Classifications

• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B40/00—Libraries per se, e.g. arrays, mixtures
  • C40B40/04—Libraries containing only organic compounds
  • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1034—Isolating an individual clone by screening libraries
  • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2881—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD71
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

• Transferrin receptor-binding molecules conjugates thereof and their uses
• the invention relates to Transferrin receptor (TfR)-binding molecules and the uses thereof.
• the invention particularly relates to Variable Domain of Camelid Heavy Chain-only (VHH) molecules, which bind TfR at the surface of cell membranes such as the blood-brain barrier (BBB), and the uses thereof e.g., to transport molecules of pharmaceutical or diagnostic interest into cells of the central nervous system or TfR-expressing tissues or organs, such as cancers.
• VHH Variable Domain of Camelid Heavy Chain-only
• the brain is protected from potentially toxic substances by the presence of two principal physiological barrier systems: the BBB, and the blood-cerebrospinal fluid barrier (BCSFB).
• BBB is regarded as the principal route for the uptake of plasma ligands. Its surface area is approximately 5000 times larger than that of the BCSFB.
• the overall length of the constitutive blood vessels of the BBB is approximately 600 km.
• Each cm 3 of cerebral cortex contains the equivalent of 1 km of blood vessels.
• the total surface area of the BBB is estimated at 20 m 2 (De Boer et al., 2007, Clin. Pharmacokinet, 46(7), 553-576).
• the cerebral endothelium which constitutes the BBB, represents a large surface of potential exchange between the blood and nervous tissue.
• this cerebral endothelium because of its specific properties, is also a major obstacle to the use of drugs to treat CNS disorders.
• the BBB is composed of brain capillary endothelial cells (BCECs) that present unique properties, not found in the fenestrated endothelial cells that compose the vascular system of other organs.
• BCECs form tight junctions, they are surrounded by a basal lamina, astrocyte end-feet, pericytes and microglial and neuronal cells that all together compose a very selective barrier, that controls molecular exchanges between the blood and the brain, that maintains brain homeostasis and that very efficiently protects the brain from toxins and pathogens.
• the drawback is that the BBB is also impermeable to most molecules, including drugs and imaging agents.
• the BBB is thus regarded as a major obstacle to overcome in the development of novel therapies for treating CNS disorders ⁇ Neuwelt et ah, 2008, Lancet Neurol., 7, 84-96).
• One of the research priorities to be associated with the discovery of molecules for treating, diagnosing or imaging CNS pathologies is the development of strategies that will allow/increase the passage of active substances across the BBB.
• One approach to avoid the BBB is to administer drugs by direct injection into the CNS (e.g., intraventricular, intracerebral or intrathecal), or to disrupt the BBB.
• CNS e.g., intraventricular, intracerebral or intrathecal
• Such highly invasive approaches have drawbacks (such as costs, short effect) and potential risks.
• TfR transferrin receptor
• Tf ligand transferrin
• Anti-TfR monoclonal antibodies have been studied as vectors for brain delivery, including the 0X26 antibody that targets the rat TfR (Moos and Morgan, 2001; Pardridge et al., 1991 ; Ulbrich et al., 2009), or the 8D3 (Pardridge, 2015; Zhang and Pardridge, 2005; Zhou et al., 2010) and R17-217 antibodies (Lee et al., 2000; Pardridge, 2015; Ulbrich et al., 2009) that target the mouse TfR (see also W02012075037, WO2013177062, WO201275037, W02016077840, WO2016208695).
• drawbacks of these antibodies include their absence of cross-species reactivity, and especially their absence of binding to the human TfR, which precludes preclinical or clinical studies. Also, the ability of such antibodies to effectively transport drugs across BBB still remains of debate.
• the present invention provides novel binding molecules, which can be used to effectively transport molecules across the BBB. More particularly, the invention discloses VHH molecules that bind both human and non-human TfR and which can deliver drugs to the CNS through transcytosis. The invention demonstrates that VHH molecules of the invention can effectively transmigrate through the CNS and deliver conjugated drugs or imaging agents in vivo. Such VHH thus represent valuable molecules for use in therapeutic or diagnostic approaches.
• An object of the invention thus relates to VHH molecules that bind a human and a non human TfR.
• a further object of the invention relates to VHH molecules that bind both a human and a non-human (e.g., rodent, such as murine or rat) TfR with substantially similar affinity.
• a further object of the invention is a VHH molecule that binds a human and a non- human TfR and can cross the human blood-brain barrier (“BBB”).
• BBB human blood-brain barrier
• Preferred VHH of the invention bind both a human and a murine TfR, can cross the human BBB, and have an affinity for TfR (Kd) below 10 mM, preferably comprised between 0.1 nM and 10 mM.
• the invention also relates to chimeric agents (also interchangeably called herein “conjugates”) comprising one or more VHH as defined above conjugated to at least one molecule or scaffold.
• the molecule conjugated to VHH may be e.g., any active compound useful in medicine, such as a drug, virus, diagnostic agent, tracer, etc.
• the chimeric agent may also contain, in addition to or instead of said active compound, a stabilizing group (e.g., a Fc or IgG for instance) to increase the plasma half-life of the VHH or conjugate.
• Particular chimeric agents of the invention thus comprise at least one VHH, a stabilizing group, and an active compound, in any order (for example a conjugate VHH-Fc-therapeutic agent).
• the invention further provides pharmaceutical or diagnostic compositions comprising a chimeric agent as defined above and, optionally, a suitable excipient.
• the invention further provides nucleic acids, vectors, and host cells encoding a VHH or chimeric agent as defined above.
• the invention also provides methods for making a VHH or chimeric agent, comprising culturing a host cell as defined above under conditions allowing expression of the nucleic acid.
• the invention further provides methods for making a chimeric agent, comprising conjugating one or more VHH as defined above to a molecule or agent or scaffold, covalently or non-covalently.
• Another object of the invention relates to a VHH molecule or chimeric agent as defined above for use as a medicament or diagnostic agent.
• Another object of the invention relates to the use of a VHH molecule as defined above for increasing the biological activity and/or CNS delivery of a substance of interest.
