Transferrin receptor binding molecules, conjugates thereof, and uses thereof

Abstract

The present invention relates to variable domains of camelid heavy chain-only (VHH) molecules that bind to TfR and their use for delivering molecules of pharmaceutical or diagnostic interest to cells and organs affected by pathological conditions, including, for example, cancer.

Description

[Technical field]

[0001] The present invention relates to transferrin receptor (TfR) binding molecules and uses thereof, in particular variable domains of camelid heavy chain only (VHH) molecules that bind to TfR at cell membrane surfaces, such as the blood-brain barrier (BBB), and their use for the delivery of molecules of pharmaceutical or diagnostic interest to TfR expressing tissues or organs, such as cells of the central nervous system or cancers. [Background technology]

[0002] According to Global Industry Analysts, the global market for drugs to treat central nervous system (CNS, brain and spinal cord) pathologies was approximately $100 billion in 2015, of which approximately $9 billion represented products derived from drug delivery technologies (Non-Patent Document 1). Thus, today, neurology is considered one of the three major therapeutic areas, along with cardiovascular medicine and oncology. Although the number of people suffering from CNS disorders and pathologies worldwide is greater than the number of people suffering from cardiovascular disease or cancer, neurology is an untapped market. This is explained by the fact that 98% of potential drugs for treating CNS pathologies do not cross the BBB (Non-Patent Document 2).

[0003] In fact, the brain is protected from potentially toxic substances by two major physiological barrier systems: the BBB and the blood-cerebrospinal fluid barrier (BCSFB). The BBB is considered the main route of plasma ligand uptake. Its surface area is approximately 5000 times greater than that of the BCSFB. The total length of the blood vessels that make up the BBB is approximately 600 km. Each cm of the cerebral cortex is 3 The total surface area of ​​the BBB is 20 m 2 (Non-Patent Document 3). Thus, the cerebral endothelium, which constitutes the BBB, represents a large potential exchange surface between blood and nervous tissue. However, due to its specific properties, the cerebral endothelium also poses a major obstacle to the use of drugs to treat CNS disorders.

[0004] In fact, the BBB is composed of brain capillary endothelial cells (BCECs) that exhibit unique properties not found in the fenestrated endothelial cells that compose the vasculature of other organs. BCECs form tight junctions and are surrounded by basement membranes, astroglial endfeet, pericytes, microglia and neuronal cells, which together constitute a highly selective barrier that controls the exchange of molecules between the blood and the brain, maintains brain homeostasis and protects the brain from toxins and pathogens very effectively. The difficulty is that the BBB is also impermeable to most molecules, including drugs and imaging agents. As a rule, only a few small lipophilic molecules of approximately 450 to 600 daltons (only 2% of drug candidates) are able to cross the BBB, and most, but not all, high molecular weight molecules, such as therapeutic peptides, proteins and antibodies that show promising results in in vitro and animal studies for the treatment of CNS disorders, do not cross the BBB.

[0005] Thus, the BBB is considered a major obstacle to be overcome in the development of new therapies to treat CNS disorders (Non-Patent Document 4). One of the research priorities related to molecular detection for treating, diagnosing, or imaging CNS pathologies is the development of methods that enable / increase the passage of active substances across the BBB.

[0006] One approach to circumventing the BBB is to administer drugs by direct injection into the CNS (e.g., intracerebroventricularly, intracerebrally, or intrathecally) or to disrupt the BBB, however, such highly invasive approaches have drawbacks (cost, short duration of action, etc.) and potential risks.

[0007] Pharmacological methods based on the addition of fatty or lipophilic groups to active substances (transcellular lipophilic diffusion, TLD) or the use of liposomes have been considered (Non-Patent Document 5). However, the addition of fatty or lipophilic groups or the use of liposomes can result in non-specific complexes with large molecular weights above the optimal limit of 450 daltons that are less effective at crossing the BBB (Non-Patent Document 6; Non-Patent Document 7).

[0008] Among the methods evaluated for delivering protein therapeutics to the brain, hijacking the cellular machinery involved in the transport of natural nutrients and endogenous ligands across the BBB is recognized as the safest and most effective (Fang et al., 2017; Jones and Shusta, 2007; Pardridge et al., 1992). Transport of macromolecules across the BBB can be facilitated by receptor-mediated transcytosis (RMT), a physiological process involving binding of ligands to receptors expressed by BCECs, internalization by endocytosis, intracellular trafficking and dissociation from the receptor in sorting endosomes, followed by release at the abluminal side of the BBB (Tuma and Hubbard, 2003; Xiao and Gan, 2013). In this regard, US Pat. No. 6,233,633 and US Pat. No. 6,233,633 disclose various peptides with high affinity for the LDL receptor that allow the transport of drugs or other molecules across the BBB.

[0009] Another receptor that has been studied to transport drugs across the BBB is the transferrin receptor (TfR), which is involved in iron transport to the brain by its ligand, transferrin (Tf) (Fishman et al., 1987). This receptor is also abundant in blood cells and lungs (Chan and Gerhardt, 1992), but has been shown to be highly expressed in brain endothelium (Jefferies et al., 1984; Pardridge et al., 1987). The use of Tf as a transporter has been investigated (Chang et al., 2009; Jain et al., 2011; Yan et al., 2013), but the transport mechanism of this molecule is saturable and competitive with endogenous Tf. Anti-TfR monoclonal antibodies, including the OX26 antibody (Moos and Morgan, 2001; Pardridge et al., 1991; Ulbrich et al., 2009) targeting rat TfR, or 8D3 (Pardridge, 2015; Zhang and Pardridge, 2005; Zhou et al., 2010), and the R17-217 (Lee et al., 2000; Pardridge, 2015; Ulbrich et al., 2009) antibody targeting mouse TfR, have been investigated as vectors for brain delivery (see also U.S. Pat. No. 5,393,633; U.S. Pat. No. 5,433,363; U.S. Pat. No. 5,433,363; U.S. Pat. No. 5,433,363). However, the drawbacks of these antibodies include a lack of cross-species reactivity, which prevents preclinical or clinical studies, and in particular a failure to bind to human TfR. Also, the ability of such antibodies to effectively transport drugs across the BBB remains under debate. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Publication No. 2010 / 046588 [Patent Document 2] International Publication No. 2011 / 131896 [Patent Document 3] International Publication No. 2012 / 075037 [Patent Document 4] International Publication No. 2013 / 177062 [Patent Document 5] WO 2012 / 75037 [Patent Document 6] International Publication No. 2016 / 077840 [Patent Document 7] International Publication No. 2016 / 208695 [Non-patent literature]

[0011] [Non-Patent Document 1] Jain,Jain PharmaBiotech Report,Drug Delivery in CNS disorders,2008 [Non-Patent Document 2] Pardridge,Mol.Interv.,2003,3,90-105 [Non-Patent Document 3] De Boer et al.,Clin.Pharmacokinet.,2007,46(7),553-576 [Non-Patent Document 4] Neuwelt et al.,Lancet Neurol.,2008,7,84-96 [Non-Patent Document 5] Zhou et al.,J.Control.Release,1992,19,459-486 [Non-Patent Document 6] Levin, J.Med.Chem.,1980,23,682-684 [Non-Patent Document 7] Schackert et al.,Selective Cancer Ther.,1989,5,73-79 Summary of the Invention [Problem to be solved by the invention]

[0012] Thus, despite advances in the field, there remains a need in the art for more effective methods and agents that can enhance delivery of drugs to the CNS. [Means for solving the problem]

[0013] The present invention provides new binding molecules that can be used to effectively transport molecules across the BBB. More specifically, the present invention discloses VHH molecules that bind to both human and non-human TfR and can deliver drugs to the CNS via transcytosis. The present invention shows that the VHH molecules of the present invention can effectively migrate through the CNS and deliver conjugated drugs or imaging agents in vivo. Thus, such VHHs represent useful molecules for use in therapeutic or diagnostic procedures.

[0014] Thus, an object of the present invention relates to VHH molecules that bind to human and non-human TfR.

[0015] A further object of the present invention relates to VHH molecules that bind to both human and non-human (e.g. rodent, such as mouse or rat) TfR with substantially similar affinity.

[0016] A further object of the present invention is a VHH molecule that binds to human and non-human TfR and is capable of crossing the human blood-brain barrier ("BBB").

[0017] Preferred VHHs of the present invention bind to both human and mouse TfR, are able to cross the human BBB, and have an affinity (Kd) for TfR less than 10 μM, preferably comprised between 0.1 nM and 10 μM.

[0018] The present invention also relates to a chimeric agent (also referred to herein interchangeably as "conjugate") comprising one or more VHHs as described above conjugated to at least one molecule or scaffold. The molecule conjugated to the VHH may be any active compound useful in medicine, for example a drug, a virus, a diagnostic agent, a tracer, etc. The chimeric agent may also comprise a stabilizing group (e.g. Fc or IgG) in addition to or instead of the active compound to increase the plasma half-life of the VHH or conjugate. Thus, a particular chimeric agent of the present invention comprises at least one VHH, a stabilizing group and an active compound in any order (e.g. a conjugate VHH-Fc-therapeutic agent).

[0019] Furthermore, the present invention provides a pharmaceutical or diagnostic composition comprising a chimeric agent as described above, and optionally a suitable excipient.

[0020] Furthermore, the present invention provides nucleic acids, vectors and host cells encoding a VHH or a chimeric agent as described above.

[0021] The present invention also provides a method for producing a VHH or a chimeric agent comprising culturing a host cell as described above under conditions allowing expression of the nucleic acid.

[0022] Furthermore, the present invention provides a method for making a chimeric drug comprising covalently or non-covalently conjugating one or more VHHs as described above to a molecule or drug or scaffold.

[0023] Another object of the invention relates to a VHH molecule or a chimeric agent as described above for use as a pharmaceutical or diagnostic agent.

[0024] Another object of the present invention relates to the use of a VHH molecule as described above for increasing the biological activity and / or CNS delivery of a substance of interest.

[0025] Another object of the present invention relates to a method for enhancing or enabling the passage of a molecule across the BBB, comprising coupling said molecule to a VHH as described above.

[0026] Another object of the invention is a method for treating a pathology in a subject, comprising administering to said subject a conjugate as described above.

[0027] Another object of the invention is a method for imaging a specific cell type, target tissue or organ in a subject, comprising administering to said subject a conjugate as described above.

[0028] Another object of the present invention is an improved method for treating a pathology in a subject with a drug, which improvement consists in coupling said drug to a VHH molecule as described above.

[0029] The present invention may be used in any mammal, particularly any human. [Brief description of the drawings]

