice with 2% PBS / BSA and incubated for 1 hr with Alexa647-conjugated anti-M13 antibody. After the last two washes with 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). GraphPad Prism was used. ® The software uses nonlinear fitting to fit the experimental data.

[0425] The concentration-dependent loss of phage-H1 signaling induced by VHH E8 and C10a indicates that their binding to TfR hinders phage-H1 binding. These three VHHs (E8, C10a, and H1) were selected via phage display on rhTfR, and these results suggest they should bind to nearby epitopes. Furthermore, VHHs B6 and C5 do not interfere with phage-H1 binding. Therefore, H1, E8, and C10a should bind to different epitopes than B6 and C5. Figure 17 -A). Figure 17 -B confirms this, showing that neither E8 nor C10a VHH interferes with phage -C5 binding. (As...) Figure 17As shown in -C, VHH C3 and F2a induced concentration-dependent inhibition of phage-B6 binding. As these results illustrate, these three VHHs (C3, F2a, and B6) bind to the apical domain of the TfR at proximal epitopes. VHH C10a and C5 do not interfere with phage-B6 binding, confirming the above results. Therefore, C3, F2a, and B6 should bind to proximal epitopes, unlike the epitopes of C10a and C5. It is noteworthy that VHHB7, H7, C12b, and E4 were not evaluated here because they have nearly the same CDR3 as B6 and bind to the same epitopes, and they can be considered part of the same family. Figure 17 -D also indicates that there is no VHH interference with phage-C5 binding.

[0426] Overall, these results demonstrate that the various VHHs of the present invention (e.g., B6 and its variants; C5 and its variants; or E8, C10a, H1 and their variants) possess different epitopes. These results further highlight the diversity of the VHHs of the present invention, allowing for the targeting of several TfR epitopes with potentially different effects.

[0427] Example XVIII: Competition assay between purified VHH with affinity for TfR and the natural ligand (Tf)

[0428] To evaluate the ability of the selected VHH to compete with transferrin (Tf) (the natural ligand of TfR) for receptor binding, a competition assay was performed using flow cytometry. All experiments were performed in 96-well plates at 2 x 10⁵ cells / well with shaking at 4 °C. CHO cell lines expressing hTfR fused to EGFP were saturated with 2% PBS / BSA for 30 min to avoid nonspecific binding, and then incubated for 1 hr with gradually increasing concentrations of the competitor (VHH or Tf). Then, EC80 tracer (VHH or Tf-A647) was added and incubated for another 1 hr. With Tf-A647 as the tracer, cells were washed three times with 2% PBS / BSA, fixed by incubation with 2% PBS / PFA for 15 min, washed once with PBS, and finally resuspended in PBS. Cells were washed once with 2% PBS / BSA and incubated for 1 hour with anti-6His-tagged antibody (mouse) using VHH as a tracer. After washing twice with 2% PBS / BSA, cells were incubated for 1 hour with an anti-mouse secondary antibody conjugated to Alexa647. Cells were finally fixed using Tf-A647 as a tracer. Fluorescence levels were assessed using an Attune NxT flow cytometer (ThermoFisher Scientific). Experimental data were fitted using nonlinear fitting with GraphPad Prism® software.

[0429] TfR-bound VHH was used as a tracer ( Figure 18 -A) and competitors ( Figure 18 -B). In both cases, there is no competition between VHH and the ligand Tf, indicating that VHH binds to TfR at an epitope different from that of Tf. Maintaining the binding of Tf to its receptor is a prerequisite for limiting side effects and allowing TfR to perform its natural function.

[0430] Example XIX: Brain uptake of the homodimeric 13C3-HC-VHH fusion with a divalent TfR binding mode in C57Bl / 6 mice compared to uncarrierized 13C3.

[0431] To evaluate the potential of some VHH variants of the present invention with different mTfR binding properties to promote brain uptake of mouse 13C3 monoclonal antibodies, homodimeric fusions 13C3-HC-C5h20, 13C3-HC-C5h9, 13C3-HC-C5h16, 13C3-HC-C5h24, or unvectored 13C3 were first generated, and their epigenetic binding affinity (Kb) to mTfR was evaluated as described in Example XI (Design and Production of Therapeutic Antibodies Fusion with VHH). d app ) and maximum binding level (B max Unlike the fusion complex 13C3-HC-C5h20 / C5h9 / C5h16 / C5h24, which specifically and efficiently binds to mTfR, no significant binding was observed using the control 13C3. Due to B... max and K d app The parameters were determined so that the binding potential of different fusion modulosomes with mTfR was calculated as a normalized B. max / K d app Ratio x 100. For example... Figure 19 As shown in -A, 13C3-HC-C5h20 has the strongest mTfR binding potential (436.8), while 13C3-HC-C5h16 has about 3 times lower mTfR binding potential (128.2).

[0432] Next, the homodimeric fusions 13C3-HC-C5h20 / C5h9 / C5h16 / C5h24 or uncarrierized 13C3 were injected into the tail vein of C57Bl / 6 mice at a dose of 35 nanomoles / kg. Blood was collected and the mice were perfused with saline solution at 6 and 18 hours post-injection (pi). The brains were collected, and half of the brain was treated using the capillary depletion method described in Example XII (In vivo brain uptake of 13C3-HC-VHH and 13C3-LC-VHH fusions) to separate the capillary network from the brain parenchyma and extracellular fluid (ECF). The other half of the mouse brains were directly processed (homogenized and lysed) for whole-brain quantification. The amount of 13C3-HC-VHH in the whole brain, brain parenchyma, and ECF was measured using an in-house qualified Meso Scale Discovery (MSD) direct coating (Abeta) immunoassay (CV < 20%, recovery ± 30%). The results were presented as concentrations (nM) Figure 19 -B, C, E, F) or the percentage ratio of brain tissue to plasma ( Figure 19 -D).