• Another object of the invention relates to a method for improving or enabling passage of a molecule across the BBB, comprising coupling said molecule to a VHH as defined above.
• Another object of the invention is a method for treating a pathology in a subject comprising administering to the subject a conjugate as defined above.
• Another object of the invention is a method for imaging a particular cell type, target tissue or organ in a subject comprising administering to the subject a conjugate as defined above.
• Another object of the invention is an improved method for treating a pathology in a subject with a drug, the improvement consisting in coupling said drug to a VHH molecule as defined above.
• the invention can be used in any mammal, in particular any human being.
• FIG. 1 TfR expression at the BBB.
• Western blots were performed on the membrane fraction of brain microvessels (BMVs) and brain microvessel endothelial cells (BMEC) from mouse, rat, pig and non-human primate (NHP; rhesus macaque). The amount of protein loaded is indicated under the picture n-d: non-digested; dig-: digested.
• Map of the plasmid construct used to generate the CHO-hTfR-EGFP cell line (B) Representative confocal photomicrographs of CHO-hTfR-EGFP cells (green) incubated 1 hr at 37 °C with Tf-Alexa647 (250 mg/ml, red). Cell nuclei were labeled with Hoechst#33342 at 0.5 mg/mL (blue). Co-labeling appears in yellow in the merged picture.
• FIG. 3 Cell surface binding and endocytosis of VHH A and VHH Z on CHO cells expressing hTfR and mTfR.
• Cell nuclei were labeled with Hoechst#33342 at 0.5 mg/ml (blue). Co-labeling appears in yellow/orange in the merged pictures.
• FIG. 4 Apparent K d determination of VHHs on hTfR- and mTfR- expressing CHO cell lines.
• A CHO-hTfR-EGFP and CHO-mTfR-EGFP cells were incubated 1 hr at 4 °C with various concentrations of VHHs, detected with a mouse anti-6His (1/1000) and an Alexa647-conjugated anti-mouse secondary antibody (1/200 or 1/400). Measurements were performed using flow cytometry. The ratio of fluorescence intensity for each point was normalized with the corresponding EGFP signal (receptor expression) and gave rise to the arbitrary unit. Data are presented as mean ⁇ SEM of 3 independent experiments.
• VHHs Characteristics of selected VHHs: Molecular Weight (Da); Theoretical pi; Apparent K d on human TfR (nM); Apparent K d on mouse TfR (nM). Data are presented as mean ⁇ SEM of 3 independent experiments. NB: no binding.
• FIG. 5 Competition assays between VHHs and Tf.
• A Principle of competition test. In a first step, CHO-hTfR-EGFP cells were incubated 1 hr at 4 °C with the competitor in dilution series. Second, the tracer at EC90 was added and incubated for 1 hr at 4 °C. Tracer was then revealed with the appropriate revelation system. Measurements were performed using flow cytometry. The ratio of fluorescence intensity for each point was normalized with the corresponding EGFP signal (receptor expression) and gave rise to the arbitrary unit.
• B CHO-hTfR-EGFP cells were incubated with the competitor (Tf).
• VHHs at EC90 were then added and detected with a mouse anti-cMyc antibody (1/50) and an Alexa647-conjugated anti mouse secondary antibody (1/200).
• C CHO-hTfR-EGFP cells were incubated with competitors (VHHs). Tracer (Tf-Alexa647) at EC90 was then added and detected directly. Data are presented as mean ⁇ SEM of 3 independent experiments.
• VHH conjugation strategies Using either chemical conjugation or recombinant fusion, VHHs can be used to vectorize all kinds of molecules, including non-exhaustively peptides, siRNAs, dyes, nanoparticles (NPs), liposomes, imaging agents and antibodies. Moreover, VHHs can be used to vectorize a molecule as a monovalent (VHH) or multivalent (VHH n ) conjugate.
• FIG. 7 Cell surface binding and endocytosis of VHH A-Fc and VHH Z-Fc fusion proteins on hTfR- and mTfR-expressing CHO cells.
• Cell nuclei were labeled with Hoechst#33342 at 0.5 mg/ml (blue). Co labeling appears in yellow/orange in the merged pictures.
• FIG. 8 Apparent K d determination of VHH-Fcs and Fc-VHHs on hTfR- and mTfR- expressing CHO cell lines.
• A CHO-hTfR-EGFP and CHO-mTfR-EGFP cells were incubated 1 hr at 4 °C with various concentrations of VHH-Fcs or Fc-VHHs, detected with an Alexa647-conjugated anti-hFc antibody (1/400). Measurements were performed using flow cytometry. The ratio of fluorescence intensity for each point was normalized with the corresponding EGFP signal (receptor expression) and gave rise to the arbitrary unit. Data are presented as mean ⁇ SEM of 3 independent experiments.
• VHH Z Characteristics of selected VHH-Fcs and Fc-VHHs: Molecular Weight (Da); Apparent K d on human TfR (nM); Apparent K d on mouse TfR (nM). Data are presented as mean ⁇ SEM of 3 independent experiments. NB: no binding for the control VHH (VHH Z).
• FIG. 9 Uptake and transport of VHH A-Fc and VHH B-Fc fusion proteins in an in vitro BBB model.
• rBMEC rat brain microvascular endothelial cell
• FIG. 1 Schematic representation of the in vitro BBB model, a co-culture system with primary rBMECs plated on collagen type IV/fibronectin- coated filter 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 rBMEC monolayers 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 hrs and transport to the abluminal compartment was evaluated (named 24 hrs).
• FIG. 10 Distribution of VHH-Fc fusion proteins in WT C57B1/6 mice at 2 and 24 hrs post-injection (p.i.).
• VHH A-Fc, VHH A-Fc-Agly and VHH Z-Fc fusions were injected into the tail vein at 5 mg/kg and mice were perfused with saline at either 2 or 24 hrs p.i., after collection of plasmas.
• Intermediate plasma samples were also collected using retro-orbital sampling at 15 min and 6 hrs p.i.
• Brains were processed to isolate brain parenchyma from capillary. Amounts of VHH-Fcs in each tissue were 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 ratio (D), and microvessel-to-plasma ratio (E). (4 ⁇ n ⁇ 12 per group per time point; * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001).