[0030] [Figure 1] Figure 1 shows TfR expression in the BBB. Western blots were performed on membrane fractions of brain microvessels (BMVs) and brain microvascular endothelial cells (BMECs) from mice, rats, pigs, and non-human primates (NHPs, rhesus monkeys). The amount of protein loaded is shown below the figure. nd indicates undigested, dig indicates digested. [Diagram 2]Figure 2 shows validation of CHO cell lines expressing human or mouse TfR. (A) Map of plasmid constructs used to generate CHO-hTfR-EGFP cell lines. (B) Representative confocal micrographs of CHO-hTfR-EGFP cells (green) incubated with Tf-Alexa647 (250 μg / ml, red) for 1 h at 37°C. Cell nuclei were labeled with 0.5 μg / ml Hoechst #33342 (blue). Co-labeling appears in yellow in merged images. (C) Western blots on cell membrane preparations of CHO cells expressing hTfR-EGFP and mTfR-EGFP compared to CHO wild type using rabbit anti-TfR antibody (1 / 1000) or mouse anti-GFP antibody (1 / 1000) followed by HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (1 / 10000). [Diagram 3] Figure 3 shows cell surface binding and endocytosis of VHH A and VHH Z in CHO cells expressing hTfR and mTfR. Representative confocal micrographs of CHO-hTfR-EGFP and CHO-mTfR-EGFP cells (green) incubated with 20 μg / ml VHH A (A, B) and control VHH Z (C, D) for 1 h at 37°C and detected with mouse anti-cMyc (1 / 1000) and Alexa594-conjugated anti-mouse secondary antibodies (1 / 800, red) after PFA fixation and with or without subsequent Triton X-100 permeabilization of the cell membrane. Cell nuclei were labeled with 0.5 μg / ml Hoechst #33342 (blue). Co-labeling appears in yellow / orange in merged images. [Figure 4]Figure 4 shows the apparent Kd measurements of VHHs in hTfR and mTfR expressing CHO cell lines. (A) CHO-hTfR-EGFP and CHO-mTfR-EGFP cells were incubated with various concentrations of VHHs for 1 h at 4°C and detected by mouse anti-6His (1 / 1000) and Alexa647-conjugated anti-mouse secondary antibodies (1 / 200 or 1 / 400). Measurements were performed using flow cytometry. The fluorescence intensity ratios of each point were normalized with the corresponding EGFP signal (receptor expression) to obtain arbitrary units. Data are presented as mean ± SEM of three independent experiments. (B) The molecular weight (Da), theoretical pI, apparent Kd (nM) at human TfR, and apparent Kd (nM) at mouse TfR of the selected VHHs are shown. Data are presented as mean ± SEM of three independent experiments. NB is no binding. [Diagram 5] Figure 5 shows the competitive assay between VHH and Tf. (A) The principle of the competitive test is shown. In the first step, CHO-hTfR-EGFP cells were incubated with a dilution series of competitors at 4°C for 1 h. Then, a tracer at EC90 was added and incubated at 4°C for 1 h. The tracer was then detected with an appropriate colorimetric system. Measurements were performed using flow cytometry. The fluorescence intensity ratio of each point was normalized with the corresponding EGFP signal (receptor expression) to obtain arbitrary units. (B) CHO-hTfR-EGFP cells were incubated with competitors (Tf). Then, a tracer at EC90 (VHH) was added and detected by 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, an EC90 tracer (Tf-Alexa647) was added for direct detection. Data are presented as the mean ± SEM of three independent experiments. [Figure 6]Figure 6 shows the VHH conjugation method. Using either chemical conjugation or recombinant fusion, VHHs can be used to vector any kind of molecule, including non-exhaustively peptides, siRNAs, dyes, nanoparticles (NPs), liposomes, imaging agents, and antibodies. Furthermore, VHHs can be used to vector molecules as monovalent (VHH) or multivalent (VHHn) conjugates. [Figure 7] Figure 7 shows cell surface binding and endocytosis of VHH A-Fc and VHH Z-Fc fusion proteins in hTfR- and mTfR-expressing CHO cells. Representative confocal micrographs of CHO-hTfR-EGFP and CHO-mTfR-EGFP cells (green) incubated with 50 nM VHH A-Fc (A, B) and control VHH Z-Fc (C, D) for 1 h at 37°C and detected by Alexa594-conjugated anti-hFc antibody (1 / 1000, red) after PFA fixation and with or without subsequent Triton X-100 permeabilization of the cell membrane. Cell nuclei were labeled with 0.5 μg / ml Hoechst #33342 (blue). Co-labeling appears in yellow / orange in merged images. [Figure 8] Figure 8 shows the apparent Kd measurements of VHH-Fc and Fc-VHH in hTfR and mTfR expressing CHO cell lines. (A) CHO-hTfR-EGFP and CHO-mTfR-EGFP cells were incubated with various concentrations of VHH-Fc and Fc-VHH for 1 h at 4°C and detected by Alexa647-conjugated anti-hFc antibody (1 / 400). Measurements were performed using flow cytometry. The fluorescence intensity ratio of each point was normalized with the corresponding EGFP signal (receptor expression) to obtain arbitrary units. Data are presented as mean ± SEM of three independent experiments. (B) The molecular weight (Da), apparent Kd (nM) at human TfR, and apparent Kd (nM) at mouse TfR characteristics of selected VHH-Fc and Fc-VHH are shown. Data are presented as mean ± SEM of three independent experiments. NB, no binding to the control VHH (VHH Z). [Figure 9]Figure 9 shows uptake and transport of VHH A-Fc and VHH B-Fc fusion proteins in an in vitro BBB model. (A) Representative micrographs of rat brain microvascular endothelial cell (rBMEC) monolayers probed for uptake of 500 nM VHH A-Fc and VHH B-Fc after incubation with 200 nM Tf-Alexa647 (red) in live cells for 2 h, PFA fixation, and Triton X-100 permeabilization of the cell membrane, as detected by Alexa488-conjugated anti-fFc antibody (1 / 50, green). Cell nuclei were labeled with 0.5 μg / ml Hoechst #33342 (blue). Co-labeling appears in yellow in the merged image. (B) Schematic of an in vitro BBB model, a co-culture system containing primary rBMECs seeded on collagen IV / fibronectin-coated filters in the upper compartment (1) and primary astrocytes in the lower compartment (2). (C, D) Transport of VHH A-Fc, VHH B-Fc, and VHH Z-Fc fusion proteins across the rBMEC monolayer from the luminal (upper) to the abluminal (lower) compartment. (C) VHH A-Fc, VHH B-Fc, and VHH Z-Fc were incubated at 10 nM in the luminal compartment for 24 h and transport to the abluminal compartment was assessed (indicated as 24hrs). The inserts containing the VHH-Fc solutions were then transferred to another 96-well plate containing fresh transport buffer for an additional 48-h transport interval to the abluminal compartment (indicated as +48hrs). (D) Kinetic representation of the experiment described in (C) (72-h transport is the sum of the 24-h and 48-h transport intervals). Fc fragment content in the abluminal compartment was quantified using an in-house anti-Fc ELISA assay. Absorbance units were converted to femtomoles per insert (surface area of ​​0.143 cm2 for an insert in a 96-well plate). For each conjugate, three independent experiments with at least 12 inserts were evaluated. Data are presented as mean ± SEM (***p ≤ 0.001). [Figure 10]Figure 10 shows the distribution of VHH-Fc fusion proteins in wild-type C57Bl / 6 mice at 2 and 24 hours post-injection (pi). VHH A-Fc, VHH A-Fc-Agly, and VHH Z-Fc fusions were injected into the tail vein at 5 mg / kg, and after plasma collection, mice were perfused with saline at 2 or 24 hours post-injection. Intermediate plasma samples were also collected at 15 minutes and 6 hours post-injection using retro-orbital sampling. Brains were processed to isolate brain parenchyma from capillaries. VHH-Fc content in each tissue was assessed using an in-house anti-Fc ELISA assay. Data are presented as mean ± SEM of VHH-Fc concentrations in plasma (A), parenchyma (B), and microvessels (C), or by mean ± SEM of parenchyma-to-plasma ratios (D) and microvessels-to-plasma ratios (E) (40% for each group per time point). <n<12、*p≦0.05、**p≦0.01、***p≦0.001)。 [Figure 11] FIG. 11 shows the apparent Kd measurements of VHH A1-A9 in CHO cell lines stably expressing hTfR and mTfR. (A) CHO-hTfR-EGFP and CHO-mTfR-EGFP cells were incubated with various concentrations of VHH for 1 h at 4° C. and detected by mouse anti-6His (1 / 1000) and Alexa647-conjugated anti-mouse secondary antibodies (1 / 400). Measurements were performed using flow cytometry. The fluorescence intensity ratios of each point were normalized with the corresponding EGFP signal (receptor expression) to obtain arbitrary units. Data are presented as mean ± SEM of three independent experiments. (B) The characteristics of VHH are shown: molecular weight (Da), theoretical pI, apparent Kd (nM) at human TfR, and apparent Kd (nM) at mouse TfR. Data are presented as mean ± SEM of three independent experiments. NB is no binding and LB is low binding. [Figure 12]Figure 12 shows the apparent Kd measurements of VHH A10-A19 in CHO cell lines stably expressing hTfR and mTfR. (A) CHO-hTfR-EGFP and CHO-mTfR-EGFP cells were incubated with various concentrations of VHH for 1 h at 4°C and detected by mouse anti-6His (1 / 1000) and Alexa647-conjugated anti-mouse secondary antibodies (1 / 400). Measurements were performed using flow cytometry. The fluorescence intensity ratios of each point were normalized with the corresponding EGFP signal (receptor expression) to obtain arbitrary units. Data are presented as mean ± SEM of three independent experiments. (B) The characteristics of VHH are shown: molecular weight (Da), theoretical pI, apparent Kd (nM) at human TfR, and apparent Kd (nM) at mouse TfR. Data are presented as mean ± SEM of three independent experiments. NB is no binding. [Figure 13] Figure 13 shows the apparent Kd measurement of 13C3-HC-VHH fusions in hTfR and mTfR expressing CHO cell lines. (A) CHO-hTfR-EGFP and CHO-mTfR-EGFP cells were incubated with various concentrations of 13C3 fusions at 4°C for 1 hour and detected by Alexa647-conjugated anti-mouse antibody (1 / 400). Measurements were performed using flow cytometry. The fluorescence intensity ratio of each point was normalized to the corresponding EGFP signal (receptor expression) to obtain arbitrary units. Data are presented as mean ± SEM of three independent experiments. (B) The molecular weight (Da), apparent Kd (nM) at human TfR, and apparent Kd (nM) at mouse TfR characteristics of 13C3 fusions are shown. Data are presented as mean ± SEM of three independent experiments. NB is no binding, LB is low binding. [Figure 14]FIG. 14 shows the distribution of 13C3 monoclonal antibody and 13C3-HC-VHH fusions in wild-type C57Bl / 6 mice at 2 and 6 hours post-injection (pi). 13C3, 13C3-HC-VHH A, and 13C3-HC-VHH A1 were injected into the tail vein at 35 nmoles / kg and mice were perfused with saline at 2 or 6 hours post-injection. Brains were processed and brain parenchyma was isolated from capillaries. The amount of 13C3 and 13C3-HC-VHH A / A1 in each tissue / compartment was assessed using a certified Meso Scale Discovery (MSD) direct coating (Aβ) immunoassay (%CV<20% and recovery±30%). Data are presented as mean±SEM of 13C3 and 13C3-HC-VHH A / A1 concentrations in whole brain (A) and parenchyma (B) (100% for each time point and group). <n<4、*p≦0.05、**p≦0.01、***p≦0.001)。 [Figure 15]FIG. 15 shows the in vitro gene silencing activity of VHH-siGFPst1 bioconjugates. (A) VHH A-siGFPst1 and VHH B-siGFPst1 bioconjugates bind to hTfR. CHO-hTfR-EGFP cells were incubated with various concentrations of compounds as indicated at 4° C. for 1 h. Detection of VHHs was performed using a primary mouse anti-6His (1 / 1000) and an Alexa647-conjugated anti-mouse secondary antibody (1 / 400). Measurement of cell surface signals associated with VHHs was performed using flow cytometry. Results are expressed as the ratio of Alexa647-associated fluorescence intensity of the test compound to background fluorescence intensity. (B) VHH A-siGFPst1 bioconjugates show gene silencing activity. CHO-hTfR-EGFP cells were transfected with 25 nM of the indicated compounds at 37° C. for 72 h using Dharmafect1 (Dharmacon). The total fluorescence associated with EGFP protein was then quantified using flow cytometry and normalized to that of untreated (control) cells (set to 100%). ***p<0.001. (C) VHH A-siGFPst1 bioconjugates show endogenous gene silencing activity in the picomolar range when delivered directly into the cytosol. CHO-hTfR-EGFP cells were transfected with various concentrations of VHH A-siGFPst1 bioconjugates using Dharmafect1 (Dharmacon) for 120 hours at 37°C. The total fluorescence associated with EGFP protein was then quantified using flow cytometry and normalized to that of untreated (control) cells (set to 100%). Data were fitted using nonlinear regression using GraphPad Prism software (solid line) to estimate IC50 (concentration that reduces GFP protein levels by 50%) and maximum effect (bottom plateau). (D) VHH A-siGFPst1 bioconjugates induce specific and efficient TfR-mediated gene silencing. CHO-hTfR-EGFP cells were incubated with 1 μM of the indicated compounds for 120 h at 37° C. Data were processed and analyzed as described in (B).***p<0.001 vs. untreated cells. (E) hTfR-mediated binding and uptake of VHH A-siGFPst1 bioconjugate allows delivery to the cytosol and subsequent gene silencing at nanomolar concentrations. CHO-hTfR-EGFP cells were incubated with various concentrations of VHH A-siGFPst1 bioconjugate for 120 h at 37°C. The total fluorescence associated with EGFP protein was then quantified using flow cytometry and normalized to that of untreated (control) cells (set to 100%). Data were processed and analyzed as described in (C). (F) The gene silencing effect of VHH A-siGFPst1 bioconjugate is inhibited by incubation with excess of free TfR-binding VHH A and B, but not the irrelevant VHH Z. CHO-hTfR-EGFP cells were incubated with 30 nM VHH A-siGFPst1 alone or in the presence of 100× excess of free VHH A, B, or Z for 120 h at 37° C. Data were processed and analyzed as described in (C). (G) Exposure of cells to a brief 6-h pulse of VHH A-siGFPst1 bioconjugate is sufficient to induce effective gene silencing. CHO-hTfR-EGFP cells were incubated with various concentrations of VHH A-siGFPst1 bioconjugate for a brief 6-h pulse and then chased in ligand-free medium for up to 120 h. Data were processed and analyzed as described in (B). (H) The gene silencing effect of VHH B-siGFPst1 bioconjugate was similar to that observed with VHH A-siGFPst1. CHO-hTfR-EGFP cells were incubated with 30 nM (saturating concentration based on IC50 obtained with VHH A-siGFPst1) of VHH A-siGFPst1 or VHH B-siGFPst1 for 120 h at 37° C. Data were processed and analyzed as described in (C). [Figure 16]Figure 16 shows PET imaging of VHH A-68Ga bioconjugates in a subcutaneous mouse model of glioblastoma tumors. (A) VHH A-NODAGA and VHH A-68Ga bioconjugates bind hTfR as effectively as unconjugated VHH A compounds. CHO-hTfR-EGFP cells were incubated for 1 h at 4°C with various concentrations of compounds as indicated. Detection of VHHs was performed using a primary mouse anti-6His (1 / 1000) and an Alexa647-conjugated anti-mouse secondary antibody (1 / 400). Measurement of cell surface signal associated with VHHs was performed using flow cytometry. Results are expressed as the ratio of Alexa647-associated fluorescence intensity of test compound to background fluorescence intensity. (B) PET imaging of mice administered VHH A-68Ga 28 days after implantation of U87-MG cells (2 h post-injection). Glioblastoma tumors are circled in the sagittal view. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] The present invention provides new TfR binding agents that can be used to transport molecules, such as therapeutic, imaging or diagnostic agents, across the BBB. More specifically, the present invention discloses improved VHH molecules that bind to TfR and uses thereof.

[0032] TfR is involved in the uptake of iron transported by the transferrin ligand and in the regulation of cell proliferation (Neckers and Trepel 1986, Ponka and Lok 1999). There are two transferrin receptors: the TfR1 receptor and the homologous TfR2 receptor, which is expressed mainly in the liver. In the context of the present invention, the term TfR is used to refer to the TfR1 homologue.

[0033] TfR is a type II homodimeric transmembrane glycoprotein consisting of two identical 90 kDa subunits linked by two disulfide bridges (Jing and Trowbridge 1987, McClelland et al., 1984). Each monomer has a short cytoplasmic N-terminal domain of 61 amino acids containing a YTRF (tyrosine-threonine-arginine-phenylalanine) internalization motif, a single hydrophobic transmembrane segment of 27 amino acids, and a large C-terminal extracellular domain of 670 amino acids that contains 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 of which is particularly important for proper folding and trafficking of the receptor to the cell surface (Hayes et al., 1997). There are also palmitoylation sites in the intramembrane domain, which presumably anchor the receptor and allow its endocytosis (Alvarez et al., 1990; Omary and Trowbridge, 1981), as well as intracellular phosphorylation sites whose function is unclear and which have no role in endocytosis (Rothenberger et al., 1987).

[0034] The TfR receptor is expressed at high levels by highly proliferating cells, both healthy and neoplastic (Gatter et al., 1983). Numerous studies have demonstrated high levels of TfR expression in cancer cells compared to healthy cells. Thus, pathologies 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) show increased TfR expression associated with tumor aggressiveness and disease stage or prognosis.

[0035] Thus, drug targeting to TfR may be suitable for cancer treatment and BBB crossing.

[0036] Using purified membrane preparations from cells expressing high levels of hTfR and mTfR, VHH molecules that bind to both human and non-human TfRs were generated and selected. When these VHH molecules were fused to the Fc region of human IgG1 or a drug (such as an antibody, siRNA, etc.) or an imaging agent, they retained their TfR-binding ability, migrated across an in vitro BBB model, and exhibited brain targeting properties in vivo. Also, when these VHH molecules were fused to siRNA or a NODAGA scaffold, they retained their TfR-binding ability and demonstrated effective delivery to cells and organs in vivo. The VHH molecules exhibit an appropriate level of affinity and specificity to perform proper endocytosis following TfR binding. Thus, the present invention provides novel TfR-binding molecules corresponding to drugs useful in drug targeting.

[0037] Thus, the object of the present invention relates to VHH molecules that bind to TfRs of both human and non-human (e.g., rodents such as rats or mice) origin. Preferably, the VHH can cross the human BBB or bind to TfR-expressing tissues such as cancer. The present invention also relates to chimeric drugs containing such VHHs, their manufacture, compositions containing them, and their use.