[0433] The results showed that the plasma concentrations of the TfR-binding conjugates 13C3-HC-C5h20 / C5h9 / C5h16 / C5h24 were lower than those of the 13C3 control antibody at 18 hours, which was consistent with the target-mediated clearance of the TfR-targeting conjugates. Figure 19 -B). However, compared with the uncarrierized 13C3 control antibody, 13C3-HC-C5h20 / C5h9 / C5h16 / C5h24 showed a significant advantage in brain uptake at 6 hr pi ( Figure 19 -C), and this advantage remained significant for 13C3-HC-C5h9 at 18 hr pi. When observing the brain-to-plasma ratio, a significant advantage was confirmed for all TfR-binding conjugates 13C3-HC-C5h20 / C5h9 / C5h16 / C5h24, particularly at 18 hr pi.

[0434] The BBB-crossing ability of 13C3-HC-C5h20 / C5h9 / C5h16 / C5h24 in the posterior compartment of the brain was further confirmed by the fact that, at 6 hr pi, the concentrations of 13C3-HC-C5h20 / C5h9 / C5h16 / C5h24 in the brain parenchyma and ECF were 7.5 to 9 times and 2 to 7.2 times that of the uncarrierized 13C3 antibody, respectively, and at 18 hr pi, the concentration of 13C3-HC-C5h9 / C5h24 in the brain parenchyma was still significantly 2.2 to 2.5 times that of 13C3. Interestingly, both of these constructs exhibited moderate mTfR binding potential.

[0435] Overall, these results demonstrate that the TfR-targeting VHH of the present invention can be used for efficient delivery of therapeutic antibodies to the brain. Furthermore, due to the TfR binding potential of the VHH of the present invention, the intensity and duration of antibody delivery can be modulated.

[0436] Example XX: Brain uptake of the heterodimer VHH-Fc fusion with a monovalent TfR binding mode in C57Bl / 6 mice

[0437] To evaluate the potential of certain VHH variants of the present invention, which have a monovalent TfR binding mode, to promote brain uptake of prototype proteins (e.g., human IgG1 Fc fragments without effector function), heterodimers (VHHs) were first generated using a mortar and pestle technique. 1k -hFc LALA A fusion.

[0438] To produce the fusion protein, a protein encoding (VHH) was synthesized. 1k -hFc LALA Fc-type IgG1 (containing Fc-type IgG1 with T366W and L234A / L235A mutations to reduce effector function, wherein the amino acid numbering is based on the EU index in the Kabat of full-length hIgG1) and hFc LALAA DNA fragment of mortar (carrying T366S, L368A, Y407V and L234A, L235A mutations) was cloned into the pCDNA3.4 vector (ThermoFisher Scientific) to encode a human IgG1 Fc fragment containing a single VHH in its N-ter region. The fusion protein was prepared using the Expi293 expression system, following the manufacturer's instructions (LifeTechnologies), by co-transfecting plasmids encoding VHH hFc mortar and mortar at a 1:4 ratio. 72 hours post-transfection, the supernatant was first recovered and purified using affinity chromatography (HiTrap Prot A, Cytiva) on an AKTA purification system (GE Healthcare), followed by size exclusion chromatography (Hiload 16 / 600 Superdex 200, Cytiva). The purified fusion protein was validated by SDS-PAGE, SEC-HPLC, and LC-MS to ensure its purity (over 92%) and correct identity.

[0439] Secondly, as described in Example XIX, the TfR-targeting heterodimer (C5h18) was evaluated. 1k -hFc LALA (C5h19) 1k -hFc LALA Fusion and (C5neg) 1k -hFc LALA The binding potential of the negative control fusion to mTfR. Figure 20 As shown in -A, (C5h18) 1k -hFc LALA (C5h19) 1k -hFc LALA Both exhibited a low mTfR binding potential of approximately 1, consistent with the low apparent affinity of uncoupled VHH C5h18 / C5h19 for mTfR (see Table 5). (Using (C5neg)) 1k -hFc LALA No significant TfR binding was observed in the control group.

[0440] Third, the heterodimer (C5h18) targeting TfR. 1k -hFc LALA (C5h19) 1k -hFc LALA Fusion and (C5neg) 1k -hFc LALAThe negative control fusion was administered subcutaneously to the tail vein of C57Bl / 6 mice at a dose of 70 nanomoles / kg. Plasma, brain, posterior vascular compartment (i.e., depleted cerebral microvascular parenchyma and ECF), and liver were recovered at 6, 18, and 42 hr p.m. VHH in each tissue was measured using an internal anti-Fc antibody ELISA assay. 1k -hFc LALA The amount of the fusion compound. The result is presented as concentration (nM).

[0441] (C5h18) 1k -hFc LALA (C5h19) 1k -hFc LALA The plasma pharmacokinetics of the fusion compound were nearly negative (C5neg). 1k -hFc LALA The control group showed a low clearance rate mediated by TfR, which is consistent with (C5h18). 1k -hFc LALA and (C5h19) 1k -hFc LALA The low TfR binding potential of the fusion is consistent ( Figure 20 -B). Interestingly, in the posterior compartment of the brain and cerebral blood vessels, (C5h18) 1k -hFc LALA and (C5h19) 1k -hFc LALA Fusion and (C5neg) 1k -hFc LALA Compared to the negative control fusion, it showed significant and sustained uptake until 42 hours after injection. Figure 20 -C, D, E). (C5h19) 1k -hFc LALA It reaches its peak brain concentration (4 nM) at 42 hr pi, while (C5h18) 1k -hFc LALA It reached its peak value (4.8 nM) at 18 hr pi (C5neg) 1k -hFc LALA The control group remained stable at 1.7 nM between 18 and 42 hours. In the liver, the concentration was (C5h18 / C5h19). 1k -hFc LALA Control (C5neg) 1k -hFc LALA No cumulative differences were observed between them. Figure 20 -F).