• FIG. 11 Apparent K d determination of VHH A1 to A9 on CHO cell lines stably expressing hTfR and mTfR.
• A CHO-hTfR-EGFP and CHO-mTfR-EGFP cells were incubated 1 hr at 4 °C with various concentrations of VHHs, detected with a mouse anti-6His (1/1000) and an Alexa647-conjugated anti-mouse secondary antibody (1/400). Measurements were performed using flow cytometry. The ratio of fluorescence intensity for each point was normalized with the corresponding EGFP signal (receptor expression) and gave rise to the arbitrary unit. Data are presented as mean ⁇ SEM of 3 independent experiments.
• VHHs Molecular Weight (Da); Theoretical pi; Apparent K d on human TfR (nM); Apparent K d on mouse TfR (nM). Data are presented as mean ⁇ SEM of 3 independent experiments. NB: no binding, LB: low binding.
• FIG. 12 Apparent K d determination of VHH A10 to A19 on CHO cell lines stably expressing hTfR and mTfR.
• A CHO-hTfR-EGFP and CHO-mTfR-EGFP cells were incubated 1 hr at 4 °C with various concentrations of VHHs, detected with a mouse anti-6His (1/1000) and an Alexa647-conjugated anti-mouse secondary antibody (1/400). Measurements were performed using flow cytometry. The ratio of fluorescence intensity for each point was normalized with the corresponding EGFP signal (receptor expression) and gave rise to the arbitrary unit. Data are presented as mean ⁇ SEM of 3 independent experiments.
• VHHs Molecular Weight (Da); Theoretical pi; Apparent K d on human TfR (nM); Apparent K d on mouse TfR (nM). Data are presented as mean ⁇ SEM of 3 independent experiments. NB: no binding.
• FIG. 13 Apparent K d determination of 13C3-HC-VHH fusions on hTfR- and mTfR- expressing CHO cell lines.
• A CHO-hTfR-EGFP and CHO-mTfR-EGFP cells were incubated 1 hr at 4 °C with various concentrations of 13C3 fusions and detected with an Alexa647-conjugated anti-mouse antibody (1/400). Measurements were performed using flow cytometry. The ratio of fluorescence intensity for each point was normalized with the corresponding EGFP signal (receptor expression) and gave rise to the arbitrary unit. Data are presented as mean ⁇ SEM of 3 independent experiments.
• FIG. 14 Distribution of 13C3 monoclonal antibody and 13C3-HC-VHH fusions in WT C57B1/6 mice at 2 and 6 hrs post-injection (p.i). 13C3, 13C3-HC-VHH A, and 13C3-HC-VHH Al, were injected into the tail vein at 35 nmoles/kg and mice were perfused with saline at either 2 or 6 hrs p.i. Brains were processed to isolate brain parenchyma from capillary.
• Amounts of 13C3 and 13C3-HC-VHH A/Al in each tissue/compartment were assessed using a qualified Meso Scale Discovery (MSD) direct coating (Abeta) immunoassay (%CV ⁇ 20 % and recovery ⁇ 30 %).
• MSD Meso Scale Discovery
• Abeta direct coating
• %CV ⁇ 20 % and recovery ⁇ 30 % were assessed using a qualified Meso Scale Discovery (MSD) direct coating (Abeta) immunoassay (%CV ⁇ 20 % and recovery ⁇ 30 %).
• Data are presented as mean ⁇ SEM of 13C3 and 13C3-HC-VHH A/Al concentration in total brain (A) and parenchyma (B) (1 ⁇ n ⁇ 4 per group per time point; * p ⁇ 0.05, ** p ⁇
• FIG. 1 In vitro gene silencing activity of VHH-siGFPstl bioconjugates.
• VHH A-siGFPstl and VHH B-siGFPstl bioconjugates bind hTfR.
• CHO-hTfR-EGFP cells were incubated 1 hr at 4 °C with various concentrations of the indicated compounds.
• Detection of VHHs was performed using a primary mouse anti-6His (1/1000) and an Alexa647-conjugated anti-mouse secondary antibody (1/400). Measurements of cell- surface signal associated to VHH were performed using flow cytometry. The results are expressed as the ratio of Alexa647-associated fluorescence intensity of test compounds to that of background fluorescence.
• the VHH A-siGFPstl bioconjugate displays gene silencing efficiency.
• CHO-hTfR-EGFP cells were transfected with the indicated compound at 25 nM using Dharmafect 1 (Dharmacon) during 72h at 37°C.
• the total fluorescence associated to the EGFP protein was then quantified using flow cytometry and rationalized to that of untreated (control) cells (set at 100%) *** p ⁇ 0.001.
• the VHH A-siGFPstl bioconjugate displays an intrinsic gene silencing activity in the picomolar range upon direct delivery into the cytosol.
• CHO-hTfR-EGFP cells were transfected with various concentrations of the VHH A-siGFPstl bioconjugate using Dharmafect 1 (Dharmacon) during 120 hrs at 37°C.
• the total fluorescence associated with the EGFP protein was then quantified using flow cytometry and rationalized to that of untreated (control) cells (set at 100%). Data were fit using a nonlinear regression using GraphPad Prism® software (solid line) to estimate the IC50 (concentration allowing 50% reduction of GFP protein levels) and the maximum effect (bottom plateau).
• D The VHH A-siGFPstl bioconjugate triggers specific and efficient TfR-mediated gene silencing. CHO-hTfR-EGFP cells were incubated with the indicated compounds at 1 mM during 120 hrs 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 the VHH A-siGFPstl bioconjugate allows cytosol delivery and subsequent gene silencing at nanomolar concentrations.
• CHO-hTfR-EGFP cells were incubated with various concentrations of the VHH A-siGFPstl bioconjugate during 120 hrs at 37°C.
• the total fluorescence associated with the EGFP protein was then quantified using flow cytometry and rationalized to that of untreated (control) cells (set at 100%). Data were processed and analyzed as described in (C).
• VHH A-siGFPstl bioconjugate was incubated with various concentrations of the VHH A-siGFPstl bioconjugate during a short 6-hour pulse followed by chase up to 120 hrs in ligand-free medium. Data were processed and analyzed as described in (B).