[0038] <VHH molecule> VHH molecules correspond to the variable domains of camelid antibodies, which are heavy chains only and naturally lack light chains. VHHs have an extremely small size of about 15 kDa. They comprise a single chain molecule capable of binding to its cognate antigen using a single domain. The antigen-binding surface of VHHs is usually convex (or protruding) rather than that of normal antibodies, which are usually flat or concave. More specifically, VHHs are composed of four framework regions (or FRs), whose sequences and structures are defined as conserved, and three complementarity determining regions (or CDRs), which show high variability in both sequence content and structural conformation involved in antigen binding and conferring antigen specificity. Compared to the VHs of normal human antibodies, few amino acids are substituted in the FR2 regions and the complementarity determining regions (or CDRs) of VHHs. For example, highly conserved hydrophobic amino acids in the FR2 region (such as Val47, Gly49, Leu50, and / or Trp52) may be substituted with hydrophilic amino acids (Phe42, Glu49, Arg50, Gly52), making the overall structure more hydrophilic and contributing to increased stability, solubility, and aggregation resistance.

[0039] A VHH molecule according to the present invention is a polypeptide comprising (or consisting of, or consisting essentially of) the antigen-binding domain of a heavy chain-only antibody (HcAb).

[0040] To generate VHH molecules with suitable properties, the inventors tested more than 700 TfR-binding VHHs from a library of VHHs generated by immunization of llamas with TfR immunogens. After analyzing the clones for binding and specificity, the inventors further selected about 100 clones with the required affinity, specificity and cross-species binding properties. All the clones were sequenced and their structures were analyzed and compared. Furthermore, VHHs with controlled / improved binding properties were generated by mutagenesis. The relevant domains and sequences of the preferred VHHs are listed in the experimental section and sequence listing. The properties of the VHHs and their conjugates are also shown in the experimental section.

[0041] The VHH molecule of the invention typically comprises comprising or consisting of the formula: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FRn refers to framework region and CDRn refers to complementarity determining region.

[0042] In a particular embodiment, a VHH molecule of the 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 or 69, or a variant thereof having at least 75% amino acid identity, preferably at least 85% amino acid identity over its entire length to any one of the aforementioned sequences and retaining TfR binding ability. A preferred VHH molecule of the invention comprises a CDR1 domain having an amino acid sequence selected from SEQ ID NO: 1, 5, 9, 13, 17, 19, 67 or 69, or a variant thereof having at most one amino acid modification.

[0043] The "% identity" between amino acid (or nucleic acid) sequences can be determined by techniques known per se in the art. Typically, the % identity between two nucleic acid or amino acid sequences is determined by computer programs such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1996, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman, SB and Wunsch, CD, (1970), Journal of Molecular Biology, 48, 443-453). The % identity between two sequences refers to the identity over the entire length of the sequences.

[0044] Particular examples of VHH molecules of the invention comprise a CDR1 sequence comprising or consisting essentially of SEQ ID NO: 1, 5, 9, 13, 17, 19, 67 or 69.

[0045] In a further particular embodiment, the VHH molecule of the invention comprises a CDR2 domain comprising or consisting of an amino acid sequence selected from SEQ ID NO: 2, 6, 10, 14, 21, 23, 71, 73 or 75, or a variant thereof having at least 70% amino acid identity, preferably 85% amino acid identity over its entire length to any one of the aforementioned sequences and retaining TfR binding ability. Preferred VHH molecules of the invention comprise a CDR2 domain having an amino acid sequence selected from SEQ ID NO: 2, 6, 10, 14, 21, 23, 71, 73 or 75, or a variant thereof having at most one amino acid modification.

[0046] Particular examples of VHH molecules of the invention comprise a CDR2 sequence comprising or consisting essentially of SEQ ID NO: 2, 6, 10, 14, 21, 23, 71, 73 or 75.

[0047] In a further particular embodiment, the VHH molecule of the invention comprises a CDR3 domain comprising or consisting of an amino acid sequence selected from SEQ ID NO: 3, 7, 11, 15, 25, 27, 29, 31, 33, 77, 79, 81, 83 or 85, or a variant thereof having at least 60% amino acid identity, preferably 80% amino acid identity, more preferably 85% amino acid identity over its entire length to any one of the aforementioned sequences and retaining TfR binding ability. Preferred VHH molecules of the invention comprise 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 or 85, or a variant thereof having at most one amino acid modification.

[0048] Particular examples of VHH molecules of the invention comprise a CDR3 sequence comprising or consisting essentially of SEQ ID NO: 3, 7, 11, 15, 25, 27, 29, 31, 33, 77, 79, 81, 83, or 85.

[0049] In a further particular embodiment, the VHH molecule of the 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, or 69, or a variant thereof having at least 75% amino acid identity, preferably at least 85% amino acid identity, more preferably at least 95% amino acid identity over its entire length to any one of the aforementioned sequences; a CDR2 domain comprising or consisting of an amino acid sequence selected from SEQ ID NO: 2, 6, 10, 14, 21, 23, 71, 73, or 75, or a variant thereof having at least 70% amino acid identity, preferably at least 85% amino acid identity, more preferably at least 95% amino acid identity over its entire length to any one of the preceding sequences; a CDR3 domain comprising or consisting of an amino acid sequence selected from SEQ ID NO: 3, 7, 11, 15, 25, 27, 29, 31, 33, 77, 79, 81, 83, or 85, or a variant thereof having at least 60% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 95% amino acid identity over its entire length to any one of the preceding sequences, The aforementioned VHHs have TfR binding ability.

[0050] In a preferred embodiment, the VHH molecule of the invention comprises: a CDR1 domain having an amino acid sequence selected from SEQ ID NO: 1, 5, 9, 13, 17, 19, 67, or 69, or a variant thereof having at most one amino acid modification; a CDR2 domain having an amino acid sequence selected from SEQ ID NO: 2, 6, 10, 14, 21, 23, 71, 73, or 75, or a variant thereof having at most one amino acid modification; and 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, or 85, or a variant thereof having at most one amino acid modification.

[0051] In a more preferred embodiment, the VHH molecule of the invention comprises CDR1, CDR2 and CDR3, the CDR1, CDR2 and CDR3 domains being respectively: SEQ ID NOs: 1, 2, and 3, or SEQ ID NOs: 17, 2, and 3, or SEQ ID NOs: 19, 2, and 3, or SEQ ID NOs: 67, 2, and 3, or SEQ ID NOs: 69, 2, and 3, or SEQ ID NOs: 1, 21, and 3, or SEQ ID NOs: 1, 23, and 3, or SEQ ID NOs: 1, 71, and 3, or SEQ ID NOs: 1, 73, and 3, or SEQ ID NOs: 1, 75, and 3, or SEQ ID NOs: 1, 2, and 25, or SEQ ID NOs: 1, 2, and 27, or SEQ ID NOs: 1, 2, and 29, or SEQ ID NOs: 1, 2, and 31, or SEQ ID NOs: 1, 2, and 33, or SEQ ID NOs: 1, 2, and 77, or SEQ ID NOs: 1, 2, and 79, or SEQ ID NOs: 1, 2, and 81, or SEQ ID NOs: 1, 2, and 83, or SEQ ID NOs: 1, 2, and 85, or SEQ ID NOs: 5, 6, and 7, or SEQ ID NOs: 9, 10, and 11, or SEQ ID NOs: 13, 14, and 15, or It comprises or consists of a variant thereof as described above.

[0052] A preferred VHH molecule of the present invention comprises a FR domain as defined below.

[0053] In a particular embodiment, the FR1 domain comprises or consists of SEQ ID NO: 35 as shown below, or a variant thereof having at least 85% amino acid identity, preferably at least 90% amino acid identity, more preferably at least 95% amino acid identity to this sequence over its entire length. E[VQL]VE[SGG]GL[V]QP[G]G[SLKL]S[CA]A[S] (SEQ ID NO: 35)

[0054] More preferably, the amino acid residues in the tortoiseshell brackets are present and variable only at other positions. In certain embodiments, the first E may be replaced with Q. In certain embodiments, the fifth V may be substituted with Q. In certain embodiments, the sixth E may be substituted with Q. In certain embodiments, the 10th G may be replaced with K or A. In certain embodiments, the 11th L may be substituted with V or E. In certain embodiments, the 23rd A may be substituted with V or T.

[0055] More preferably, FR1 contains at most 4 amino acid modifications, even more preferably at most 3 amino acid modifications, even more preferably at most 2 amino acid modifications in amino acid residues that are not bolded for this sequence.

[0056] In further specific embodiments, FR1 has an amino acid sequence selected from any one of the amino acid sequences set forth below. EVQLVESGGGVVQPGGSLKLSCVAS (SEQ ID NO: 36) EVQLVESGGGVVQPGGSLRLSCAAS (SEQ ID NO: 37) EVQLVESGGGLVQPGGSLRLSCTAS (SEQ ID NO: 38) EVQLVESGGGEVQPGGSLKLSCVAS (SEQ ID NO: 39)

[0057] In a particular embodiment, the VHH molecule of the invention comprises a FR2 domain comprising or consisting of SEQ ID NO: 40 as shown below, or a variant thereof having at least 85% amino acid identity, preferably at least 90% amino acid identity, or at least 95% amino acid identity to this sequence over its entire length. MR[W]Y[R]QA[PGK]Q[RE]L[VA]T (SEQ ID NO: 40)

[0058] More preferably, the amino acid residues in the tortoiseshell brackets are present and variable only at other positions. In certain embodiments, the first M may be substituted with I or V. In certain embodiments, the second R may be replaced with G. In certain embodiments, the fourth Y may be substituted with F. In certain embodiments, the sixth Q may be replaced with R. In certain embodiments, the seventh A may be substituted with R. In certain embodiments, the 11th Q may be substituted with E. In certain embodiments, the 14th L may be substituted with F or W. In certain embodiments, T at position 17 may be substituted with G or S.

[0059] More preferably, FR2 contains at most 6 amino acid modifications, even more preferably at most 5, at most 3 amino acid modifications, even more preferably at most 2 amino acid modifications at amino acid residues that are not bolded for this sequence.

[0060] In a particular embodiment, the VHH molecule of the invention comprises at least one of the following amino acids in the FR2 domain: Phe42, Glu49, Arg50, or Gly52.

[0061] In further specific embodiments, FR2 has an amino acid sequence selected from any one of the amino acid sequences set forth below. IRWYRQAPGKQREFVAG (SEQ ID NO: 41) MRWYRQAPGKQREWVAG (SEQ ID NO: 42) MGWFRRAPGKERELVAS (SEQ ID NO: 43) VRWYRQRPGKQREWVAG (SEQ ID NO: 44)

[0062] In a particular embodiment, the VHH molecule of the invention comprises a FR3 domain comprising or consisting of SEQ ID NO: 45 as shown below, or a variant thereof having at least 85% amino acid identity, preferably at least 90% amino acid identity, more preferably at least 95% amino acid identity to this sequence over its entire length. YYAD[S]V[K]G[RF]T[I]S[RDNA]K[N]T[V]Y[LQ]MNS[LK]PE[DTA]V[Y]Y[C] (SEQ ID NO: 45)

[0063] More preferably, the amino acid residues in the tortoiseshell brackets are present and variable only at other positions. In certain embodiments, the first Y may be substituted with N. In certain embodiments, the second Y may be substituted with A. In certain embodiments, the third A may be substituted with P or I. In certain embodiments, the fourth D may be replaced with S.

[0064] More preferably, FR3 contains at most 7 amino acid modifications, even more preferably at most 6, at most 3 amino acid modifications, even more preferably at most 2 amino acid modifications in amino acid residues that are not bolded for this sequence.

[0065] In further specific embodiments, FR3 has an amino acid sequence selected from any one of the amino acid sequences set forth below. NYADSMKGRFTISRDNTKNAVYLQIDSLKPEDTAVYYC (SEQ ID NO: 46) NYPDSAKGRFTISRDNAKNTVYLQIDSLKPEDTAVYYC (SEQ ID NO: 47) YAISSVKGRFTISRDNAENTVFLQMNSLKPDDTAVYYC (SEQ ID NO: 48) NYPDSMKGRFTISRDNAKNTVYLQINSLKSEDTAVYYC (SEQ ID NO: 49)

[0066] In a particular embodiment, the VHH molecule of the invention comprises a FR4 domain comprising or consisting of SEQ ID NO: 50 as shown below, or a variant thereof having at least 85% amino acid identity, preferably at least 90% amino acid identity, more preferably at least 95% amino acid identity to this sequence over its entire length. W[G]Q[GTQVT]V[S]S (SEQ ID NO:50)

[0067] More preferably, the amino acid residues in the tortoiseshell brackets are present and variable only at other positions.

[0068] More preferably, FR4 contains at most 4 amino acid modifications, even more preferably at most 3 amino acid modifications, even more preferably at most 2 amino acid modifications at amino acid residues that are not bolded for this sequence.

[0069] A specific example of a FR4 sequence is SEQ ID NO:50.

[0070] Specific examples of TfR binding VHH molecules of the invention are those represented by SEQ ID NOs: 4 (VHHA), 8 (VHHB), 12 (VHHC), 16 (VHHD), 18 (VHHA1), 20 (VHHA2), 22 (VHHA3), 24 (VHHA4), 26 (VHHA5), 28 (VHHA6), 30 (VHHA7), 32 (VHHA8), 34 (VHHA9), 68 (VHHA10), 70 (VHHA11), 72 (VHHA12), 74 (VHHA13), 76 (VHHA14), 78 (VHHA15), 79 (VHHA16), 80 (VHHA17), 82 (VHHA18), 83 (VHHA19), 84 (VHHA20), 85 (VHHA21), 86 (VHHA22), 87 (VHHA23), 88 (VHHA24), 89 (VHHA25), 90 (VHHA26), 91 (VHHA27), 92 (VHHA28), 93 (VHHA29), 94 (VHHA30), 95 (VHHA31), 96 (VHHA32), 97 (VHHA33), 98 (VHHA34), 99 (VHHA35), 100 (VHHA36), 102 (VHHA37), 104 (VHHA38), 106 (VHHA39), 108 (VHHA40), 108 (VHHA41), 109 (VHHA42), 110 (VHHA43), 112 (VHHA44), 113 (VHHA45), 114 ( and x is 0.

[0071] In a particular embodiment, the VHH of the invention is humanized.

[0072] In humanization, one or more FR and / or CDR domains may be (further) modified by one or more amino acid substitutions.

[0073] In this regard, in a particular embodiment, the VHH is humanized by modification (e.g., amino acid substitution) of the FR1 domain. Exemplary humanized positions in FR1 are selected from 19R and 23A, or both (e.g., for any one of SEQ ID NOs: 35-39 or variants thereof). Thus, a particular example of such a humanized FR1 includes SEQ ID NO: 36, in which K19 and / or V23 have been modified to 19R and 23A, respectively.

[0074] In another particular embodiment, the VHH is humanized by modification of the CDR1 domain. A typical humanized position in CDR1 is 8A (e.g. with respect to any one of SEQ ID NOs: 1, 5, 9, 13, 17, 19, 67, or 69 or variants thereof).