[0442] Overall, these results demonstrate that the present invention’s TfR-targeting VHH, with its low TfR binding potential, can be used to efficiently deliver proteins of interest across the BBB to the brain without significantly altering their plasma pharmacokinetics.

[0443] Example XXI: Targeted delivery of neurotensin peptide (NT(8-13)) in the CNS in C57Bl / 6 and B-hTfR mice

[0444] Neurotensin is a 13-amino acid neuropeptide that can induce a variety of central effects, including hypothermia. However, upon systemic administration, neurotensin peptides do not induce any central effects due to inefficient BBB crossing. The potential of certain VHH variants of this invention to promote brain uptake of shorter active fragments (NT(8-13)) of neurotensin peptides and induce central hypothermia was evaluated in wild-type and human TfR extracellular domain-expressing mice (C57Bl / 6 and B-hTfR (Biocytogen) mice, respectively).

[0445] To generate the fusion protein, a DNA fragment encoding VHH-NT(8-13) was synthesized and cloned into the pHEN1 vector to encode a protein containing a single copy of VHH at its N-terminus and an NT(8-13) peptide at its C-terminus. A sequence encoding a hexahistine affinity tag, followed by a (G4S)2 linker, was introduced between the sequences encoding VHH and NT(8-13). VHH-NT(8-13) was then expressed in *Escherichia coli* (strain BL21-DE3) and purified based on the His tag by immobilized metal affinity chromatography. Endotoxin was removed by purification steps including size exclusion chromatography and filtration on a Mustang Q or E membrane (Pall). The purified fusion protein was validated by SDS-PAGE, SEC-HPLC, and LC-MS to ensure its purity (over 95%) and correct identity. A LAL assay (Pierce) was also performed to determine endotoxin levels.

[0446] VHH B8h1-NT(8-13) (K) was evaluated in a cell-based assay. d app 2.8 nM) and B8V1-NT(8-13) (K d app 8.3 nM) for mTfR ( Figure 21 -A) and C5V30-NT(8-13) (K d app 16.7 nM), C5h18-NT(8-13) (K d app 16.8 nM), B8V31-NT(8-13) (K d app 25.6 nM) and C12b-NT(8-13) (Kd app 1865 nM) for hTfR ( Figure 21 The apparent affinity of -B) was observed. No significant m / hTfR binding was observed using the control C5neg-NT (8-13).

[0447] Next, in C57Bl / 6 or B-hTfR mice, following a single intravenous injection of 500 nanomolars / kg, rectal temperature measurement was used to monitor the hypothermia induction of some VHH-NT(8-13) fusions of the present invention. Figure 21 -C, D). The results are presented on a graph as mouse body temperature (in °C) as a function of time (minutes after injection), or on a bar graph as the area under the curve (AUC) relative to the mouse's mean basal body temperature (38 °C).

[0448] The negative control C5neg-NT (8-13) had no significant effect on the body temperature of C57Bl / 6 or B-hTfR mice. Conversely, B8h1-NT (8-13) and B8V1-NT (8-13) induced a significant decrease in body temperature in C57Bl / 6 mice, reaching a maximum of -3 to -4°C at 60–75 min pi. C5h18-NT (8-13), B8V31-NT (8-13), C5V30-NT (8-13), and C12b-NT (8-13) induced a significant decrease in body temperature in B-hTfR mice, reaching a maximum of -5 to -7°C at 45–60 min pi. Interestingly, C5h18, which was shown in Example XX to be able to direct the IgG1 Fc fragment to the brain of C57Bl / 6 (wild-type) mice, was also shown in this example to be effective in delivering NT(8-13) to the brain of mice (B-hTfR) expressing the extracellular domain of hTfR, thus validating its cross-species TfR targeting potential.

[0449] Overall, these results indicate that in mice expressing human or mouse versions of TfR, the TfR-targeting VHH of the present invention allows NT (8-13) to cross the BBB and reach its brain target in pharmacologically active amounts.

[0450] Example XXII: Brain uptake of the heterodimer VHH-Fc-NT fusion with a monovalent TfR binding mode in C57Bl / 6 and B-hTfR mice

[0451] To evaluate the potential of certain VHH variants of the present invention with a monovalent TfR binding mode to promote brain uptake of human IgG1 Fc fragments with no-effect function fused to NT(8-13) peptides, heterodimers (VHHs) were generated using a mortar and pestle technique. 1k -hFc LALA-NT(8-13) fusion variant. To generate the fusion protein, a protein encoding (VHH) was synthesized. 1k -hFc LALA Fc-type glutamate (which has Fc-type glutamate with T366W mutation and L234A, L235A mutation to reduce effector function, wherein the amino acid numbering is based on the EU index in the Kabat of full-length hIgG1) and hFc LALA A DNA fragment of NT(8-13) (containing T366S, L368A, Y407V and L234A, L235A mutations, plus a sequence encoding a (G4S)3 adapter followed by NT(8-13) added to its C-terminus) was cloned into the pCDNA3.4 vector (ThermoFisher Scientific) to encode a non-effector-functional human IgG1-Fc fragment containing a single VHH copy in the N-terminus and a single NT(8-13) copy in the C-terminus. The fusion protein was prepared as described in Example XIX.