• H The gene silencing effect of the VHH B-siGFPstl bioconjugate was similar to that observed with VHH A-siGFPstl .
• CHO-hTfR-EGFP cells were incubated with VHH A-siGFPstl or VHH B-siGFPstl at 30 nM (saturating concentration based on the IC50 obtained with VHH A-siGFPstl) during 120 hrs at 37°C. Data were processed and analyzed as described in (C).
• FIG. 16 PET imaging of VHH A-68Ga bioconjugate in a subcutaneous mouse model of glioblastoma tumor.
• the VHH A-NODAGA and VHH A-68Ga bioconjugates bind hTfR as efficiently as the non-conjugated VHH A compound.
• CHO- hTfR-EGFP cells were incubated 1 hr at 4 °C with various concentrations of the indicated compounds.
• Detection of VHHs was performed using a primary mouse anti- 6His (1/1000) and an Alexa647-conjugated anti-mouse secondary antibody (1/400). Measurements of cell-surface signal associated with VHH were performed using flow cytometry.
• the present invention provides novel TfR-binding agents which can be used to transport molecules, such as therapeutic, imaging or diagnostic agents, across the BBB. More particularly, the invention discloses improved VHH molecules which bind TfR, and the uses thereof.
• the TfR is involved in the incorporation of iron, transported by its transferrin ligand, and in the regulation of cell growth (Neckers and Trepel 1986, Ponka and Lok 1999).
• transferrin receptors There are two types of transferrin receptors: the TfRl receptor and a homologous receptor, TfR2, expressed primarily in the liver.
• TfR is used to designate the TfRl homologue.
• 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 broad C -terminal extracellular domain of 670 amino acids, containing a trypsin cleavage site and a transferrin binding site (Aisen, 2004).
• YTRF Tyrosine-Threonine- Arginine-Phenylalanine
• Each subunit is capable of binding a transferrin molecule.
• the extracellular domain has one O-glycosylation site and three N-glycosylation sites, the latter being particularly important for the proper folding and transport of the receptor to the cell surface (Hayes et al., 1997).
• an intracellular phosphorylation site is present, whose functions are uncertain, and which plays no role in endocytosis (Rothenberger et al., 1987).
• the TfR receptor is expressed at high level by highly proliferating cells, whether healthy or neoplastic (Gatter et al., 1983). Many studies have shown high levels of TfR expression in cancer cells compared to healthy cells. Thus, pathologies such as breast cancer (Yang et al., 2001), gliomas (Prior et al., 1990), pulmonary 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, correlated with tumor grade and stage of disease or prognosis.
• pathologies such as breast cancer (Yang et al., 2001), gliomas (Prior et al., 1990), pulmonary adenocarcinoma (Kondo et al., 1990), chronic lymphocytic leukemia (Das Gupta and Shah
• Targeting drugs to TfR may thus be suitable for cancer treatment, as well as for crossing the BBB.
• VHH molecules that bind both the human and non human TfR.
• a human IgGl Fc region or drug (such as an antibody, siRNA) or imaging agent these VHH molecules retain TfR binding capacity, transmigrate across an in vitro BBB model, and demonstrate brain-targeting properties in vivo.
• a siRNA or NODAGA scaffold these VHH molecules retain TfR. binding capacity and efficient cell and organ delivery in vivo.
• the VHH molecules exhibit suitable levels of affinity and specificity to undergo proper endocytosis following TfR. binding.
• the invention thus provides novel TfR-binding molecules which represent valuable agents for drug targeting.
• An object of the invention thus relates to VHH molecules, wherein said VHH molecules bind both a human and a non-human (e.g., rodent, such as rat or murine) TfR.
• the VHH can cross the human BBB or bind TfR-expressing tissues such as cancers.
• the invention also relates to chimeric agents comprising such VHH, their manufacture, compositions comprising the same and the use thereof.
• VHH molecules correspond to the variable region of heavy chain only camelid antibodies that are naturally devoid of light chains.
• VHH have a very small size of around 15 kDa. They contain a single chain molecule that can bind its cognate antigen using a single domain.
• the antigen-binding surfaces of VHHs are usually more convex (or protruding) than those of conventional antibodies, which are usually flat or concave.
• VHHs are composed of 4 Framework Regions (or FRs) whose sequences and structures are defined as conserved, and three Complementarity Determining Regions (or CDRs) showing high variability both in sequence content and structure conformation, which are involved in antigen binding and provide antigen specificity.
• VHH molecules according to the present invention are polypeptides comprising (or consisting of, or consisting essentially of) an antigen-binding domain of a heavy chain only antibody (HcAb).
• HcAb heavy chain only antibody
• the inventors tested over 700 TfR-binding VHH from a library of VHH produced by lama immunization with a TfR immunogen. Following analysis of said clones for binding and specificity, the inventors further selected about 100 clones which had the required affinity, specificity and cross species binding. Said clones were all sequenced and their structure was analyzed and compared. Further VHH with controlled/improved binding properties were produced by mutagenesis. The sequences of the relevant domains and preferred VHH are provided in the experimental section and sequence listing. The properties of the VHH and conjugates thereof are also illustrated in the experimental section.
• VHH molecules of the invention typically comprise or consist of the formula:
• FR1 -CDR1 -FR2-CDR2-FR3 -CDR3 -FR4 wherein FRn designates framework regions and CDRn designates complementarity determining regions.
• VHH molecules of the invention comprise a CDR1 domain comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 1, 5, 9, 13, 17, 19, 67 or 69, or variants thereof having at least 75% amino acid identity to anyone of said sequences over the entire length thereof, preferably at least 85%, said variants retaining a TfR binding capacity.
• Preferred VHH molecules of the invention contain a CDR1 domain having an amino acid sequence selected from SEQ ID NOs: 1, 5, 9, 13, 17, 19, 67 or 69, or variants thereof having at most 1 amino acid modification.
• The“% identity” between amino acid (or nucleic acid) sequences may be determined by techniques known per se in the art.
• the % identity between two nucleic acid or amino acid sequences is determined by means of 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, S.B. and Wunsch, C.D., (1970), Journal of Molecular Biology, 48, 443-453).