[0075] In other particular embodiments, the VHH is humanized by modification of the FR2 domain. Exemplary humanized positions in FR2 are selected from 1M, 2S or 2H, 4V, 11G, 12L, 14W, or combinations thereof (e.g., for any one of SEQ ID NOs: 40-44 or variants thereof). Thus, a particular example of such a 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.

[0076] In another particular embodiment, the VHH is humanized by modification of the CDR2 domain. A typical humanized position in CDR2 is 1I (e.g. with respect to any one of SEQ ID NOs: 2, 6, 10, 14, 21, 23, 71, 73, 75 or variants thereof).

[0077] In other particular embodiments, the VHH is humanized by modification of the FR3 domain. Exemplary humanized positions in FR3 are selected from 6V, 17A, 20T, 21L, 25M, 26N, 29R, or combinations thereof (e.g., for any one of SEQ ID NOs: 45-49 or variants thereof). Thus, a particular example of such a 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.

[0078] In other particular embodiments, the VHH is humanized by modification of the CDR3 domain. Exemplary humanized positions in CDR3 are 1A or 2R, or both (e.g. with respect to any one of SEQ ID NOs: 3, 7, 11, 15, 25, 27, 29, 31, 33, 77, 79, 81, 83, 85 or variants thereof).

[0079] In a further particular embodiment, the FR1 and / or FR2 and / or FR3 and / or CDR1 and / or CDR2 and / or CDR3 domains are humanized.

[0080] Particular examples of humanized TfR-binding VHH molecules of the invention are molecules comprising or consisting of an amino acid sequence selected from any one of SEQ ID NOs: 87 (VHHA20), 88 (VHHA21), 89 (VHHA22), 90 (VHHA23), 91 (VHHA24) and 92 (VHHA25), wherein x is 0.

[0081] In further particular embodiments, the VHH molecule may further comprise one or more tags, e.g. suitable for purification, coupling, etc. Examples of such tags include a His-tag (e.g. His6), a Q-tag (LQR) or a myc-tag (EQKLISEEDL). Typically, the one or more tags are located at the C-terminus of the VHH.

[0082] As an example, the VHH may comprise an additional sequence at the C-terminus which is AAA(EQKLIS[E]EDL)NGA[A][HHHHHH]GS (sequence number 51), where the parentheses are a myc tag and the square brackets are a His tag (the remaining residues are linkers or result from cloning).

[0083] Specific examples of such tagged TfR binding VHH molecules of the invention are SEQ ID NOs: 4 (VHHA), 8 (VHHB), 12 (VHHC), 16 (VHHD), 18 (VHHA1), 20 (VHHA2), 22 (VHHA3), 24 (VHHA4), 26 (VHHA5), 28 (VHHA6), 30 (VHHA7), 32 (VHHA8), 34 (VHHA9), 68 (VHHA10), 70 (VHHA11), 72 (VHHA12). , 74 (VHHA13), 76 (VHHA14), 78 (VHHA15), 80 (VHHA16), 82 (VHHA17), 84 (VHHA18), 86 (VHHA19), 87 (VHHA20), 88 (VHHA21), 89 (VHHA22), 90 (VHHA23), 91 (VHHA24), and 92 (VHHA25), wherein x is 1.

[0084] As another example, a VHH of the invention may comprise a Q-tag of sequence LQR, preferably located at the C-terminus of the VHH.

[0085] As a further example, the VHH of the present invention may comprise a Gly linker, preferably located at the C-terminus of the VHH. The Gly linker may comprise a Gly repeat of, for example, 2-7 Gly residues, 3-6 Gly residues, etc. Particular examples of Gly linkers include Gly3, Gly4, Gly5, or SerGlySerGly5.

[0086] In a particular embodiment, the VHH of the present invention may comprise a Gly linker and a Q-tag, preferably located at the C-terminus. A more specific example of such a VHH comprises the structure VHH-Gly linker-Q-tag, in which the Gly linker comprises 2-6 Gly residues and the Q-tag comprises or consists of LQR.

[0087] As an example, a VHH may comprise an additional sequence at the C-terminus that is [GGG](LQR), where the parentheses are the Q-tag and the tortoiseshell brackets are the Gly linker.

[0088] In further specific embodiments, the VHH of the invention may comprise an Ala linker, a His tag, a Gly linker, and a Q tag. Preferably, the linker and the tag are located at the C-terminus of the VHH. In other embodiments, the Q tag can be located at least at the N-terminus of the VHH. A more specific example of such a VHH comprises the structure VHH-Ala linker-His tag-Gly linker-Q tag, where the Ala linker comprises 3 residues, the His tag comprises 2-7 His residues, the Gly linker comprises 2-6 Gly residues, and the Q tag comprises or consists of LQR.

[0089] As an example, a VHH may comprise an additional sequence at the C-terminus where: [AAA][HHHHHH][GGG](LQR), the parentheses are a Q tag, the tortoiseshell brackets are an Ala linker and a Gly linker and the square brackets are a His tag.

[0090] Further specific examples of TfR-binding VHH molecules of the invention are VHH molecules that competitively inhibit the binding of VHHs as described above to human and non-human TfR. The term "competitively inhibits" indicates that the VHH is capable of reducing or inhibiting or replacing the binding of such a reference VHH to TfR in vitro or in vivo. Competitive assays can be performed using standard techniques, such as, for example, competitive ELISA or other binding assays. Typically, competitive binding assays involve recombinant cells or membrane preparations expressing TfR, unlabeled test VHH (or a phage expressing it), and a labeled reference VHH (or a phage expressing it), optionally bound to a solid substrate. Competitive inhibition is measured by determining the amount of labeled VHH that binds in the presence of the test VHH. Typically, the test VHH is present in excess, such as about 5-500 times the amount of the reference VHH. Typically, in an ELISA, the test VHH is in 100-fold excess. A test VHH present in excess is considered to competitively inhibit a reference VHH if it inhibits or displaces at least 70% of the binding of the reference VHH to TfR. Preferred competing VHHs bind to epitopes that share common amino acid residues.

[0091] As shown in the experimental section, the VHH molecules are capable of binding to TfR in vitro and in vivo. This shows sufficient affinity with apparent Kd comprised between 0.1 nM and 10 μM, in particular between 1 μM and 1 nM. Moreover, all of the molecules bind to both human and mouse TfR. Moreover, the binding of the aforementioned VHHs of the 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 said ligand. Conjugates made with such VHH molecules have been further shown to bind to TfR in vitro and be transported across the BBB to the CNS in vivo, demonstrating transcytosis. Thus, such VHHs represent powerful agents in drug delivery or targeting.

[0092] The VHH of the invention can be synthesized by any technique known to the person skilled in the art, such as chemical, biological or genetic synthesis. It can be stored as is or formulated in the presence of the substance of interest or any acceptable excipient.

[0093] Chemical synthesis uses commercially available constructs into which natural and unnatural amino acids, e.g. D enantiomers and residues with side chains that differ in hydrophobicity and steric hindrance from those of their natural homologues (so-called exotic, i.e. non-coded amino acids), or VHH sequences containing one or more peptide-like bonds that may include, inter alia, the insertion of a methylene (-CH2-) or phosphate (-PO2-) group, a secondary amine (-NH-) or oxygen (-O-) or an N-alkyl peptide.

[0094] During synthesis, various chemical modifications can be introduced, for example at the N- or C-terminus or at the side chains, such as by incorporating lipid (or phospholipid) derivatives or liposome or nanoparticle constructs, so that the VHH of the invention can be incorporated into lipid membranes, such as those of liposomes or nanoparticles composed of one or more lipid layers or lipid bilayers.

[0095] A VHH of the invention may also be derived from a nucleic acid sequence encoding it, as further described below.

[0096] <Conjugate> A further object of the present invention relates to a conjugate (also referred to herein interchangeably as "chimeric agent") comprising one or more VHH molecules as described above conjugated to at least one molecule or scaffold of interest.

[0097] The molecule of interest may be any molecule, such as a medicine or drug, a diagnostic agent, an imaging molecule, a tracer, etc. Examples of conjugate molecules of interest include, but are not limited to, any chemical moiety, such as small chemical molecules (antibiotics, antivirals, immunomodulators, antineoplastic agents, anti-inflammatory agents, adjuvants, etc.); peptides, polypeptides, and proteins (enzymes, hormones, neurotrophic factors, neuropeptides, cytokines, apolipoproteins, growth factors, antigens, antibodies or parts of antibodies, adjuvants, etc.); nucleic acids (e.g. RNA or DNA, such as of human, viral, animal, eukaryotic or prokaryotic, plant, or synthetic origin, including coding genes, inhibitory nucleic acids such as ribozymes, antisense, interfering nucleic acids, whole genomes or parts thereof, plasmids, etc.); lipids, viruses, markers, or tracers, etc. In general, the "molecule of interest" may be any pharmaceutical active ingredient, whether a chemical compound, a biochemical compound, a natural compound, or a synthetic compound. In general, the term "small chemical molecule" refers to a molecule of pharmaceutical interest, having a maximum molecular weight of 1000 Daltons, typically between 300 Daltons and 700 Daltons.

[0098] The conjugated compound is typically a pharmaceutical (a small molecule drug, a nucleic acid, or a polypeptide, such as an antibody or a fragment thereof), or an imaging agent, preferably suitable for the treatment or detection of a neurological, infectious, or cancerous pathology of the CNS, such as the brain.

[0099] The chimeric agent may also contain a stabilizing group in addition to or instead of the aforementioned compounds of interest to increase the plasma half-life of the VHH or conjugate. Thus, a particular chimeric agent of the invention comprises at least one VHH, a stabilizing group, and an active compound in any order.

[0100] The stabilizing group may be any group known to have a reasonable plasma half-life (e.g., at least 1 hour) and to have substantially no adverse biological activity. Examples of such stabilizing groups include, for example, Fc fragments of immunoglobulins or variants thereof, albumin, large human serum proteins such as HSA, or IgG or PEG molecules. In a particular embodiment, the stabilizing group is an Fc fragment of human IgG1. More preferably, the stabilizing group is a deglycosylated Fc fragment of IgG1.

[0101] The VHH may be conjugated to the N-terminus or C-terminus, or both, of the stabilizing group. When the stabilizing group is an Fc fragment, the conjugation is typically by genetic fusion. Depending on the nature of the stabilizing group, the resulting protein may remain a monomeric entity or may be a multimeric entity. In the case of an Fc fragment, the fusion protein Fc-VHH or VHH-Fc usually forms a homodimer.

[0102] In the conjugate compounds of the present invention, coupling can be performed by any acceptable binding means, taking into account the chemical nature, disorder and number of the components to be conjugated. That is, coupling can be performed by one or more covalent, ionic, hydrogen, hydrophobic or van der Waals bonds, cleavable or non-cleavable in a physiological medium or inside a cell. Furthermore, coupling can be performed at various reactive groups, in particular at one or more terminal and / or one or more internal or lateral reactive groups. Coupling can also be performed using genetic engineering.

[0103] The interaction is preferably strong enough so that the VHH does not dissociate from the active substance before it has reached its site of action. For this reason, the preferred coupling in the present invention is a covalent coupling, although non-covalent coupling may also be used. The compound of interest can be coupled to the VHH either at one of its termini (N-terminus or C-terminus) or at the side chain of one of the constituent amino acids of its sequence (Majumdar and Siahaan, Med Res Rev., Epub ahead of print). The compound of interest can be coupled directly to the VHH or indirectly by means of a linker or spacer. Means of covalent chemical coupling with or without a spacer include those selected from bi- or polyfunctional substances, including, for example, esters, aldehydes or alkyl, aryl or peptide groups with alkyl or aryl acids, anhydrides, sulfhydryls or carboxyl groups, groups derived from cyanogen bromide or chloride, carbonyldiimidazole, succinimide esters or sulfonyl halides.

[0104] An exemplary method for conjugating a VHH of the invention to a molecule or scaffold is disclosed in FIG.

[0105] In a particular embodiment, the coupling (or conjugation) is by gene fusion. Such a method can be used when the coupling molecule is a peptide or polypeptide. In such a case, a nucleic acid molecule encoding the VHH fused to said molecule is prepared and expressed in any suitable expression system to generate the conjugate.

[0106] In another particular embodiment, the coupling (or conjugation) is by enzymatic reaction. In particular, the transglutaminase enzyme (TGase) can be used to perform site-specific conjugation to the VHH. TGase catalyzes the formation of a stable isopeptide bond between (i) the side chain of a glutamine residue inserted in a tag sequence (i.e. Q-tag) that is specifically recognized by TGase, and (ii) a donor substrate bearing an amino functional group. In this regard, the inventors have developed a specific tag sequence (called "Q-tag") that is recognized by TGase and can be used to couple the VHH of the invention to any molecule of interest, in particular a chemical agent or substance. For this purpose, the VHH is prepared by genetic fusion to add in tandem the following tags (typically at its C-terminus): first an optional trialanine, then an optional His-tag, an optional small triglycine linker, and finally a Q-tag. The triglycine linker distances the Q tag to allow better access for the TGase to the glutamine, while the His tag aims to facilitate purification of the VHH and its further functionalized versions.

[0107] The general conjugation methodology that has been developed is a convergent synthesis based on a process that includes the following: 1) A reactive moiety is introduced into the glutamine of the Q-tag of the VHH for further conjugation to a molecule of interest. For this purpose, a heterobifunctional linker can be processed by TGase, which has two different reactive ends, one of which is a suitable primary amine group for TGase and the other is an orthogonal reactive moiety. Representative examples of such orthogonal reactive groups include azide, constrained alkynes such as DBCO (dibenzocyclooctyne) or BCN (bicyclo[6.1.0]nonyne), tetrazine, TCO (trans-cyclooctene), free or protected thiols, etc. 2) Introducing into the molecule of interest a reactive moiety complementary to that incorporated into the Q-tag of the VHH. Representative examples of such orthogonal reactive groups include azides, constrained alkynes such as DBCO or BCN, tetrazines, TCO, free or protected thiols, etc. 3) Conjugation of both the functionalized VHH and the molecule via their complementary reactive groups.

[0108] Such a conjugation method represents a further object of the present invention. In particular, an object of the present invention is a method for coupling two molecules using a Q-tag as described above via a TGase coupling reaction. A further object of the present invention is a VHH comprising a Q-tag. A further object of the present invention is a VHH molecule comprising a linker, such as a Gly linker, and a Q-tag. A preferred VHH of the present invention is VHH-Linker-His m -linker-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 6.

[0109] In a particular embodiment, the present invention relates to a conjugate comprising a VHH covalently linked to a chemical moiety. A preferred variant of such a conjugate comprises one VHH and one chemical moiety.