[0452] Secondly, the TfR-targeting heterodimer (C5h18) was evaluated as described in Example XIX. 1k -hFc LALA -NT(8-13), (C10a) 1k -hFc LALA -NT(8-13), (H1) 1k -hFc LALA -NT(8-13) fusion and (C5neg) 1k -hFc LALA -NT(8-13) negative control fusion's epigenetic affinity for hTfR. For example... Figure 22 As shown in the table for -A, (C10a) 1k -hFc LALA -NT(8-13) and (H1) 1k -hFc LALA Both -NT(8-13) and (C5h18) exhibit similar apparent affinity for hTfR, approximately 16 nM. 1k -hFc LALA -NT(8-13) exhibited slightly lower apparent affinity, at 22 nM. (Using (C5neg)) 1k -hFc LALA No significant TfR binding was observed in the control group -NT(8-13).

[0453] Third, the heterodimer (C5h18) targeting TfR. 1k -hFc LALA -NT(8-13), (C10a) 1k -hFcLALA -NT(8-13), (H1) 1k -hFc LALA -NT(8-13) fusion and (C5neg) 1k -hFc LALA The -NT(8-13) negative control fusion was intravenously injected into the tail vein of B-hTfR mice (n=4) at a dose of 20 nanomoles / kg. B-hTfR mice were also treated with 150 µl as (VHH). 1k -hFc LALA -NT(8-13) formulation buffer was injected with PBS buffer. 24 hours after injection, blood from previously injected mice was collected and the mice were perfused with saline solution. Brains were collected, and one half of the brain was treated using the capillary depletion method described in Example XII (In vivo brain uptake of 13C3-HC-VHH and 13C3-LC-VHH fusions) to separate the capillary network from the brain parenchyma and extracellular fluid (ECF). The other half of the mouse brain was directly processed (homogenized and lysed) for whole-brain quantification. (VHH) 1k-hFc in each tissue was measured using an internal anti-Fc antibody ELISA assay. LALA The amount of -NT(8-13) fusion complexes. The results are presented as concentrations (nM) ( Figure 22 -B, C, D, E).

[0454] (C5h18) 1k -hFc LALA -NT(8-13), (C10a) 1k -hFc LALA -NT(8-13), (H1) 1k -hFc LALA The plasma concentration of -NT(8-13) fusion variants is lower than that of negative C5neg variants. 1k -hFc LALA In contrast, the clearance rate was consistent with that mediated by TfR and was consistent with its hTfR apparent affinity.

[0455] Interestingly, in the brain and the posterior compartment of cerebral blood vessels, (C5h18) 1k -hFc LALA -NT(8-13), (C10a) 1k -hFc LALA -NT(8-13) and (H1) 1k -hFc LALA -NT(8-13) fusion and (C5neg) 1k -hFc LALA -NT(8-13) negative control fusions showed significantly higher concentrations ( Figure 22-C, D, E). Specifically, in the whole brain (C10a) 1k -hFc LALA The accumulation of -NT(8-13) is (C5neg). 1k -hFc LALA -NT(8-13) 6.4 times ( Figure 22 -C), and in substance (H1) 1k -hFc LALA -NT(8-13), (C10a) 1k -hFc LALA -NT(8-13) and (C5h18) 1k -hFc LALA The cumulative effect of -NT(8-13) is 17 to 7 times ( Figure 22 -E).

[0456] Finally, in order to study (C5h18) 1k -hFc LALA The potential of -NT(8-13) to induce CNS-dependent hypothermia was demonstrated by injecting the molecule into B-hTfR mice at a dose of 20 nanomoles / kg. (C5neg) 1k -hFc LALA -NT(8-13) negative control fusion and PBS buffer were used as controls. Rectal temperature was monitored until 4 hours after injection. Figure 22 -F). The control molecule and the molecular formulation buffer (PBS buffer) did not induce any low temperatures, while (C5h18) 1k -hFc LALA -NT(8-13) induced a maximum decrease in body temperature of -5.8°C in mice 90 min after injection, which persisted until approximately 200 min after injection, confirming that the C5h18 VHH of this invention crosses the blood-brain barrier and releases Fc LALA -NT(8-13) has the ability to be rapidly transported to the brain at pharmacologically active levels.

[0457] Overall, these results confirm that the TfR-targeting VHHs of the present invention, with moderate TfR affinity, can be used to efficiently deliver proteins of interest across the BBB to the brain. Furthermore, different VHHs of the present invention targeting different epitopes can be used to vectorize payloads and facilitate their brain delivery.

[0458] Example XXIII: Oligonucleotide Sequences and Their Coupling with TfR-Binding VHH

[0459] The potential of some VHH variants of the present invention to promote the functional delivery of oligonucleotides in the CNS was evaluated using VHs coupled with siRNA or gapmer ASO sequences listed in Table 7 below. The chemically modified siRNA sequences with RNAi activity target ubiquitous superoxide dismutase 1 (SOD1) mRNA in mice and rats (siSOD1m) or humans and non-human primates (NHPs, including East African baboons, rhesus monkeys, and cynomolgus monkeys) (which are adapted from siSOD1m to match human and NHP-derived SOD1 mRNA (siSOD-1)). The single-stranded gapmer, rBase H-active antisense oligonucleotides (ASOs) used in the examples below target the ubiquitously expressed long noncoding RNA (lncRNA) MALAT-1, which is preferentially enriched in nucleoli, where it regulates posttranscriptional RNA processing (…). MALAT1 -ASO1), and adapted from "AS1" by Tripathi et al. by introducing the modification "mc" (i.e., 2'-O-methoxyethyl-5-methyl-cytidine; 2'MOE meC) in the sequence of SEQ ID NO: 405 in Table 7 below to replace 2'-MOE-C (i.e., 2'-O-methoxyethyl-cytidine).