• GAP Global Program for the Wisconsin Package, Version 8, August 1996, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711
• the % identity between two sequences designates the identity over the entire length of said sequences.
• 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.
• VHH molecules of the invention comprise a CDR2 domain comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 2, 6, 10, 14, 21, 23, 71, 73 or 75, or variants thereof having at least 70% amino acid identity to anyone of said sequences over the entire length thereof, preferably at least 85%, said variants retaining a TfR binding capacity.
• Preferred VHH molecules of the invention contain a CDR2 domain having an amino acid sequence selected from SEQ ID NOs: 2, 6, 10, 14, 21, 23, 71, 73 or 75, or variants thereof having at most 1 amino acid modification.
• 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.
• VHH molecules of the invention comprise a CDR3 domain comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 3, 7, 11, 15, 25, 27, 29, 31, 33, 77, 79, 81, 83, or 85, or variants thereof having at least 60% amino acid identity to anyone of said sequences over the entire length thereof, preferably at least 80%, more preferably at least 85%, said variants retaining a TfR binding capacity.
• Preferred VHH molecules of the invention contain a CDR3 domain having an amino acid sequence selected from SEQ ID NOs: 3, 7, 11, 15, 25, 27, 29, 31, 33, 77, 79, 81, 83, or 85, or variants thereof having at most 1 amino acid modification.
• VHH molecules of the invention comprise a CDR3 sequence comprising, or consisting essentially of SEQ ID NOs: 3, 7, 11, 15, 25, 27, 29, 31, 33, 77, 79, 81, 83, or 85.
• VHH molecules of the invention comprise:
• a CDR1 domain comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 1, 5, 9, 13, 17, 19, 67 or 69, or variants thereof having at least 75% amino acid identity to anyone of said sequences over the entire length thereof, preferably at least 85%, more preferably at least 95%;
• a CDR2 domain comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 2, 6, 10, 14, 21, 23, 71, 73 or 75, or variants thereof having at least 70% amino acid identity to anyone of said sequences over the entire length thereof, preferably at least 85%, more preferably at least 95%;
• a CDR3 domain comprising or consisting of an amino acid sequence selected from SEQ ID NOs: 3, 7, 11, 15, 25, 27, 29, 31, 33, 77, 79, 81, 83, or 85, or variants thereof having at least 60% amino acid identity to anyone of said sequences over the entire length thereof, preferably at least 80%, more preferably at least 95%,
• VHH having a TfR-binding capacity.
• VHH molecules of the invention comprise:
• a CDR1 domain having an amino acid sequence selected from SEQ ID NOs: 1, 5, 9,
• a CDR2 domain having an amino acid sequence selected from SEQ ID NOs: 2, 6, 10,
• a CDR3 domain having an amino acid sequence selected from SEQ ID NOs: 3, 7, 11,
• the VHH molecules of the invention comprise a CDR1, a CDR2 and a CDR3, wherein said CDR1, CDR2 and CDR3 domains comprise or consist of, respectively:
• VHH molecules of the invention comprise FRs domains as defined below.
• the FR1 domain comprises or consists of SEQ ID NO: 35 as represented below, or variants thereof having at least 85% amino acid identity to this sequence over the entire length thereof, preferably at least 90%, more preferably at least 95%:
• the bold amino acid residues are present and the variability occurs only on the other positions.
• the E in position 1 may be replaced with Q.
• V in position 5 may be replaced with Q.
• the E in position 6 may be replaced with Q.
• the G in position 10 may be replaced with K or A.
• the L in position 11 may be replaced with V or E.
• the A in position 23 may be replaced with V or T.
• the FR1 contains at most 4 amino acid modifications by reference to this sequence, even more preferably at most 3, even more preferably at most 2 amino acid modifications in non-bold amino acid residues.
• the FR1 has an amino acid sequence selected from anyone of the amino acid sequences listed below:
• VHH molecules of the invention comprise a FR2 domain comprising or consisting of SEQ ID NO: 40 as represented below, or variants thereof having at least 85% amino acid identity to this sequence over the entire length thereof, preferably at least 90%, or at least 95%:
• MRWYRQAPGKQRELVAT (SEQ ID NO: 40) More preferably, the bold amino acid residues are present and the variability occurs only on the other positions.
• the M in position 1 may be replaced with I or V.
• the R in position 2 may be replaced with G.
• the Y in position 4 may be replaced with F.
• the Q in position 6 may be replaced with R.
• the A in position 7 may be replaced with R.
• the Q in position 11 may be replaced with E.
• the L in position 14 may be replaced with F or W.
• the T in position 17 may be replaced with G or S.
• the FR2 contains at most 6 amino acid modifications by reference to this sequence, even more preferably at most 5, at most 3, even more preferably at most 2 amino acid modifications in non-bold amino acid residues.
• VHH molecules of the invention comprise at least one of the following amino acids in the FR2 domain: Phe42, Glu49, Arg50 or Gly52.
• the FR2 has an amino acid sequence selected from anyone of the amino acid sequences listed below:
• VHH molecules of the invention comprise a FR3 domain comprising or consisting of SEQ ID NO: 45 as represented below, or variants thereof having at least 85% amino acid identity to this sequence over the entire length thereof, preferably at least 90%, more preferably at least 95%:
• the Y in position 1 may be replaced with N.
• the Y in position 2 may be replaced with A.
• the A in position 3 may be replaced with P or I.
• the D in position 4 may be replaced with S.
• the FR3 contains at most 7 amino acid modifications by reference to this sequence, even more preferably at most 6, at most 3, even more preferably at most 2 amino acid modifications in non-bold amino acid residues.
• the FR3 has an amino acid sequence selected from anyone of the amino acid sequences listed below:
• VHH molecules of the invention comprise a FR4 domain comprising or consisting of SEQ ID NO: 50 as represented below, or variants thereof having at least 85% amino acid identity to this sequence over the entire length thereof, preferably at least 90%, more preferably at least 95%:
• the bold amino acid residues are present and the variability occurs only on the other positions.
• the FR4 contains at most 4 amino acid modifications by reference to this sequence, even more preferably at most 3, even more preferably at most 2 amino acid modifications in non-bold amino acid residues.