[0110] In another particular embodiment, the present invention relates to a conjugate comprising a VHH covalently linked to a nucleic acid. The nucleic acid may be an antisense oligo, a ribozyme, an aptamer, an siRNA, etc. A preferred variant of such a conjugate comprises one VHH and one nucleic acid molecule.

[0111] In another particular embodiment, the present invention relates to a conjugate comprising a VHH covalently linked to a peptide. The peptide may be an active molecule, a bait, a tag, a ligand, etc. A preferred variant of such a conjugate comprises one VHH and one peptide.

[0112] In another embodiment, the present invention relates to a conjugate comprising a VHH covalently linked to a dye.

[0113] In another embodiment, the present invention relates to a conjugate comprising a VHH covalently linked to a nanoparticle or liposome. The nanoparticle or liposome may be loaded or functionalized with an active agent. A preferred variant of such a conjugate comprises multiple VHH molecules coupled to each nanoparticle or liposome.

[0114] In a further embodiment, the conjugate comprises an antibody or fragment thereof coupled to one or more VHH molecules. Typically, the VHH molecules are coupled to the C-terminus or N-terminus of the heavy or light chain, or both, or to the C-terminus or N-terminus of the Fc fragment.

[0115] The present invention also relates to a method for preparing a conjugate compound as described above, characterized in that it comprises a step of coupling the VHH to a molecule or scaffold, preferably by chemical, biochemical or enzymatic pathway or by genetic engineering.

[0116] When multiple VHHs are present in a chimeric agent of the invention, they may have the same or different binding specificities.

[0117] <Nucleic acids, vectors, and host cells> A further aspect of the invention relates to a nucleic acid encoding a VHH as described above, or a conjugate thereof (when the conjugate moiety is an amino acid sequence). The nucleic acid may be single-stranded or double-stranded. The nucleic acid may be DNA (cDNA or gDNA), RNA, or a mixture thereof. It may be in single-stranded or double-stranded form or a mixture of the two. It may contain modified nucleotides, for example containing modified bonds, modified purine or pyrimidine bases, or modified sugars. It may be prepared by any method known to the person skilled in the art, including chemical synthesis, recombination, and / or mutagenesis. The nucleic acid according to the invention may be deduced from the amino acid sequence of the VHH molecule according to the invention, and the codon usage may be adapted depending on the host cell in which the nucleic acid is transcribed. These steps may be performed according to methods known to the person skilled in the art, some of which are described in the reference manuals of Sambrook et al. (Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual, Third Edition Cold Spring Harbor)

[0118] Specific examples of such nucleic acid sequences include sequences comprising any one of SEQ ID NOs: 52-64 and 95-110, and their complementary sequences, as well as fragments thereof without any tag-coding portion. The domains encoding CDR1, CDR2, and CDR3 are underlined. The tag-coding portion is shown in bold.

[0119] The present invention also relates to vectors comprising such nucleic acids, optionally under the control of regulatory sequences (e.g., promoters, terminators, etc.). The vectors can be plasmids, viruses, cosmids, phagemids, artificial chromosomes, etc. In particular, the vectors can comprise the nucleic acids of the present invention operably linked to a regulatory region, i.e., a region comprising one or more regulatory sequences. Optionally, the vectors can comprise multiple nucleic acids of the present invention operably linked to multiple regulatory regions.

[0120] The term "control sequence" refers to a nucleic acid sequence necessary for expression by a coding region. Control sequences can be endogenous or heterologous. Control sequences that are known and currently used by those skilled in the art are preferred. Such control sequences include, but are not limited to, promoters, signal peptide sequences, and transcription terminators.

[0121] The term "operably linked" refers to a configuration in which a control sequence is positioned appropriately relative to a coding sequence such that the control sequence controls expression by the coding region.

[0122] The present invention further relates to the use of a nucleic acid or vector according to the invention for transforming, transducing or transducing a host cell.

[0123] The invention also provides a host cell comprising one or more nucleic acids of the invention and / or one or more vectors of the invention.

[0124] The term "host cell" also includes any progeny of a parent host cell that is not identical to the parent host cell due to mutations that occur during replication. Suitable host cells can be prokaryotic (e.g., bacteria) or eukaryotic (e.g., yeast, plant, insect, or mammalian cells). Specific examples of such cells include E. coli strains, CHO cells, Saccharomyces strains, plant cells, sf9 insect cells, and the like.

[0125] <Use> The VHH molecules of the present invention are capable of binding to TfR and thus targeting / delivering the molecules to TfR expressing cells or organs.

[0126] Within the context of the present invention, the binding is preferably specific, and the binding to the TfR occurs with higher affinity than the binding to any other antigen of the same species. Preferred VHH molecules of the present invention bind to human TfR1 and mouse or rat TfR. More preferably, the VHH molecules bind to the human and mouse receptors with substantially similar affinity.

[0127] Thus, the present invention relates to a method for targeting / delivering a compound to / through a TfR expressing cell or organ comprising coupling said compound to at least one VHH of the present invention.

[0128] The present invention further relates to the use of a VHH as described above as a vector for the transport of compounds to / through a TfR expressing cell or organ.

[0129] The present invention also relates to the use of a VHH as described above for the preparation of a drug capable of crossing the BBB.

[0130] The present invention also relates to a method for allowing or enhancing the passage of a molecule across the BBB comprising coupling said molecule to a VHH of the present invention.

[0131] The VHHs of the present invention can be used to transport or deliver any compound, such as small molecule drugs, proteins, polypeptides, peptides, amino acids, lipids, nucleic acids, viruses, liposomes, etc.

[0132] The present invention also relates to a pharmaceutical composition, characterized in that it comprises at least one VHH or VHH-drug conjugate as described above and one or more pharma- ceutically acceptable excipients.

[0133] The present invention also relates to a diagnostic composition, characterized in that it comprises a VHH or a VHH-diagnostic or medical imaging agent conjugate compound as described above.

[0134] The conjugate can be used in the form of any pharma- ceutically acceptable salt. The term "pharma-ceutically acceptable salt" refers, for example and without limitation, to pharma-ceutically acceptable base or acid addition salts, hydrates, esters, solvates, precursors, metabolites or stereoisomers of the aforementioned vectors or conjugates loaded with at least one substance of interest.

[0135] The expression "pharmaceutically acceptable salts" refers to non-toxic salts which may generally be prepared by reacting the free bases with suitable organic or inorganic acids, and which retain the biological effectiveness and properties of the free bases. Representative examples of such salts include water soluble and water insoluble salts, such as acetate, N-methylglucamine ammonium, amsonate (4,4-diaminostilbene-2,2'-disulfonate), benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, hydrobromide, bromide, butyrate, camsylate, carbonate, hydrochloride, chloride, citrate, clavulanate, dichlorohydrate, diphosphate, edetate, calcium edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycolylarsanilate, hexafluorophosphate, hexylresorcinate, hydrabamine, hydroxynaphthoate, iodide, isothioate, Salts include, but are not limited to, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methyl bromide, methyl nitrate, methyl sulfate, mucate, napsylate, nitrate, 3-hydroxy-2-naphthoate, oleate, oxalate, palmitate, pamoate (1,1-methylene-bis-2-hydroxy-3-naphthoate, or embonate), pantothenate, phosphate, picrate, polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate, subacetate, succinate, sulfate, sulfosalicylate, suramate, tannate, tartrate, teoclate, tosylate, triethiodide, trifluoroacetate, and valerate.

[0136] The composition of the present invention advantageously comprises a pharma- ceutically acceptable carrier or excipient.The pharma-ceutically acceptable carrier can be selected from the carriers conventionally used for each administration mode.Depending on the administration mode envisaged, the compound can be in solid, semi-solid or liquid form. For solid compositions such as tablets, pills, powders or granules loose or contained in gelatin capsules, the active substance can be combined with a) diluents such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine, b) lubricants such as silica, talc, stearic acid, its magnesium or calcium salts, and / or polyethylene glycol, c) binders such as magnesium and aluminum silicate, starch paste, gelatin, tragacanth gum, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone, d) disintegrants such as starch, agar, alginic acid or its sodium salt, or effervescent mixtures, and / or e) adsorbents, dyes, flavors and sweeteners. Excipients can be, for example, mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate and pharmaceutical grade analogues. For semi-solid compositions, e.g., suppositories, the excipients can be, for example, emulsions or oily suspensions, or polyalkylene glycols, e.g., based on polypropylene glycol.Liquid compositions, particularly those for injection or inclusion in soft capsules, can be prepared, for example, by dissolving, dispersing, etc., the active substance in a pharma- ceutically pure solvent, such as water, saline, aqueous dextrose, glycerol, ethanol, oils, and the like.

[0137] The compositions or conjugates of the invention may be administered by any suitable route, including but not limited to, by parenteral route, such as in the form of a formulation that can be injected via subcutaneous, intravenous or intramuscular routes, by oral route (or per os), such as in the form of coated or uncoated tablets, gelatin capsules, powders, pellets, suspensions or oral solutions (one such oral administration form may be with immediate release or with extended or delayed release), by rectal route, such as in the form of suppositories, by topical route, such as in the form of patches, pomades or gels, in particular by transdermal route, by intranasal route, such as in aerosol and spray form, by translingual route or by intraocular route.

[0138] The pharmaceutical composition typically comprises an effective dose of the VHH or conjugate of the invention. As described herein, a "therapeutically effective dose" refers to a dose that produces a therapeutic effect in a given condition and administration schedule. This is typically the average dose of an active agent administered to clearly improve some of the symptoms associated with a disease or pathological condition. For example, when treating cancer, CNS pathologies, lesions, or disorders of the brain or other tissues, a dose of an active agent that reduces, prevents, delays, eliminates, or stops one of the causes or symptoms of the aforementioned disease or disorder may be effective for treatment. A "therapeutically effective dose" of an active agent may not necessarily cure a disease or disorder, but may treat the disease or disorder in such a way that it delays, hinders, or prevents its appearance, or suppresses its symptoms, or changes its duration or, for example, makes it less severe, or speeds up the patient's recovery.

[0139] It will be understood that a "therapeutically effective dose" particularly for humans will depend on a variety of factors, including the activity / potency of the active substance, its frequency of administration, its route of administration, its toxicity, its rate of excretion and metabolism, drug combinations / interactions, and the severity of the disease (or disorder) being treated on a preventive or curative basis, as well as the age, weight, general health, sex and / or diet of the patient.

[0140] Depending on the substance to be coupled, the conjugates and compositions of the invention can be used for the treatment, prevention, diagnosis or imaging of numerous pathologies, especially pathologies affecting the CNS, infectious pathologies or cancer. The VHHs of the invention have the ability to target TfR expressing cells, especially cells that express the receptor significantly, such as cancer cells, neural or non-neural tissues, and / or cross cell membranes, especially those of the physiological barriers of the CNS, and more particularly those of the blood-tumor barrier (BTB) of cancer neural tissues. TfR is abundant in organs such as bone marrow, placenta, and the gastrointestinal tract. TfR is also highly expressed in brain endothelial cells, but not in endothelial cells lining blood vessels in other tissues. TfR expression has been confirmed in the plasma membrane of purified brain microvessels and cultured endothelial cells of rats, mice, pigs, and non-human primates.

[0141] In this regard, the present invention relates to the use of a pharmaceutical conjugate or composition as described above for treating or preventing CNS conditions or disorders, brain tumors or other cancerous cells, and bacterial, viral, parasitic or fungal infectious conditions of the brain or other tissues.

[0142] The present invention also relates to a VHH, conjugate or composition as described above for use in diagnosing, imaging or treating CNS conditions or disorders, brain tumors or other cancerous cells, and bacterial, viral, parasitic or fungal infectious conditions in the brain or other tissues.

[0143] The present invention also relates to a VHH, a conjugate or a composition as described above for use in treating, imaging and / or diagnosing brain tumors or other types of cancer.

[0144] The present invention also relates to a VHH, conjugate or composition as described above for use in treating, imaging and / or diagnosing neurodegenerative conditions such as, but not limited to, Alzheimer's disease, Parkinson's disease, stroke, Creutzfeldt-Jakob disease, bovine spongiform encephalopathy, multiple sclerosis, amyotrophic lateral sclerosis.

[0145] The present invention also relates to a VHH, a conjugate or a composition as described above for use in treating, imaging and / or diagnosing neurological conditions such as, but not limited to, epilepsy, migraine, encephalitis, CNS pain.

[0146] The present invention also relates to VHHs, conjugates or compositions as described above for use in treating, imaging and / or diagnosing rare diseases such as, but not limited to, lysosomal storage diseases, Farber disease, Fabry disease, gangliosidoses GM1 and GM2, Gaucher disease, various mucopolysaccharidoses.

[0147] The present invention also relates to a VHH, a conjugate or a composition as described above for use in treating, imaging and / or diagnosing neuropsychiatric conditions such as, but not limited to, depression, autism, anxiety, schizophrenia.

[0148] The present invention also relates to a VHH, conjugate or composition as described above for use in treating, imaging and / or diagnosing cancers such as, but not limited to, glioblastoma, pancreatic cancer, ovarian cancer, hepatocellular carcinoma.

[0149] The present invention also relates to a VHH, a conjugate or a composition as described above, wherein the conjugated agent is a virus, such as a recombinant virus, or a virus-like particle. The present invention can indeed be used to increase the delivery of recombinant (e.g., replication-deficient or attenuated) viruses used in gene therapy, such as adenoviruses, adeno-associated viruses, lentiviruses, retroviruses, etc., or virus-like particles, to the brain or to cancer or any TfR-rich tissue. Coupling to a virus or VLP can be performed, for example, by coupling to a viral capsid protein.

[0150] The present invention also relates to a method of treating any of the above-mentioned conditions or diseases by administering to a subject in need of treatment a VHH, a conjugate or a composition of the invention.

[0151] The present invention also relates to the use of a VHH, a conjugate or a composition of the invention for the manufacture of a medicament for the treatment of any of the above mentioned pathologies or diseases.

[0152] Other aspects and advantages of the present invention will become apparent from the following examples, which are merely illustrative in nature and are not intended to limit the scope of the present application. EXAMPLES

[0153] Example I Verification of TfR expression in the BBB We analyzed the plasma membrane TfR expression profile in brain endothelium of various species. We used the ProteoExtract® Cell Fractionation Proteome Extraction Kit (Calbiochem, La Jolla, CA, USA) to prepare membrane extracts of digested or undigested brain microvessels (BMVs) and primary cultures of brain microvascular endothelial cells (BMECs) from rat, mouse, pig, and non-human primate (NHP, rhesus monkey) (Figure 1).

[0154] Membrane extracts were quantified using BioRad's 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 onto nitrocellulose membranes (ThermoFisher Scientific). Proteins were probed with a primary antibody against TfR (Genetex GTX102596; 1 / 1000) and then with an HRP-conjugated donkey anti-rabbit IgG secondary antibody diluted 1 / 10000 (Jackson ImmunoResearch). Finally, proteins were detected using chemiluminescence.