[0460] Table 7:

[0461] siSOD1m: A double-stranded siRNA targeting mouse and rat superoxide dismutase 1 (SOD1) mRNA; hMALAT1-ASO: A gapmer antisense oligonucleotide (ASO) targeting human MALAT-1 long non-coding RNA; lowercase indicates modification of the 2'-O-methyl (2'-OMe) sugar of adenosine, cytidine, guanosine, or uridine nucleotides, respectively; italic uppercase indicates modification of the 2'-deoxy-2'-fluoro (2'F) sugar of adenosine, cytidine, guanosine, or uridine nucleotides, respectively; dN indicates deoxynucleotide (e.g., dT indicates deoxythymidine). The symbol indicates a link between phosphate thioester (PS) nucleosides; L indicates a linker (coupling arm); VP indicates a vinyl-phosphonate; lowercase italics indicate modification of adenosine, guanosine, or thymidine nucleotides with 2'-O-methoxyethyl (2'-MOE) sugar; mc indicates 2'-O-methoxyethyl-5-methylcytidine (2'MOE meC).

[0462] The oligonucleotides were synthesized or purchased from Horizon Discovery Biosciences Ltd. or GeneLink Inc., and had coupling arms, such as hexylamino linkers, introduced into the 3'-terminus (3'SS) of the sense strand of the siRNA duplex or the 5'-terminus of the single-stranded ASO.

[0463] The overall coupling strategy involves pooling synthesis, such as the strategy described in WO 2020 / 144233, which consists 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 previous section]). Figure 23 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 24 ).

[0464] Example XXIV: Evaluation of the binding of VHH-oligonucleotides and VHH-hFc-oligonucleotide conjugates to mTfR and hTfR in living cells

[0465] The binding of VHH-oligonucleotide and VHH-hFc-oligonucleotide conjugates to mTfR and hTfR, respectively, was evaluated using a competitive assay on live cells expressing the receptors of interest (i.e., murine neuroblastoma Neuro-2a cells (N2a) or human breast ductal carcinoma MCF-7 cells). MCF-7 and N2a cells were grown in Dulbecco modified Eagle medium (DMEM) GlutaMAX supplemented with 10% v / v FBS at 37°C and 5% CO2. Cells were seeded at a density of 50,000 cells / well in 96-well plates two days prior to the experiments. On the day of the experiment, free VHH, VHH-oligonucleotide, or VHH-hFc-oligonucleotide conjugates diluted at various concentrations were incubated at different concentrations on each cell line with a subsaturated concentration of the fluorescent reference compound C5-Alexa680 (100 nM) at 37°C for 3 hours in DMEM supplemented with 1% bovine serum albumin (BSA). After treatment, the cells were washed with D-PBS and dissociated by incubation with trypsin / EDTA at 37°C for 5 min, followed by the addition of cold medium (4°C) to inhibit trypsin activity. The resuspended cells were transferred to 96-well deep-well plates (V-bottom) 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 the cells were fixed at room temperature for 15 min. PFA was removed by centrifugation (5 min, 3000 rpm, 4°C), and the cells were resuspended in 5 mM EDTA in D-PBS. Cells were then used with an Attune setting. TM Attune for NxT v3.1.2 TM Quantification of A680-related fluorescence signals in cells was performed using an NxT flow cytometer (Thermo Fisher Scientific). A GraphPad Prism was used. ® The software uses nonlinear fitting to fit the experimental data (three parallel experiments) to determine the apparent... K i Suppression constant. Figure 25 The figure shows a representative inhibition curve, and the table shows the mean ± SD of at least two independent experiments.

[0466] In expressing mouse ( Figure 25 A) or TfR ( Figure 25In cells of B), all tested conjugates (e.g., C5-siSOD1m-5'VP, C5-hFc-siSOD1m-5'VP, B8-MALAT1-ASO1, C5-siSOD1h, or B8-siSOD1h) induced concentration-dependent inhibition of binding to and uptake of the reference compound, epigenetically. K i The values ​​are similar to those of free VHH (e.g., C5 or B8), confirming the efficient binding of VHH-oligonucleotide conjugates to TfR.

[0467] Example XXV: In vitro functional delivery and gene silencing potential of TfR-binding VHH-oligonucleotides and VHH-hFc-oligonucleotide conjugates after free uptake in mouse and human cells.

[0468] Mouse neuroblastoma (Neuro-2a) and human glioblastoma (LN229) cells 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 in 96-well plates at densities of 2000 cells / well (Neuro-2a) and 10000 cells / well (LN229). 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) or the Nucleospin RNA XS Kit (Macherey-Nagel) 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 or MALAT-1 genes (Applied Biosystems). Expression data were analyzed using the DDCq method, normalized to the expression of the reference RpL13 or GAPDH genes, 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 26 A, 26C, and 26D show representative inhibition curves.