• VHH A A specific illustrative example of a FR4 sequence is SEQ ID NO: 50.
• TfR-binding VHH molecules of the invention are molecules comprising or consisting of an amino acid sequence selected from anyone of SEQ ID NOs: 4 (VHH A), 8 (VHH B), 12 (VHH C), 16 (VHH D), 18 (VHH Al), 20 (VHH A2), 22 (VHH A3), 24 (VHH A4), 26 (VHH A5), 28 (VHH A6), 30 (VHH A7), 32
• VHH A8 34 (VHH A9), 68 (VHH A10), 70 (VHH Al 1), 72 (VHH A12), 74 (VHH A13), 76 (VHH A14), 78 (VHH A15), 80 (VHH A16), 82 (VHH A17), 84 (VHH A18), 86 (VHH A 19), 87 (VHH A20), 88 (VHH A21), 89 (VHH A22), 90 (VHH A23), 91 (VHH A24), and 92 (VHH A25) wherein x is 0.
• the VHH of the invention are humanized.
• one or more of the FR and/or CDR domains may be (further) modified by one or more amino acid substitutions.
• the VHH are humanized by modification (e.g., amino acid substitution) of the FR1 domain.
• a typical humanized position in FR1 is selected from 19R and 23 A, or both (by reference to e.g., anyone of SEQ ID NOs: 35-39 or variants thereof).
• a specific example of such a humanized FR1 thus comprises SEQ ID NO: 36 wherein K19 and/or V23 are respectively modified into 19R and 23 A.
• the VHH are humanized by modification of the CDR1 domain.
• a typical humanized position in CDR1 (by reference to e.g., anyone of SEQ ID NO: 1, 5, 9, 13, 17, 19, 67 or 69 or variants thereof) is 8A.
• the VHH are humanized by modification of the FR2 domain.
• a typical humanized position in FR2 is selected from 1M, 2S or 2H, 4V, 11G, 12L, 14W, or combinations thereof (by reference to e.g., anyone of SEQ ID NOs: 40- 44 or variants thereof).
• a specific example of such a humanized FR2 thus comprises
• SEQ ID NO: 41 wherein one or more or all of II, R2, Y4, Q 11 , R12, and F14 are respectively modified into 1M, 2S or 2H, 4V, 11G, 12L, and 14W.
• the VHH are humanized by modification of the CDR2 domain.
• a typical humanized position in CDR2 (by reference to e.g., anyone of SEQ ID NO: 2, 6, 10, 14, 21, 23, 71, 73, 75 or variants thereof) is II.
• the VHH are humanized by modification of the FR3 domain.
• a typical humanized position in FR3 is selected from 6V, 17A, 20T, 21L, 25M, 26N, 29R, or combinations thereof (by reference to e.g., anyone of SEQ ID NOs: 45-49 or variants thereof).
• a specific example of such a humanized FR3 thus comprises SEQ ID NO: 46 wherein one or more or all of M6, T17, A20, V21, 125, D26, and K29, are respectively modified into 6V, 17A, 20T, 21L, 25M, 26N, and 29R.
• the VHH are humanized by modification of the CDR3 domain.
• a typical humanized position in CDR3 (by reference to e.g., anyone of SEQ ID NO: 3, 7, 11, 15, 25, 27, 29, 31, 33, 77, 79, 81, 83, 85 or variants thereof) is 1A or 2R, or both.
• the FR1 and/or FR2 and/or FR3 and/or CDR1 and/or CDR2 and/or CDR3 domains are humanized.
• humanized TfR-binding VHH molecules of the invention are molecules comprising or consisting of an amino acid sequence selected from anyone of SEQ ID NOs: 87 (VHH A20), 88 (VHH A21), 89 (VHH A22), 90 (VHH A23), 91 (VHH A24), and 92 (VHH A25), wherein x is 0.
• the VHH molecules may further comprise one or several tags, suitable for e.g., purification, coupling, etc.
• tags include a His tag (e.g., His 6 ), a Q-tag (LQR), or a myc tag (EQKLISEEDL).
• His tag e.g., His 6
• Q-tag LQR
• myc tag EQKLISEEDL
• the one or several tags are located C-ter of the VHH.
• the VHH may comprise, at the C-ter end, the following additional sequence AAAEOKLISEEDLNGAAHHHHHHGS (SEQ ID NO: 51), wherein simple underline is a myc tag and double underline is a His tag (the remaining residues being linkers or resulting from cloning).
• VHH A molecules comprising or consisting of an amino acid sequence selected from anyone of SEQ ID Nos: 4 (VHH A), 8 (VHH B), 12 (VHH C), 16 (VHH D), 18 (VHH Al), 20 (VHH A2), 22 (VHH A3), 24 (VHH A4), 26 (VHH A5), 28 (VHH A6), 30 (VHH A7), 32 (VHH A8), 34 (VHH A9), 68 (VHH A10), 70 (VHH Al l), 72 (VHH A12), 74 (VHH A13), 76 (VHH A14), 78 (VHH A15), 80 (VHH A16), 82 (VHH A17), 84 (VHH A18), 86 (VHH A19), 87 (VHH A20), 88 (VHH A21), 89 (VHH A22), 90 (VHH A23), 91 (VHH A24), and 92 (VHH A25), wherein
• the VHH of the invention may comprise a Q-tag of sequence LQR, preferably located C-ter of the VHH.
• the VHH of the invention may comprise a Gly linker, preferably located C-ter of the VHH.
• the Gly linker may comprise a Gly repeat of e.g., 2-7 Gly residues, such as 3 to 6.
• Specific examples of Gly linkers include Gly3, Gly4, Gly5 or SerGlySerGly5.
• VHH of the invention may comprise a Gly linker and a Q-tag, preferably located C-terminally. More specific examples of such VHH comprise the following structure: VHH-GlyLinker-Qtag, wherein the GlyLinker comprises 2-6 Gly residues and the Q tag contains or consists of LQR.
• the VHH may comprise, at the C-ter end, the following additional sequence GGGLQR wherein underline is the Q-tag and bold is a Gly linker.
• VHH of the invention may comprise an Ala linker, a His tag, a Gly linker and a Q-tag.