[0155] As shown in Figure 1, TfR is expressed in digested and undigested brain microvessels of rats, mice, pigs, and non-human primates. TfR is also expressed in brain endothelial cells of mice, rats, and pigs (note that only 1 μg of membrane protein was loaded onto SDS-PAGE for brain microvascular endothelial cells, versus 10 μg or 5 μg for brain microvessels). TfR expression is elevated in digested brain microvessels of NHPs.

[0156] These data indicate that the TfR represents a valid target for the design of molecules for in vivo applications.

[0157] Example II Generation of CHO cell lines stably expressing human and mouse TfR A prerequisite for the identification and characterization of TfR-binding VHHs was the generation of stable cell lines in eukaryotic cells (Chinese Hamster Ovary cells, CHO) that constitutively and rapidly express hTfR and mTfR. These cell lines were used to i) identify and characterize drugs that bind to the receptors expressed on the cell surface in their natural configuration, and ii) test whether the receptors can internalize such drugs by endocytosis.

[0158] For the generation of these cell lines, the cDNA encoding hTfR was cloned using sequence information available in the database (accession number NM_003234.3). The primers necessary for the amplification of the cDNA by RT-PCR were selected (see table below), which contain at their ends (bold) the restriction sites (EcoRI and SalI) necessary for cloning in the pEGFP-C1 expression vector (Clontech) (Figure 2-A). [Table 1]

[0159] Total RNA prepared 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 enzymes and ligated into pEGFP-C1 expression vector (Clontech), digested with the same restriction enzymes. After transfection into eukaryotic cells, this vector allows the expression of hTfR fused to EGFP at its N-terminus, i.e., at the end of its intracellular domain, under the control of the CMV promoter. Competent E. coli DH5α bacteria were transformed to obtain isolated colonies, plasmid DNA was prepared, and both strands of the construct were fully sequenced for verification.

[0160] A plasmid encoding mTfR fused to EGFP was purchased from GeneCopoeia (plasmid reference number: EX-Mm05845-M29).

[0161] CHO-K1 cells were transiently transfected and used to select stable transfectants by limiting dilution and resistance to antibiotics (G418). These cell lines were expanded while maintaining selection pressure.

[0162] Confocal micrographs taken after immunocytochemistry in fixed (PFA) cell lines using Alexa647-conjugated transferrin (Tf-Alexa647) confirm in FIG. 2B that EGFP (green) and Tf-Alexa647 (red) are colocalized, thus confirming good expression and functional receptor binding.

[0163] The expression of the receptor of the expected size on the membrane was confirmed by Western blot in cell membranes extracted with the ProteoExtract Cell Fraction Proteome Extraction Kit. Antibodies were raised against GFP or TfR. A protein corresponding to the combined size of EGFP and h / mTfR (170 kDa) was recognized by 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 antibodies did not detect any protein.

[0164] These data confirmed the expression of functional receptors on the cell surface of CHO cell lines.

[0165] Example III Generation of VHHs that bind TfR Llamas (Lama glama) were immunized subcutaneously four times with membrane preparations of stable CHO cell lines expressing the human and mouse receptors of interest. VHH library construction was performed as described previously (Alvarez-Rueda et al., 2007, Behar et al., 2009). Shortly, VHH-encoding mRNAs were amplified by RT-PCR from total RNA of peripheral blood mononuclear cells isolated by Ficoll density gradient and cloned into pHEN1 phagemid. Recursive selection allowed the isolation of phages displaying VHHs with strong affinity to the cell surface expressed TfR.

[0166] In total, over 700 clones were screened for their ability to bind to TfR and approximately 100 clones were sequenced.

[0167] VHHs were obtained with improved binding properties (to both mouse and human cell lines), cell penetration and transport properties. Exemplary VHHs are VHH A, VHH B, VHH C, VHH D (see also sequence listing). These VHHs do not bind to cells of the control CHO cell line.

[0168] Furthermore, suitable TfR-binding VHHs with improved binding properties were generated by site-directed mutagenesis. More specifically, site-directed mutagenesis was performed to introduce a single alanine substitution in complementarity determining regions (CDRs) 1, 2, and 3 of VHH A, resulting in VHHs A1-A9. VHHs A1 and A2 were mutated in CDR1, VHHs A3 and A4 in CDR2, and VHHs A5-A9 in CDR3. Furthermore, a single site-directed mutagenesis was performed by replacing some CDR amino acids with structurally similar amino acids. VHHs A10-A19 were obtained, where VHHs A10 and A11 were mutated in CDR1, VHHs A12-A14 in CDR2, and VHHs A15-A19 in CDR3.

[0169] Furthermore, humanized TfR-binding VHHs were generated to improve in vivo efficacy, for example by avoiding immunogenicity, designated VHH A20-A25.

[0170] Additionally, tagged VHH molecules were generated to facilitate purification and / or coupling.

[0171] The amino acid sequences of each of these VHHs are set forth in the sequence listing.

[0172] Example IV Binding and endocytosis of purified VHHs of the invention To confirm the ability of selected VHH molecules to bind to TfR and to be endocytosed, immunocytochemical experiments were performed involving incubation of VHHs in a live CHO cell line expressing TfR fused to EGFP, detected using a mouse anti-cMyc primary antibody (ThermoFisher) followed by an Alexa594-conjugated donkey anti-mouse secondary antibody (Jackson ImmunoResearch), and observed by confocal microscopy. Results obtained with VHH A are shown as an example.

[0173] As shown in Figure 3, the VHHs bound to CHO-hTfR-EGFP (Figure 3B) and CHO-mTfR-EGFP (Figure 3A) cell lines and were taken up by endocytosis and accumulated intracellularly as shown using Triton permeabilization, but not the control VHH (VHH Z) (Figure 3C,D).

[0174] Example V Measurement of binding affinity Flow cytometry was used to test the binding properties of VHHs with affinity for TfR and to measure apparent affinity (K d app ) was measured in a 96-well plate. 5 All experiments were performed with 1000 cells / well at 4°C with shaking. CHO cell lines expressing TfR fused to EGFP or CHO wild type cells were saturated with PBS / BSA 2% for 30 min to eliminate non-specific binding and then incubated with purified VHHs at concentrations ranging from 2 μM to 1 pM for 1 h. After one wash in PBS / BSA 2%, cells were incubated with anti-6His tag antibody (mouse) for 1 h, washed twice with PBS / BSA 2% and incubated with Alexa647-conjugated anti-mouse secondary antibody for 45 min. After two final washes in PBS / BSA 2%, cells were either 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).

[0175] In the control condition, where cells were incubated with a control VHH (VHH Z), there was no nonspecific labeling. All VHHs tested produced a concentration-dependent change in signal, confirming binding to the receptor of interest (Figure 4-A). No labeling of CHO wild-type control cells was detectable with all VHHs tested (not shown). The K d appwas calculated using GraphPad Prism software (Figure 4-B). d app was in the same range for all VHHs, ranging from 7.5 nM (VHH B) to 56 nM (VHH D) for mTfR and from 1.6 nM (VHH B) to 2.7 nM (VHH A) for hTfR.

[0176] Example VI Competition assay between purified VHHs with affinity for TfR and natural ligands To assess the ability of selected VHHs to compete with the TfR natural ligand transferrin (Tf) for binding to the receptor, a competition assay was performed using flow cytometry experiments. In a first step, a dilution series of competitors was incubated for 1 h at 4° C. in CHO cells expressing the receptor of interest fused to EGFP. Then, an EC90 tracer was added, incubated for another 1 h and detected with an appropriate colorimetric system (FIG. 5).

[0177] TfR-binding VHHs were used as tracers (Figure 5B) and competitors (Figure 5C). In all conditions, there was no competition between VHHs and the ligand Tf, suggesting that VHHs bind to TfR at an epitope distinct from that of Tf.

[0178] Example VII Measurement of binding affinity of VHH A1–A19 Flow cytometry was used to test the binding properties of VHH A1-A19 for TfR and to determine the apparent affinity (K d app ) was measured in a 96-well plate. 5All experiments were performed with 1000 cells / well with shaking at 4°C. CHO cell lines stably expressing hTfR or mTfR fused to EGFP or CHO wild-type cells were saturated with PBS / BSA 2% for 30 min to eliminate non-specific binding, and then incubated with purified VHHs at concentrations ranging from 50 μM to 5 pM for 1 h. After one wash in PBS / BSA 2%, cells were incubated with anti-6His tag antibody (mouse) for 1 h, washed twice with PBS / BSA 2%, and incubated with Alexa647-conjugated anti-mouse secondary antibody for 45 min. After two final washes in PBS / BSA 2%, cells were fixed by incubation in 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).

[0179] All VHHs A1-A19 induced concentration-dependent changes in signal in both cell lines (except for VHH A12), confirming their effective binding to the receptors of interest (Figures 11 and 12). VHH A, VHHs A1-A4, and VHHs A10-A15 showed similar B max (plateau of the curve), whereas VHH A6-A9 and VHH A16-A19 showed slightly lower to significantly lower B max VHH A12 alone showed a lower B in hTfR-expressing cell lines compared to the other VHH Ax. max and large curve shifts. Labeling of CHO wild-type control cells was not detectable with any of the VHHs tested (not shown).

[0180] VHH's K d appwas calculated using GraphPad Prism software (Figure 11-B, Figure 12-B). VHH A, A1-A4, A6, A9-A11, and A13-A17 all had similar K of approximately 3-4 nM for binding to human TfR. d app In contrast, VHHs A5, A8, A18, and A19 showed slightly lower affinities of 9.2 to 25 nM, while VHHs A7 and A12 showed significantly lower affinities of 255 nM and 363 nM, respectively.

[0181] For binding to mouse TfR, VHH A and A9 have similar K d app All other VHH A showed slightly lower affinities ranging from 131 to 259 nM, with the exception of VHH A5, A8, and A18, which showed significantly lower affinities of 604 nM, 427 nM, and 416 nM, respectively.

[0182] Example VIII Binding and endocytosis of purified VHH-Fc fusion molecules with affinity for TfR, and affinity measurements The anti-TfRVHH molecules of the present invention were fused to an IgGFc fragment. To generate the fusion proteins, DNA fragments (without tags) encoding the VHH were amplified by PCR and cloned into the pINFUSE-IgG1-Fc2 vector (InvivoGen) to encode a human IgG1-Fc fragment containing the VHH at its N-terminus or C-terminus. The fusion proteins were prepared using the Expi293® Expression System (Life Technologies) according to the manufacturer's instructions. 72 hours after transfection, the supernatants were harvested and purified using a Protein A GraviTrap column (GE Healthcare). The purified fusion proteins were quantified using an in-house anti-Fc ELISA.

[0183] Immunocytochemistry experiments in a CHO cell line expressing TfR fused to EGFP, involving incubation of VHH-Fc fusion proteins in live cells, detected using an Alexa594-conjugated anti-hFc antibody (Jackson ImmunoResearch), imaged by confocal microscopy, were performed to confirm the ability of the fusion proteins to bind to their target receptors of interest.

[0184] The results show that the conjugates of the invention can bind to cells and be endocytosed (Figure 7). No binding of the control VHH-Fc conjugate (VHH Z-Fc) to cells was observed, indicating the specificity of this interaction.

[0185] The binding properties of VHH-Fc and Fc-VHH fusion proteins with affinity for TfR were tested in flow cytometry experiments, and the apparent affinities (K d app ) was measured in a 96-well plate. 5 All experiments were performed with 100 cells / well at 4°C with shaking. CHO cell lines expressing the receptor of interest fused to EGFP or CHO wild-type cells were saturated with PBS / BSA 2% and then incubated for 1 h with purified VHH-Fc or Fc-CHH at concentrations ranging from 350 nM to 0.03 pM. After washing, cells were incubated for 1 h with Alexa647-conjugated anti-hFc antibody (Jackson ImmunoResearch). After a final three washes and resuspension of cells in PBS, fluorescence was immediately measured using a MACSQuant flow cytometer (Miltenyi) and results were analyzed with MACSQuant software.

[0186] All VHH-Fc and Fc-VHH fusion proteins produced a concentration-dependent change in signal, confirming binding to the receptor of interest. d app was calculated using GraphPad Prism software (Figure 8B). d appwas significantly improved by conjugation with the Fc fragment, and K d app ranged from 0.44 nM to 51 nM for TfR-binding VHH-Fc and Fc-VHH.

[0187] Example IX Endocytosis and trafficking of the VHHs of the invention in an in vitro BBB model Co-culture models were set up using rat or mouse brain microvascular endothelial cells (BMECs) and rat or mouse astrocytes. This type of in vitro BBB model was used to evaluate the passive passage or active transport of numerous molecules, especially pharmacological agents, across BMECs and, by extrapolation, their ability to reach CNS tissues in vivo. The different models currently developed (bovine, porcine, murine, human) have the typical ultrastructural characteristics of the brain endothelium, especially the absence of tight junctions, fenestrations, low permeability to hydrophilic molecules, and high electrical resistance. Moreover, these models show a good correlation between the measurements obtained for the different molecules evaluated in vitro and in vivo for their properties crossing the BBB. All the data obtained so far show 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.

[0188] For example, an in vitro rat BBB model utilizes co-culture of BMECs and astrocytes (Molino et al., 2014, J. Vis. Exp. 88, e51278). Prior to cell culture, membrane inserts (Corning, Transwell, 1.0 μm porosity in 96-well plates or 12-well plates) were treated with type IV collagen and fibronectin on top to optimize BMEC adhesion and create basement membrane conditions. Primary cultures of mixed astrocytes were obtained from neonatal rat cerebral cortex. Briefly, meninges were removed and cortical pieces were mechanically dissociated and then enzymatically dissociated in trypsin solution. Dissociated cells were seeded in glial medium (GCM) containing DMEM supplemented with 10% fetal bovine serum in cell culture flasks and frozen in liquid nitrogen for later use. Primary cultures of BMECs were prepared from 5-6 week old Wistar rats. Briefly, cortical pieces were mechanically dissociated and then enzymatically dissociated in collagenase / dispase solution. Digested tissue was separated by density-dependent centrifugation in 25% bovine serum albumin. Microvessel pellets were seeded in endothelial cell medium (ECM) containing DMEM / F12 supplemented with 20% bovine platelet-poor plasma-derived serum and basic fibroblast growth factor (bFGF) 2ng / ml in culture flasks previously coated with collagen type IV and fibronectin. Five days prior to coculture establishment, astrocytes were thawed and plated in 12-well or 96-well plates (abluminal compartment). BMECs were then distributed on the upper surface of the filter in coculture (luminal compartment). Under these conditions, the in vitro model differentiated and expressed junction-associated proteins within 3 days and maintained optimal differentiation for another 3 days.