[0469] Although uncoupled siSOD1m-5'VP affects mouse Neuro-2a cells mSOD1 mRNA levels were not shown to be affected, but mTfR-binding VHH-siSOD1m-5'VP and VHH-hFc-siSOD1m-5'VP conjugates (e.g., C5-siSOD1m-5'VP or C5-hFc-siSOD1m-5'VP) showed potent and concentration-dependent effects. mSOD1mRNA downregulation had average IC50 values ​​of 7.4 nM and 8.7 nM, respectively. Figure 26 A). On the same Neuro-2a cell line, various VHH-siSOD1m conjugates (i.e., conjugates containing the following VHH molecules, wherein the VHH molecules are B8 or C5 or variants thereof, such as B8h1, C5, C5V1, C5V7, C5V8, C5V13, C5h18, C5h19 listed in Table 1) and VHH- MALAT1 -ASO conjugates (containing VHH molecules as shown in Table 1, such as B8) exhibited similar specific and efficient knockdown effects (respectively...). Figure 26 B and 26C). Additionally, on the human LN229 cell line, targeted human... SOD1 The hTfR binding of mRNA to the VHH-siSOD1h conjugate showed a similar nanomolar IC50. Figure 26 D). These results demonstrate that the VHH-oligonucleotides or VHH-hFc-oligonucleotide conjugates of the present invention efficiently bind to mouse and human TfRs on the cell surface, followed by TfR-mediated endocytosis, endosome escape, and cytosol or nuclear delivery of the active oligonucleotides, thereby allowing for efficient regulation of target gene expression using different oligonucleotide forms.

[0470] EXAMPLE XXVI: Distribution of TfR in brain microvessels and parenchymal cells

[0471] The distribution of TfR in the brains of wild-type C57Bl / 6 mice was assessed by immunohistochemistry (IHC) on frozen sections of the brain prepared from quick-frozen (dry ice) mouse hemispheres. Mouse TfR was detected using the 8D3 antibody (Novus Biologicals, NB100-64979), and its colocalization with microvessels stained with anti-collagen IV antibody (United States Biological, C7510-50H) or neurons stained with anti-NeuN antibody (Merck Millipore, MAB377) was assessed using confocal imaging. Figure 27 Extensive expression of TfR in all regions of the mouse brain is demonstrated. Examples are shown in the cortex or hippocampus, where TfR was found to be highly expressed at the BBB, as evidenced by extensive colocalization with type IV collagen-positive microvessels, and also highly expressed in parenchymal cells such as neurons, as evidenced by strong colocalization with the neuronal marker NeuN. Therefore, the distribution of TfR in the brain demonstrates that the VHH ligand of the present invention, targeting TfR, not only has the potential to cross the BBB but also the potential to mediate the endocytosis and intracellular delivery of therapeutic payloads in parenchymal cells.

[0472] Example XXVII: CNS knockdown effect of VHH-siRNA conjugate after local ICV administration in wild-type C57Bl / 6 mice

[0473] The efficacy of VHH-siSOD1m conjugate in mediating brain and spinal cord dysfunction in wild-type C57Bl / 6 mice after administration to the mid-lateral ventricle (ICV) was evaluated. SOD1Potential for mRNA knockdown. Wild-type C57Bl / 6 mice (n=4 per group) were injected with a single ICV dose (right ventricle, 5–10 µL, 0.75 µL / min) of 100 µg (molar equivalent of siRNA) PBS vector (control), unconjugated siSOD1m-5'VP, or a specified VHH-siSOD1m-5'VP conjugate. Seven days post-injection, tissue samples including the whole brain (right hemisphere), brain regions (from the left hemisphere), spinal cord, liver, and gastrocnemius muscle were harvested, flash-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 equipped with a Cryolys Evolution cooling system (Bertin Instruments). Total RNA was extracted from tissue homogenates using the rNeasy 96QIAcube HT kit (QIAGEN) in the QIAcube HT system. RNA samples were analyzed and quantified using the Fragment Analyzer RNA Kit in the 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 ΔΔCq 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. Both unconjugated siSOD1m-5'VP and unconjugated C5neg-siSOD1m-5'VP induced only moderate (brain) and mild (spinal cord) knockdown (KD) effects in CNS tissues, with no effect on the liver, indicating no retention and uptake in parenchymal cells. The use of the unconjugated C5neg-siSOD1m-5'VP conjugate also did not provide size-related improvements. In contrast, the TfR-conjugated C5-siSOD1m-5'VP conjugate induced strong KD effects in both the brain and spinal cord, with approximately 80-90% KD reduction. Figure 28(B) demonstrates the efficient distribution and TfR-dependent retention and functional uptake of TfR-binding VHH-siRNA conjugates in brain parenchymal cells and the spinal cord. TfR-binding C5-siSOD1m-5'VP conjugates, but not unconjugated siSOD1m-5'VP or unconjugated C5neg-siSOD1m-5'VP, also induce a strong KD effect in muscle tissue (e.g., gastrocnemius muscle).

[0474] Furthermore, analysis of brain regions in the left hemisphere showed that the KD effect of TfR-binding C5-siSOD1m-5'VP conjugates was very uniform (60-90% KD) across all tested regions, including deep structures such as the striatum or hippocampus, while unconjugated siSOD1m-5'VP exhibited moderate to high heterogeneity (10-60% KD). Figure 28 C). These results demonstrate the efficient distribution and TfR-mediated uptake of TfR-binding VHH-siRNA conjugates in all regions of the CNS and muscle tissue following a single local injection (e.g., ICV). This efficient TfR targeting and functional uptake potential translates into a robust and stable intracellular reservoir in all regions of the brain and spinal cord, as well as muscle, enabling sustained release of RNAi-active siRNAs from endolysosomal compartments into the cytosol, where target mRNA molecules are loaded and continuously degraded by the RNA-induced silencing complex (RISC). This CNS- and muscle-specific functional delivery modality enables these TfR-binding VHH oligonucleotide conjugates to be used to treat both CNS and neuromuscular disorders.