• the linkers and tags are located C- terminally of the VHH.
• the Qtag at least may be located N-ter of the VHH. More specific examples of such VHH comprise the following structure: VHH-AlaLinker-HisTag-GlyLinker-Qtag, wherein the AlaLinker comprises 3 residues, the HisTag comprises 2-7 His residues, the GlyLinker comprises 2-6 Gly residues and the Q tag contains or consists of LQR.
• the VHH may comprise, at the C-ter end, the following additional sequence AAAHHHHHHGGGLQR wherein underline is the Q-tag, bold are an Ala and a Gly linker, double underline is a His tag.
• TfR-binding VHH molecules of the invention are VHH molecules which competitively inhibit binding of a VHH as defined above to a human and a non-human TfR.
• the term“competitively inhibits” indicates that the VHH can reduce or inhibit or displace the binding of a said reference VHH to TfR, in vitro or in vivo.
• Competition assays can be performed using standard techniques such as, for instance, competitive ELISA or other binding assays.
• a competitive binding assay involves a recombinant cell or membrane preparation expressing a TfR, optionally bound to a solid substrate, an unlabeled test VHH (or a phage expressing the same) and a labeled reference VHH (or a phage expressing the same).
• Competitive inhibition is measured by determining the amount of labeled VHH bound in the presence of the test VHH.
• the test VHH is present in excess, such as about 5 to 500 times the amount of reference VHH.
• the test VHH is in 100-fold excess.
• Preferred competing VHH bind epitopes that share common amino acid residues.
• VHH molecules are able to bind TfR in vitro and in vivo. They show adequate affinity, with an apparent Kd comprised between O. lnM and IOmM, particularly between I mM and InM. Furthermore, all of these molecules bind both human and murine TfR. Moreover, binding of said VHH of the invention to a human TfR receptor does not compete with binding of transferrin, the endogenous TfR ligand, and thus does not affect regular functions of said ligand. Conjugates produced with such VHH molecules have further been shown to bind TfR in vitro and to be transported across the BBB into the CNS in vivo , showing transcytosis. Such VHH thus represent potent agents for drug delivery or targeting.
• the VHH of the invention can be synthesized by any technique known to those skilled in the art (chemical, biological or genetic synthesis, etc.). They can be preserved as-is, or be formulated in the presence of a substance of interest or any acceptable excipient.
• the VHH of the invention can also be obtained from a nucleic acid sequence coding for the same, as described further below.
• a further object of the invention relates to conjugates (also interchangeably called herein“chimeric agents”) comprising one or more VHH molecules as defined above, conjugated to at least one molecule or scaffold of interest.
• the molecule of interest may be any molecule such as a medicament or drug, a diagnostic agent, an imaging molecule, a tracer, etc.
• conjugated molecules of interest include, without limitation, any chemical entity such as small chemical molecules (such as an antibiotic, antiviral, immunomodulator, antineoplastic, anti-inflammatory, adjuvant, etc.); peptides, polypeptides and proteins (such as an enzyme, hormone, neurotrophic factor, neuropeptide, cytokine, apolipoprotein, growth factor, antigen, antibody or part of an antibody, adjuvant, etc.); nucleic acids (such as RNA or DNA of human, viral, animal, eukaryotic or prokaryotic, plant or synthetic origin, etc., including e.g., coding genes, inhibitory nucleic acids such as ribozymes, antisense, interfering nucleic acids, full genomes or portions thereof, plasmids, etc); lipids, viruses, markers, or tracers, for instance.
• the“molecule of interest” can be any drug active ingredient, whether a chemical, biochemical, natural or synthetic compound.
• the expression“small chemical molecule” designates a molecule of pharmaceutical interest with a maximum molecular weight of 1000 Daltons, typically between 300 Daltons and 700 Daltons.
• the conjugated compound is typically a medicament (such as a small drug, nucleic acid or polypeptide, e.g., an antibody or fragment thereof) or imaging agent suitable for treating or detecting neurological, infectious or cancerous pathologies, preferably of the CNS, such as the brain.
• a medicament such as a small drug, nucleic acid or polypeptide, e.g., an antibody or fragment thereof
• imaging agent suitable for treating or detecting neurological, infectious or cancerous pathologies, preferably of the CNS, such as the brain.
• the chimeric agent may also contain, in addition to or instead of said compound of interest, a stabilizing group to increase the plasma half-life of the VHH or conjugate.
• a stabilizing group to increase the plasma half-life of the VHH or conjugate.
• Particular chimeric agents of the invention thus comprise at least one VHH, a stabilizing group, and an active compound, in any order.
• the stabilizing group may be any group known to have substantial plasma half-life (e.g. at least 1 hour) and essentially no adverse biological activity
• examples of such stabilizing group include, for instance, a Fc fragment of an immunoglobulin or variants thereof, large human serum proteins such as albumin, HSA, or IgGs or PEGs molecules.
• the stabilizing group is a Fc fragment of a human IgGl .
• the stabilizing group is an aglycosylated Fc fragment of an IgGl .
• the VHH may be conjugated N-ter or C-ter of the stabilizing group, or both.
• the stabilizing group is a Fc fragment, conjugation is typically by genetic fusion.
• the resulting protein may remain as a monomeric agent, or multimerize, depending on the nature of the stabilizing group.
• the fusion protein Fc- VHH or VHH-Fc usually forms homodimers.
• coupling can be performed by any acceptable means of bonding taking into account the chemical nature, obstruction and number of conjugated entities. Coupling can thus be carried out by one or more covalent, ionic, hydrogen, hydrophobic or Van der Waals bonds, cleavable or non- cleavable in physiological medium or within cells. Furthermore, coupling can be made at various reactive groups, and notably at one or more terminal ends and/or at one or more internal or lateral reactive groups. Coupling can also be carried out using genetic engineering.
• the interaction is sufficiently strong so that the VHH is not dissociated from the active substance before having reached its site of action.
• the preferred coupling of the invention is covalent coupling, although non- covalent coupling may also be employed.
• the compound of interest can be coupled with the VHH either at one of the terminal ends (N-term or C-term), or at a side chain of one of the constitutive amino acids of the sequence (Majumdar and Siahaan, Med Res Rev., Epub ahead of print).