[0189] The binding / uptake of the VHH of the invention conjugated to the human Fc fragment of an IgG1 antibody (VHH-Fc) at the BBB was verified in the in vitro rat model described above (Figure 9). VHH A-Fc or VHH B-Fc were co-incubated with Tf-Alexa647 on live rBMEC monolayers for 2 hours at 37°C (Figure 9A). After this co-incubation, the cell monolayers were washed extensively and fixed with PFA 4%. The cell monolayers were permeabilized with 0.1% Triton X-100 solution. Immunostaining with an antibody against the human Fc fragment was used to detect VHH-Fc. Confocal microscopy was then used to assess the co-localization of the fluorescent signals of Tf-A647 and VHH A-Fc or VHH B-Fc (Figure 9A).

[0190] The results showed that after 2 h of co-incubation in this manner, VHH A-Fc or VHH B-Fc was rapidly endocytosed and almost completely co-localized with Tf-Alexa647.

[0191] This analysis of co-localization with different TfR ligands (VHH A-Fc, VHH B-Fc, and Tf-A647) confirmed the specificity of the VHHs of the invention for their target receptor.

[0192] To transport across the rBMEC monolayer to the abluminal compartment, VHH-Fc was incubated at 10 nM in the luminal compartment of the culture system for 24 to 72 h (Figure 9-C, Figure 9-D). Prior to the experiment, filter inserts containing rBMEC monolayers were placed in 96-well plates containing fresh transport buffer (75 μl in the luminal compartment and 250 μl in the abluminal compartment). To assess the integrity of the BBB in vitro and the lack of toxicity to endothelial cells, VHH-Fc was incubated with Lucifer Yellow (LY), a fluorescent small molecule that does not cross the BBB. After 24 h of incubation, the inserts were transferred to another 96-well plate containing fresh transport buffer for an additional 48 h interval. At the end of transport, LY accumulated in the abluminal compartment was quantified by fluorescence spectrophotometry and the results were analyzed by multiplying the LY concentration by 10 μl. -3 The endothelial surface permeability (or Pe) was expressed in cm / min. The Pe value for LY was 0.6 × 10 -3 An in vitro barrier was considered "permeable" or "open" if it was greater than ohm.cm / min. Also measured by an ohmmeter, ohm.cm 2 Transendothelial electrical resistance (TEER), expressed as , can also be used to measure BBB integrity in vitro during cross-BBB passage tests. The quality threshold is set at >400 ohm.cm 2 The carried out experiments show the absence of toxicity of VHH-Fc and no adverse effects on the permeability properties of the BBB (not shown). The content of Fc fragments of the VHH-Fc of the invention in the input (T0), the luminal compartment at the end of the transport experiment (T72, product harvest) and the abluminal compartment (24 h and +48 h transport interval) was quantified using an in-house anti-Fc ELISA assay with a sensitivity of 0.5-50 femtomoles. Absorbance units were converted to femtomoles per insert (0.143 cm for inserts in a 96-well plate). 2 surface area).

[0193] The results show that VHH B-Fc and VHH A-Fc conjugates were transported approximately 10-fold higher at 24 h and 5-fold higher at 72 h than VHH Z-Fc (negative control), and this transport clearly reached saturation between 24 and 72 h, further suggesting the involvement of a specific and saturable receptor-mediated process (Figure 9-D).

[0194] <Example X> Pharmacokinetic and organ uptake of VHH-Fc conjugates in vivo To evaluate the potential of the VHH-Fc conjugates of the invention to target receptor-rich organs 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 for various times. Plasma and brains were harvested. Brains were processed by capillary depletion method to isolate brain parenchyma from capillaries. VHH-Fc amounts in plasma, brain parenchyma, and microvessels were measured using an in-house anti-Fc ELISA. Results are shown as concentration (nM) or by organ-to-plasma ratio (Figure 10).

[0195] The TfR-binding conjugates VHH A-Fc and VHH A-Fc-Agly show significant brain targeting at 2 hours post-injection, with concentrations of 0.25 and 0.32 nM in the brain parenchyma for VHH A-Fc and VHH A-Fc-Agly, respectively, compared to 0.07 nM for the control VHH Z-Fc (Figure 10-B). Looking at the parenchyma-to-plasma ratio, a clear advantage can be seen, especially at 24 hours post-injection, where VHH A-Fc-Agly is still measurable in the brain parenchyma, while only 8 nM is present in the plasma. In the microvessels, VHH A-Fc and VHH A-Fc-Agly accumulate significantly over VHH Z-Fc at 2 hours post-injection, with concentrations 9- and 5-fold higher, respectively. Furthermore, VHH A-Fc concentration in microvessels is still 3-fold higher than VHH Z-Fc 24 hours after injection (Figure 10-C). These results were confirmed by looking at the microvessel to plasma ratio (Figure 10-E). These results indicate that the TfR-targeted VHHs of the present invention can be used to effectively deliver or improve the pharmacokinetic properties of drugs, especially protein cargoes.

[0196] Example XI Design and generation of therapeutic antibodies fused to VHHs Anti-TfR VHH A, A1, A5, A6, A7 and A8 (without tag) of the present invention were fused with mouse IfG1 13C3 monoclonal antibody, which has high specific affinity for the protofibrillar form of β-amyloid peptide (WO2009 / 065054). To generate 13C3-HC-VHH fusion proteins, DNA fragments encoding selected VHH were synthesized and cloned into 13C3 heavy chain (HC) vectors to encode 13C3-HC-VHH conjugates containing selected VHH sequences at their C-terminus fused to antibody heavy chain C-terminal amino acid residues. In another set of experiments, DNA fragments encoding selected VHH were cloned into 13C3 light chain (LC) vectors to encode 13C3 LC VHH conjugates containing selected VHH sequences at their C-terminus fused to antibody light chain C-terminal amino acid residues.

[0197] Fusion proteins were produced using the Expi293 expression system (Life Technologies) according to the manufacturer's instructions. 72 hours after transfection, supernatants were harvested and purified using HiTrap® Protein G High Performance Columns (GE Healthcare). Purified fusion proteins were quantified using 280 nm absorbance measurements.

[0198] The amino acid sequence of the 13C3-HC-VHHA conjugate is shown as SEQ ID NO:93. [QVQLQQSGPELVRPGVSVKISCKGSGYTFTDYAMHWVKQSHAKSLEWIGVISTKYGKTNYNQKFKGKATMTVDKSSSTAYMELARLTSEDSAIYYCARGDDGYSWGQGTSVTVSS](AKTTPPSVYPLAPGSAAQTNSMVT LGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQT QPREEQFNSTFRSVSELPMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEG LHNHHTEKSLSHSPG〔GGGG〕)[MA]EVQLVESGGGVVQPGGSLKLSCVASGTDFSINFIRWYRQAPGKQREFVAGFTATGNTNYADSMKGRFTISRDNTKNAVYLQIDSLKPEDTAVYYCYMLDKWGQGTQVTVSS[AAA] The tortoiseshell brackets are the 13C3 variable heavy chain sequences, the round brackets are the 13C3 constant heavy chain sequences, the tortoiseshell brackets and round brackets are Gly linkers, the MA and C-terminal AAA residues in the square brackets are derived from cloning and can be optionally removed, and the remaining part is the VHH.

[0199] The amino acid sequence of the 13C3-LC-VHHA conjugate is shown as SEQ ID NO:94. [DVVMTQTPLSLPVSLGDQASISCRSGQSLVHSNGNTYLHWYLQKPGQSPKLLIYTVSNRFSGVPDRFSGSGSGSDFTLKISRVEAEDLGVYFCSQNTFVPWTFGGGTK]LEIKR(ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDS KDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC〔SGSGGGGG〕)[MA]EVQLVESGGGVVQPGGSLKLSCVASGTDFSINFIRWYRQAPGKQREFVAGFTATGNTNYADSMKGRFTISRDNTKNAVYLQIDSLKPEDTAVYYCYMLDKWGQGTQVTVSS[AAA] The tortoiseshell brackets are the 13C3 variable kappa light chain sequence, FGGGTK are the J region, LEIKR are the multiple cloning site, the round brackets are the 13C3 constant kappa light chain sequence, the tortoiseshell brackets and round brackets are Gly linkers, the MA and C-terminal AAA residues in the square brackets are cloning derived and can be optionally removed, and the remaining portion is the VHH.

[0200] Measurement of binding affinity Flow cytometry was used to test the binding properties of the 13C3 conjugates of the present invention to the TfR and to determine the apparent affinity (K d app All experiments were performed under the same conditions as described in Example VII, with 13C3 constructs incubated at concentrations ranging from 15 μM to 7 pM and detected with Alexa647-conjugated anti-mouse antibody.

[0201] All 13C3-HC-VHH fusion proteins induced a concentration-dependent change in signal in both hTfR and mTfR expressing cell lines, confirming binding to the receptor (Figure 13). All 13C3 fusion proteins showed the same hTfR binding profile, with a slightly lower B maxAll fusion proteins showed different binding profiles at mTfR, with the exception of the VHH A7 fusion, which showed a 2-fold lower B than the 13C3-HC-VHH A. max whereas the 13C3 fusions of A5-A8 showed very low B max showed.

[0202] K of 13C3 fusion d app was calculated using GraphPad Prism software (Figure 13-B). The affinity for hTfR is approximately 10-20 nM K d app The results were similar for all fusions. max Despite the different affinities, VHH A, A1, and A6 13C3 HC fusions showed similar affinities of 10-20 nM, whereas the 13C3-HC-VHH A8 and 13C3-LC-VHH A fusions showed lower affinities of 315 nM and 106 nM, respectively.

[0203] Example XII In vivo brain uptake of 13C3-HC-VHH and 13C3-LC-VHH fusions To evaluate the potential of the VHHs of the invention to promote antibody uptake into the brain, 13C3-HC-VHH A and 13C3-HC-VHH A1 conjugates or non-vectorized 13C3 were injected into the tail vein of C57Bl6 mice at a dose of 35 nmoles / kg. Mice were perfused with saline for various times. Brains were harvested at 2 and 6 hours post-injection (pi). Half of the mouse brain was processed to isolate the capillary network from the brain parenchyma by capillary depletion method, which is based on centrifugation of the resuspended half-brain homogenate against a 20% dextran solution (Sigma Aldrich) and recovery of the parenchymal fraction. The second half of the mouse brain was directly processed (homogenized and lysed) for whole brain quantification. The amount of 13C3-HC-VHH conjugates in whole brain and brain parenchyma was measured using an in-house certified Meso Scale Discovery (MSD) direct coating (Aβ) immunoassay. (CV<20% and recovery ±30%) Results are expressed as concentration (nM) (Figure 14).

[0204] The results show that compared to the control non-vectorized 13C3 antibody, the TfR binding conjugates 13C3-HC-VHH A and 13C3-HC-VHH A1 showed a significant brain uptake advantage at 2 and 6 hours post-injection (Figure 14-A). The total brain concentrations of 13C3-HC-VHH A and 13C3-HC-VHH A1 were 8 and 5-fold greater than that of the non-vectorized 13C3 antibody, respectively, at 6 hours post-injection.

[0205] Crossing of the BBB by 13C3-HC-VHH A and 13C3-HC-VHH A1 was confirmed by concentrations measured in the brain parenchyma excluding the microcapillary network 6 hours after injection that were 10- and 9-fold greater than those of non-vectorized 13C3, respectively.

[0206] Further brain uptake studies further confirmed that 13C3-HC-VHH A and 13C3-LC-VHH A (light chain vectored type) exhibited BBB crossing at a dose of 70 nmoles / kg, with parenchymal accumulation 6- and 5-fold higher, respectively, than non-vectored 13C3 antibody at 4 hours post-injection.

[0207] Example XIII Synthesis of VHH-siRNA conjugates An anti-GFP siRNA, i.e., siGFPst1, containing chemical modifications for high resistance to nucleases, was conjugated to tagged VHH A to generate VHH A-siGFPst1 bioconjugate. Using the same conjugation method, siGFPst1 was conjugated to an unrelated VHH Z as a negative control, which has the same structure and size as VHH A-siGFPst1 conjugate but does not have the TfR targeting ability.

[0208] The conjugation method involved a convergent synthesis with parallel modification of i) VHH to site-specifically introduce an azide linker and ii) siGFPst1 to introduce a constrained azide moiety complementary to the azide functionality. In the final step, a copper-free click reaction was used to couple both functionalized VHH-azide and alkyne-siGFPst1 precursors to each other.

[0209] Synthesis of VHH-azide Site-specific conjugation with VHHs was performed using a bacterial transglutaminase (BTG)-based ligation method. The BTG enzyme catalyzes the formation of an isopeptide bond between a glutamine residue inserted into a tag sequence (i.e., a Q-tag) that is specifically recognized by the BTG enzyme and an amino-functional substrate. The amino-functional substrate introduced was a heterobifunctional linker containing an amino moiety at one end that was found to be a substrate for the BTG enzyme and an azide moiety at the other end for conjugation to siGFPst1 by copper-free click chemistry.

[0210] BTG Conjugation Protocol 3-Azido-1-propanamine (20 eq / Gln) was dissolved in PBS (1x) and added to the in-house produced Q-tagged VHH. Then, BTG (Zedira, Darmstadt, Germany) was introduced to the mixture (0.1 U / nmol of Gln), which was allowed to react overnight at 37°C. Purification of the crude mixture was performed by chromatography on a Protino Ni-ida1000 packed column according to the manufacturer's instructions to isolate the VHH-azide from the excess of starting material and potential by-products. The absorbance was read at 280 nm and the amount of purified VHH-azide construct, i.e. the amount of conjugation yield, was calculated (70-80% range).

[0211] The final VHH-azide was characterized by LCMS analysis to check its identity and purity.

[0212] Synthesis of alkyne-siGFPst1 siGFPst1 was purchased from Dharmacon, carrying a 3' amine modification (N6-siGFPst1) in the sense strand to allow further functionalization with an alkyne moiety required for conjugation with VHH-azide by click chemistry.

[0213] siGFPst1 functionalization protocol N6-siGFPst1 (1 eq) was dissolved in NaB (0.09 M, pH 8.5) conjugation buffer to obtain a final concentration of 0.3–0.8 mM. DBCO-NHS (20 eq, DMSO) was then added to this solution. The reaction mixture was stirred at room temperature for 2 h. Alkyne-siGFPst1 was purified by precipitation in cold absolute ethanol. The absorbance was read at 260 nm, and the amount of purified alkyne-siGFPst1 construct, i.e., the amount of conjugation yield, was calculated (range 40–50%).

[0214] The final alkyne-siGFPst1 was characterized by analytical HPLC to check identity and purity.

[0215] Synthesis of VHHH-siGFPst1 Both VHH-azide and alkyne-siGFPst1 precursors were finally conjugated via a copper-free click chemistry reaction to give the final conjugate VHH-siGFPst1.