[0475] Example XXVIII: CNS knockdown effect and siRNA biodistribution of VHH-siRNA conjugate after local ICV administration in wild-type C57Bl / 6 mice

[0476] Following a single ICV administration in wild-type mice, the biodistribution of the VHH-siRNA conjugate and the target of the VHH-siSOD1m conjugate were evaluated in brain and spinal cord samples using in situ hybridization (ISH). SOD1 CNS knockdown of mRNA. Wild-type C57Bl / 6 mice (n=4 per group) were injected with a single ICV (right ventricle) dose of 100 µg (molar equivalent of siRNA) PBS vector (control) or TfR-binding VHH-siSOD1m-5'VP conjugate. Seven days post-injection, the right (ipsilateral) hemisphere was removed, flash-frozen in 10 volumes of NucleoProtect RNA stabilization reagent (Macherey-Nagel), and stored at -20°C until processing for use. SOD1qPCR quantification of mRNA was performed, and the left (contralateral) cerebral hemisphere and part of the cervical spinal cord were isolated, flash-frozen on dry ice and stored at -80°C until preparation for further ISH and imaging as described below. Figure 29 A).

[0477] For RT-qPCR bioanalysis, frozen tissue samples from NucleoProtect were homogenized in QUIAzol lysis reagent using a Precellys Evolution tissue homogenizer equipped with a Cryolys Evolution cooling system (Bertin Instruments). 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 the 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 ΔΔCq 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. Mice injected with the TfR-binding C5-siSOD1m-5'VP conjugate induced mRNA levels in all tested regions of the ipsilateral (right) hemisphere of the brain (e.g., frontal cortex, parietal-temporal cortex, thalamus, hypothalamus, hippocampus, striatum, cerebellum, brainstem, and olfactory bulb) compared to mice injected with PBS. SOD1 Strong downregulation of mRNA, 70-90%.

[0478] For ISH bioassays, 20 μm sagittal frozen sections of the left hemisphere or coronal sections of the spinal cord were prepared using a Leica cryostat at -20°C, collected on Superfrost Plus slides, and stored at -80°C until processing. For RNAScope® or miRNAScope® assays, slides containing frozen sections were immediately transferred to 10% neutral buffered formalin (NBF) and fixed at RT for 1 h 30. Fluorescent RNAScope® assays for mRNA detection or chromogenic miRNAScope® assays for siRNA detection were performed according to the manufacturer’s protocol (Advanced Cell Diagnostics, ACD). RNAscope® assays were performed using the RNAscope Multiplex Fluorescence Kit v.2 (ACD), and miRNAscope® assays were performed using the miRNAscope HD Kit RED assay (ACD). After tissue pretreatment, sections were compared with mouse-specific assays. SOD1 mRNA, neuron-specific MAP2 , DAT (Dopaminergic neurons) or ChAT (cholinergic interneurons) mRNA, astrocyte-specific GJA1 mRNA, oligodendrocyte-specific OLIG2 mRNA or microglia-specific TMEM119 Hybridization was performed using probes targeting the mRNA (ACD) or probes specifically targeting the siSOD1m antisense strand (AS). After signal amplification, the slides were counterstained with DAPI (RNAscope®) or hematoxylin solution (miRNAscope®) and then images were acquired using a Zeiss Axio Scan.Z1 slide scanner. Mice injected with the TfR-binding C5-siSOD1m-5'VP conjugate showed extensive biodistribution of the siSOD1m AS strand in all brain regions on midsagittal mouse brain tissue sections, as well as in the white and gray matter of the spinal cord, as assessed using miRNAScope®. Figure 29 B and 29E (left figure). As assessed using RNAScope®, this distribution is associated with broad and potent [various factors]. SOD1 mRNA knockdown-related ( Figure 29 B and 29E (right figure). SOD1 and MAP2 , DAT or ChAT Co-detection of mRNA revealed its presence in all tested neuronal populations and all tested regions (e.g., cortical and hippocampal pyramidal neurons, cerebellar Purkinje cells, or spinal neurons). SOD1 Strong knockdown of mRNA ( Figure 29C, 29F, and 29G). A notable aspect of these results is that strong KD was observed not only in regions with strong siRNA distribution (e.g., the cortex, hippocampus, striatum, or spinal gray matter), but also in some regions with lower distribution (e.g., the thalamus, cerebellum). This is similar to the observation in the corpus callosum (…). Figure 29 D and 29G), fornix or spinal white matter ( Figure 29 As observed in F), significant [symptoms] were also observed in regions containing only a small number of neurons but a large number of non-neuronal cells (e.g., GFAP or GJA1-positive astrocytes, OLIG2-positive oligodendrocytes, or TMEM119-positive microglia). SOD1 mRNA KD.

[0479] These results demonstrate that the TfR-binding VHH-siRNA conjugate of the present invention has potent functional delivery potential in neuronal and nonneuronal cells of the brain parenchyma and spinal cord after local CNS administration, efficiently exiting from the endolysosomal compartment to the cytosol, resulting in RISC loading and potent RNAi activity, even in regions exhibiting low intracellular reservoirs.

[0480] Example XXIX: Optimization of CNS functional delivery of VHH-siRNA conjugates by systemic administration in wild-type C57Bl / 6 mice

[0481] The VHH-siSOD1m-5'VP conjugate or the heterodimer VHH-hFc-siSOD1m-5'VP conjugate were generated, and their potential to trigger BBB crossing and functional delivery in CNS tissues after systemic administration was compared. Wild-type C57Bl / 6 mice (n=4 per group) were administered 15 mg / kg (siRNA molar equivalent) of unconjugated siSOD1m-5'VP or C5-siSOD1m-5'VP conjugate at four daily SC doses (QDx4) or 1.5 mg / kg (siRNA molar equivalent) of C5-hFc-siSOD1m-5'VP conjugate at three IV doses every two days (Q2Dx3) compared to PBS-injected mice (n=8). One week after the last administration (siSOD1m-5'VP and VHH-siSOD1m-5'VP groups) or two weeks after (VHH-hFc-siSOD1m-5'VP), tissue samples including the whole brain (right hemisphere), brain regions (from the left 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 equipped with a Cryolys Evolution cooling system (Bertin Instruments). Total RNA was extracted from the tissue homogenates using the rNeasy 96QIAcube HT kit (QIAGEN) in the QIAcube HT system. RNA samples were analyzed and quantified using the Fragment Analyzer RNA Kit in the 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 target mouse SOD1 and reference RpL13 and PUM1 genes (Applied Biosystems). Expression data were analyzed using the ΔΔCq method, normalized to the expression of the reference RpL13 gene (brain region) or RpL13 and PUM1 genes (whole brain, spinal cord, liver, kidney) (multiplex qPCR), 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.

[0482] Despite its broad chemical stabilizing effect, unconjugated siSOD1m-5'VP did not show a KD effect in the brain and spinal cord 7 days after administration of a total dose of 60 mg / kg (4 x 15 mg / kg siRNA molar equivalent, i.e., 4 μmol / kg). Conversely, TfR-binding conjugates such as C5-siSOD1m-5'VP conjugate or the heterodimer C5-hFc-siSOD1m-5'VP conjugate showed target efficacy in both the whole brain and spinal cord. SOD1 Significant downregulation of mRNA was observed, reaching 30-40% KD with the same dosing regimen of the C5-siSOD1m-5'VP conjugate, and up to 65% with the C5-hFc-siSOD1m-5'VP conjugate at a 13-fold lower dose (3 x 1.5 mg / kg siRNA molar equivalent, i.e., 0.3 μmol / kg). Figure 30 B). This demonstrates the efficient BBB crossing and functional uptake of the TfR-binding conjugate of the present invention in CNS tissues, with further improved effects when using the engineered C5-hFc-siSOD1m-5'VP conjugate, which is modified to prolong the plasma half-life, compared to the C5-siSOD1m-5'VP conjugate, at a much lower dose. Furthermore, the C5-hFc-siSOD1m-5'VP conjugate does not induce or induces only slight effects in excretory organs (B). Figure 30 C). Brain region analysis of the left hemisphere of mice treated with this conjugate showed a very uniform KD effec...

Claims

1. A coupling compound comprising: (i) One or more VHH molecules of the formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and (ii) at least one therapeutic compound or a carrier containing such therapeutic compound, The VHH molecule binds to TfR on the surface of nerve cells, and The VHH molecule described herein comprises: 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 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 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.

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: 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, wherein the VHH molecule comprises an amino acid sequence selected from any one of SEQ ID NO: 242-271, 274, 275, 675, 676 and 701.

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

5. The coupling compound according to claim 1, wherein the VHH molecule comprises an amino acid sequence selected from any one 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 coupling compound according to any one of the preceding claims, wherein the VHH molecule has an affinity (K0.1 nM to 2500 nM) of the VHH molecule. d Combined with TfR.

10. The coupling compound according to any one of the preceding claims, wherein the nervous system cells are CNS cells, such as any neurons, glial cells (including astrocytes, oligodendrocytes, ependymal cells, or microglia), choroid plexus cells, pericytes, endothelial cells, or fibroblasts of the brain, spinal cord, or retina; PNS cells, such as any neurons, glial cells (including Schwann cells), satellite cells, macrophages, endothelial cells, or fibroblasts, such as nerve cells of cranial ganglia, dorsal root ganglia, or autonomic ganglia; or cancer cells of the CNS or PNS system.

11. The conjugated compound according to any one of the preceding claims, wherein the therapeutic compound is selected from peptides, polypeptides, proteins, antibodies, and nucleic acids, wherein the nucleic acid is preferably selected from mRNA, ribozymes, or oligonucleotides, 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).

12. 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 or a serum albumin 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.

13. A pharmaceutical composition comprising the coupling compound according to claim 1 and a pharmaceutically acceptable support, carrier, or excipient.

14. The coupling compound according to any one of claims 1 to 12 or the composition according to claim 13, for the treatment of neurological disorders, including CNS diseases, preferably selected from Alzheimer's disease, Parkinson's disease, stroke, Creutzfeldt-Jakob disease, dementia, multiple sclerosis, spinal muscular atrophy, amyotrophic lateral sclerosis, epilepsy, migraine, encephalitis, lysosomal storage disorders of the brain, CNS pain, and CNS cancers such as brain cancer (e.g., glioblastoma); or PNS diseases, preferably selected from peripheral neuropathy such as diabetic neuropathy, cancer and chemotherapy-induced peripheral neuropathy (CIPN), HIV-induced neuropathy, leprosy (HD), infection-induced neuropathy such as Lyme disease, trauma... Sexual nerve damage (e.g., carpal tunnel syndrome and sciatica); hereditary sensorimotor peripheral neuropathy such as peroneal muscular atrophy (CMT), Friedreich ataxia (FA), giant axonal neuropathy (GAN); autoimmune and inflammation-induced peripheral neuropathy such as Guillain-Barré syndrome, lupus, rheumatoid arthritis, chronic inflammatory demyelinating polyneuropathy (CIDP), multifocal motor neuropathy, Sjögren's syndrome; DRG cell disorders such as sensory polyneuropathy (e.g., dorsal root ganglion lesions or sensory neuron diseases), sensory polygangliopathy, sensory polyradiculopathy, central sensory axonopathy, sensory polyradiculopathy; any complication of PNS disease, such as neuropathic pain; and PNS cancer.

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

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