[0216] VHH-GFPst1 conjugation protocol Alkyne-siGFPst1 (2 eq.) was dissolved in PBS (1x) and added to VHH-azide (1 eq., final concentration in 100 μM range in PBS (1x)). The reaction mixture was stirred overnight at room temperature. The final conjugate was first purified by gel filtration chromatography on a Superdex75 column and then concentrated using an Amicon Ultra centrifugal filter (10K). The absorbance was read at 260 nm and the amount of purified VHH-siGFPst1 construct, i.e. the conjugation yield, was calculated (total yield in 30% range).

[0217] 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.

[0218] <Example XIV> In vitro gene silencing activity of VHH-siRNA bioconjugates Specific cell targeting and effective intracellular delivery of therapeutic nucleic acids, especially siRNA, oligonucleotides, remains a major challenge. The structural and physicochemical characteristics of this molecule, a multivalent hydrophilic oligomer, prevent it from entering any intracellular compartment without assistance. The VHHs of the present invention were used to transport small interfering RNA (siRNA) across the cell membrane to access the cytosol.

[0219] First, the apparent hTfR binding affinity (K d app ) were assessed in the same CHO-hTfR-GFP cells by adding concentrations ranging from 2 μM to 30 pM for 1 h at 4° C. as described in Example VII (measurement of binding affinity of VHH A1-A19). Quantification of cell surface binding molecules was performed by anti-6His immunocytochemistry and experimental data were fitted to nonlinear regression using GraphPad Prism software. As previously shown by free VHH A and VHH B, VHH A-siGFPst1 and VHH B-siGFPst1 bioconjugates showed concentration-dependent and saturable binding to the cell surface target hTfR, with K d app The values ​​were in the low nanomolar range similar to unconjugated VHH A and VHH B (Figure 15A). No significant binding was observed with the control VHH Z, thus confirming that coupling of VHH A and VHH B to siRNA does not alter their ability to specifically and efficiently bind to hTfR.

[0220] Second, the intrinsic silencing activity of the VHH-siGFPst1 bioconjugate was evaluated in a live CHO cell line stably expressing TfR fused to EGFP (CHO-hTfR-EGFP cells) by transfecting the conjugate at 25 nM using Dharmafect1 (Dharmacon) to deliver it directly to the cytosol. The total cellular amount of GFP was quantified 72 hours after transfection using flow cytometry. The results show that the VHH A-siGFPst1 conjugate reduced GFP protein levels by about 85%, in the same range as unconjugated siGFPst1 or the control VHH Z-siGFPst1 conjugate (Figure 15B). This confirms that the coupling of either VHH A or Z does not interfere with the siRNA undergoing RISC loading and exhibiting silencing activity. In another set of experiments, VHH A-siGFPst1 conjugates were transfected into CHO-hTfR-EGFP cells at concentrations ranging from 10 nM to 1 pM, and the total cellular amount of GFP was quantified 120 h post-transfection using flow cytometry, which 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 15C).

[0221] Third, the ability of VHH A to induce hTfR-mediated endocytosis and subsequently deliver in pharmacological amounts to the cytosol of target cells when conjugated with siGFPst1 was evaluated. VHH A-siGFPst1 or control VHH Z-siGFPst1 bioconjugates were incubated at 1 μM in CHO-hTfR-GFP cells for 120 h at 37° C. to allow free uptake, delivery to the cytosol, and gene silencing at the mRNA transcription and protein levels. This resulted in a significant reduction in GFP protein levels of approximately -70% for the TfR-bound VHH A-siGFPst1 bioconjugate, while no silencing was observed for the control VHH Z-siGFPst1 bioconjugate (FIG. 15D). Next, VHH A-siGFPst1 bioconjugates were incubated in CHO-hTfR-GFP cells for 120 h at concentrations ranging from 3 μM to 10 pM. This resulted in a concentration-dependent reduction 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 (FIG. 15E), indicating that binding of the VHH A-siGFPst1 bioconjugate to cell surface hTfR and subsequent endocytosis allows its delivery to the cytosol in pharmacological amounts, with an IC50 in the same nanomolar range as the hTfR binding affinity of the bioconjugate.

[0222] Fourth, the involvement of hTfR in the silencing effect of VHH A-siGFPst1 bioconjugate observed upon free uptake in CHO-hTfR-GFP cells was confirmed by a competition assay. In this experiment, VHH A-siGFPst1 was incubated at a saturating concentration of 30 nM, determined from previous experiments, alone or in the presence of 100× excess of free VHH A, B, or Z for 120 h at 37° C. The results showed that the approximately 60% decrease in GFP protein level was almost completely suppressed in the presence of free VHH A or VHH B (GFP protein level was kept at 85% and 96% of control level, respectively). Importantly, no competition was observed when an excess of irrelevant VHH Z was used (FIG. 15F). This clearly confirmed that the silencing effect of VHH A-siGFPst1 bioconjugate was indeed due to hTfR-mediated cellular uptake and subsequent delivery to the cytoplasm.

[0223] Fifth, the TfR-mediated GFP silencing effect of the VHH A-siGFPst1 bioconjugate was evaluated using a pulse-chase method. CHO-hTfR-GFP cells were briefly exposed (6 h) to VHH A-siGFPst1 at concentrations ranging from 300 nM to 1 pM and then chased for up to a total of 120 h in ligand-free medium. This experiment allowed us to evaluate the contribution of early cellular uptake to the silencing effect previously observed with continuous 120 h incubation. As observed with continuous incubation, the VHH A-siGFPst1 bioconjugate also led to 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 15G). This result suggests that the majority of the effect previously observed during the 120 h continuous incubation was due to effective TfR-mediated uptake within the first 6 h. This finding is particularly interesting because the plasma pharmacokinetic profile of such bioconjugates in vivo typically allows for therapeutic tissue exposure for only a few hours when administered by intravenous or subcutaneous bolus injection. Thus, the TfR-targeted VHHs described herein represent viable tools for targeted and effective gene silencing in vivo.

[0224] Finally, the ability of VHH B to induce hTfR-mediated endocytosis and subsequent gene silencing was assessed by incubating VHH B-siGFPst1 bioconjugate at 30 nM for 120 h in CHO-hTfR-GFP cells. Results showed a ∼-60% reduction in GFP levels similar to that obtained with VHH A-siGFPst1 bioconjugate, confirming that these VHHs exhibit similar TfR targeting and intracellular delivery potential (Figure 15H).

[0225] To the best of the inventors' knowledge, receptor-mediated hepatocyte uptake via the asialoglycoprotein receptor (ASGPR) using triantennary GalNAc as a targeting ligand is the only ligand / receptor system capable of inducing specific and effective gene silencing at nanomolar concentrations. However, since ASGPR is exclusively expressed in hepatocytes in vivo, the use of this system for in vivo therapeutic applications with therapeutic nucleic acids is limited to liver targeting. Thus, the present invention provides a new ligand / receptor system for targeting and intracytoplasmic delivery of therapeutic nucleic acids, such as siRNA, at nanomolar concentrations outside the liver organs and tissues expressing TfR.

[0226] Example XV Synthesis of VHH-NODAGA conjugates Design of Q-tagged VHH A In this example, a DNA fragment encoding VHH A with an Ala linker, a His tag, a Gly linker and a Q tag (AAA-His tag-GGG-LQR sequence) introduced at its C-terminus was synthesized and cloned into the pHEN1 vector.

[0227] BTG-based preparation of VHH A-azides 3-Azido-1-propanamine (20 eq / Gln) was dissolved in PBS (1x) and added to the in-house produced LQR tagged VHH A. BTG (Zedira, Darmstadt, Germany) was introduced to the mixture (0.1 U / nmol of Gln). The reaction mixture was then reacted overnight at 37°C. Purification of the crude mixture was performed by chromatography on a Protino Ni-ida1000 packed column according to the manufacturer's instructions to isolate the VHH A-azide from the excess of starting material and potential by-products. The absorbance was read at 280 nm and the amount of purified VHH A-azide construct, i.e. the conjugation yield, was calculated (70-80% range). The final VHH A-azide was characterized by LCMS analysis to check its identity and purity.

[0228] Click chemistry reaction of conjugated VHH A-azide to commercially available alkyne-NODAGA VHH A-azide (1 eq.) was reacted with heterobifunctional NODAGA-BCN (5 eq.) (Chematech, Dijon, France) in PBS at room temperature. The reaction was monitored by LCMS. After completion of the reaction, the final conjugate was purified by chromatography on a Protino Ni-ida1000 packed column according to the manufacturer's instructions to isolate VHH A-azide from the excess of starting material and potential by-products. The absorbance was read at 280 nm and the amount of purified VHH A-NODAGA construct, i.e. the conjugation yield, was calculated (50-60% range). The final VHH A-NODAGA was characterized by LCMS analysis to check its identity and purity.

[0229] Example XVI PET imaging of VHH-68Ga bioconjugates in a subcutaneous mouse model of glioblastoma tumors Glioblastoma is the most common primary malignant brain tumor, and the human primary glioblastoma cell line U87 is known to express high TfR levels. To evaluate the targeting of the VHHs of the present invention to glioblastoma, radiolabeled VHH A-NODAGA bioconjugates were administered intravenously to mice previously implanted with glioblastoma cells (xenograft model), and PET scan imaging was performed.

[0230] Radiolabeling and binding affinity validation of VHH A-NODAGA First, VHH A-NODAGA was radiolabeled using 68Ga chloride. Gallium was obtained in the 68Ga3+ form using a commercially available TiO2-based 68Ge / 68Ga generator (Obninsk). The radiolabeling reaction was carried out by reacting 60 μg of VHH A-NODAGA with 74–148 MBq (2–4 mCi) of 68Ga in 400 μL of ammonium acetate buffer (1 M, pH 6) at 25 °C for 10 min. The resulting VHH A-68Ga radiochemical purity (RPC) was >95%.

[0231] After radiolabeling, the apparent hTfR binding affinities (K d app ) were assessed in the same CHO-hTfR-GFP cells by adding concentrations ranging from 2 μM to 30 pM for 1 h at 4° C. as described in Example VII (measurement of binding affinity of VHH A1-A19). Quantification of cell surface bound VHH A bioconjugates was performed by anti-6His immunocytochemistry and experimental data were fitted to nonlinear regression using GraphPad Prism software. VHH A-NODAGA and VHH A-68Ga bioconjugates showed concentration-dependent and saturable binding to the cell surface target receptor hTfR, with K d app The values ​​were in the low nanomolar range, similar to unconjugated VHH A (Figure 16A). No significant binding was observed with the control VHH Z, confirming that the coupling of VHH A with the NODAGA ligand and the radiolabeling protocol did not alter its ability to specifically bind to hTfR.

[0232] <PETスキャンイメージング> Animal studies were performed according to protocols approved by the Aix-Marseille ethical code (regulation 14). Four-week-old female BALB / c nude mice were obtained from Charles River. U87-MG cells (2 × 10 6 ) were implanted subcutaneously between the shoulders in mice (n=6). At 28 days post-implantation (when tumors reached a volume comprised between 300 and 700 mm3), animals were administered a single intravenous bolus dose of 5±1 MBq of VHH A-68Ga. After administration, the biodistribution of glioblastoma cancer xenografts and other tissues was assessed using PET imaging.

[0233] PET / CT scans were acquired at 2 h in three mice and at 2 h post-injection (pi) in another three mice. 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 at 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: 20 min per FOV). For the dual-modality PET / CT, CT images (35 kVp, 350 ns exposure time, medium zoom) were acquired and anatomical registration and attenuation correction were applied to the corresponding PET scan.

[0234] Imaging of animals injected with VHH A-68Ga showed significant accumulation at the tumor site (FIG. 16B, 1.46% ID / g) with a favorable tumor / muscle ratio (4.0). Thus, the experiment shows clear selective imaging and labeling of glioblastoma cancer by VHH A-68Ga at day 28, consistent with the known high expression levels of TfR.

[0235] [Table 2] JPEG0007599425000003.jpg229155JPEG0007599425000004.jpg226152JPEG0007599425000005.jpg226152JPEG0007599425000006.jpg153153 [Table 3] JPEG0007599425000008.jpg228155JPEG0007599425000009.jpg218156JPEG0007599425000010.jpg22215 3JPEG0007599425000011.jpg227152JPEG0007599425000012.jpg248153JPEG0007599425000013.jpg29152

Claims

1. It has the formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and binds to both human and non-human animal TfR with an affinity (Kd) comprised between 1 nM and 1 μM; The combination of CDR1, CDR2 and CDR3 comprises a combination of amino acid sequences of 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, or 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, SEQ ID NO: 67, 2 and 3, or SEQ ID NO: 69, 2 and 3, or SEQ ID NO: 1, 71 and 3, or SEQ ID NO: 1, 73 and 3, or SEQ ID NO: 1, 75 and 3, or SEQ ID NO: 1, 2 and 77, or SEQ ID NO: 1, 2 and 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.

2. The VHH molecule of claim 1, which crosses the blood-brain barrier.

3. The VHH molecule of claim 1 or 2, wherein the binding of the molecule to human TfR does not compete with the binding of transferrin.

4. A VHH molecule according to any one of claims 1 to 3, which is humanized.

5. A VHH molecule described in any one of claims 1 to 4, further comprising a Q tag of sequence LQR and / or a Gly linker sequence and / or a sequence of sequence number 51.

6. Comprising the sequence of SEQ ID NO: 51, 6. The VHH molecule of claim 5, comprising or consisting of an amino acid sequence selected from any one of SEQ ID NOs: 4, 8, 12, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 87, 88, 89, 90, 91, and 92.

7. A VHH molecule according to any one of claims 1 to 6, which is conjugated with at least one molecule.

8. conjugating at least one molecule or scaffold; A chimeric drug comprising one or more VHH molecules according to any one of claims 1 to 7, wherein the at least one molecule is an active compound, a virus or virus-like particle, or a stabilising group.

9. The chimeric agent of claim 8 , wherein the active compound is a therapeutic agent, a diagnostic agent, or an imaging agent.

10. A pharmaceutical or diagnostic composition comprising the chimeric agent of claim 8 or 9.

11. A nucleic acid encoding a VHH molecule according to any one of claims 1 to 7, or a chimeric agent according to claim 8 or 9, said molecule comprising an amino acid sequence.

12. A vector comprising the nucleic acid of claim 11 operably linked to a promoter.

13. A recombinant host cell comprising the nucleic acid of claim 11 or the vector of claim 12.

14. A method for producing a VHH molecule or a conjugate thereof, comprising culturing a host cell according to claim 13 under conditions allowing expression of said nucleic acid.

15. A method for producing a chimeric agent according to claim 8 or 9, comprising covalently or non-covalently conjugating one or more VHH molecules according to any one of claims 1 to 7 to at least one molecule.

16. Use of a VHH molecule according to any one of claims 1 to 7, a chimeric agent according to claim 8 or 9, or a composition according to claim 10 for the manufacture of a therapeutic agent, a diagnostic agent or an imaging agent.

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