• the compound of interest can be coupled directly to a VHH, or indirectly by means of a linker or spacer.
• Means of covalent chemical coupling, calling upon a spacer or not include for instance those selected from bi- or multifunctional agents containing alkyl, aryl or peptide groups by esters, aldehydes or alkyl or aryl acids, anhydride, sulfhydryl or carboxyl groups, groups derived from cyanogen bromide or chloride, carbonyldiimidazole, succinimide esters or sulfonic halides.
• Fig 6. Illustrative strategies for conjugating a VHH of the invention to a molecule or scaffold are disclosed in Fig 6.
• coupling is by genetic fusion.
• Such strategy can be used when the coupled molecule is a peptide or polypeptide.
• a nucleic acid molecule encoding the VHH fused to the molecule is prepared and expressed in any suitable expression system, to produce the conjugate.
• coupling is by enzymatic reaction.
• site-specific conjugation onto the VHH can be performed using the transglutaminase enzyme (TGase).
• TGase catalyzes the formation of a stable isopeptidic bond between (i) the side chain of a glutamine residue inserted in a tag sequence specifically recognized by the TGase (namely a Q-tag) and (ii) an amino-functionalized donor substrate.
• the inventors have developed a particular tag sequence (named“Q-tag”) which is recognized by TGase and may be used to couple VHH of the invention to any molecule of interest, particularly chemical drugs or agents.
• VHHs are prepared by genetic fusion to add in tandem (typically to their C- terminus) the following tags: first an optional trialanine linker, then an optional His-tag, then an optional small triglycine linker, and finally a Q-tag.
• the triglycine linker allows to space out the Q-tag to allow a better accessibility of the TGase to the glutamine while the His-tag aims at facilitating the purification of the VHH and its further functionalized versions.
• the general conjugation strategy that was developed is a convergent synthesis that is based on a process comprising:
• a heterobifunctional linker having two different reactive ends is allowed to be processed by the TGase: one suitable primary amine-group toward the TGase and one orthogonal reactive moiety.
• orthogonal and reactive groups include azides, constraints alkynes such as DBCO (dibenzocyclooctyne) or BCN (bicyclo[6.1.0]nonyne), tetrazines, TCO (trans- cyclooctene), free or protected thiols, etc.
• orthogonal and reactive groups include azides, constrained alkynes such as DBCO or BCN, tetrazines, TCO, free or protected thiols, etc.
• an object of the invention resides in a method for coupling two molecules using a Q-tag as defined above through TGase coupling reaction.
• a further object of the invention is a VHH comprising a Q-tag.
• a further object of the invention is a VHH molecule comprising a linker, such as a Gly linker, and a Q-tag.
• Preferred VHH of the invention have the following structure:
• VHH-Linker-Hi S m -Linker-LQR wherein :
• VHH is any VHH molecule
• Linker is any molecular linker such as an Ala or Gly linker (preferably the two linkers are different); and m is an integer from 0 to 6.
• the invention relates to a conjugate comprising a VHH covalently linked to a chemical entity.
• Preferred variants of such conjugates contain 1 VHH and 1 chemical entity.
• the 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, a siRNA, etc.
• Preferred variants of such conjugates contain 1 VHH and 1 nucleic acid molecule.
• the 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.
• Preferred variants of such conjugates contain 1 VHH and 1 peptide.
• the invention relates to a conjugate comprising a VHH covalently linked to a dye.
• the 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 active agents.
• Preferred variants of such conjugates contain several VHH molecules coupled to each nanoparticle or liposome.
• the conjugate comprises an antibody or a fragment thereof to which one or several VHH molecules are coupled.
• a VHH molecule is coupled to a C- or N-ter of a heavy or light chain, or both, or to the C- or N-ter of an Fc fragment.
• the invention also relates to a method for preparing a conjugate compound such as defined above, characterized in that it comprises a step of coupling between a VHH and a molecule or scaffold, preferably by a chemical, biochemical or enzymatic pathway, or by genetic engineering.
• a chimeric agent of the invention when several VHH are present, they may have a similar or different binding specificity.
• Nucleic acids, vectors and host cells A further aspect of the invention relates to a nucleic acid encoding a VHH as defined above, or a conjugate thereof (when the conjugated moiety is an amino acid sequence).
• the nucleic acid may be single- or double-stranded.
• the nucleic acid can be DNA (cDNA or gDNA), RNA, or a mixture thereof. It can be in single stranded form or in duplex form or a mixture of the two. It can comprise modified nucleotides, comprising for example a modified bond, a modified purine or pyrimidine base, or a modified sugar. It can be prepared by any method known to one 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 molecules according to the invention and codon usage may be adapted according to the host cell in which the nucleic acid shall be transcribed. These steps may be carried out according to methods well known to one of skill in the art and some of which are described in the reference manual Sambrook et al. (Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual, Third Edition Cold Spring Harbor).
• nucleic acid sequences include the sequences comprising anyone of SEQ ID NOs: 52-64 and 95-110, and the complementary sequence thereto, as well as fragments thereof devoid of the optional tag-coding portion.
• the domains encoding CDR1, CDR2 and CDR3 are underlined.
• the tag-coding portion is in bold.
• the invention also relates to a vector containing such a nucleic acid, optionally under control of regulatory sequences (e.g., promoter, terminator, etc).
• the vector may be a plasmid, virus, cosmid, phagemid, artificial chromosome, etc.
• the vector may comprise a nucleic acid of the invention operably linked to a regulatory region, i.e. a region comprising one or more control sequences.
• the vector may comprise several nucleic acids of the invention operably linked to several regulatory regions.
• control sequences means nucleic acid sequences necessary for expression of a coding region. Control sequences may be endogenous or heterologous. Well- known control sequences and currently used by the person skilled in the art will be preferred. Such control sequences include, but are not limited to, promoter, signal- peptide sequence and transcription terminator.
• operably linked means a configuration in which a control sequence is placed at an appropriate position relative to a coding sequence, in such a way that the control sequence directs expression of the coding region.
• the present invention further relates to the use of a nucleic acid or vector according to the invention to transform, transfect or transduce a host cell.
• the present invention also provides a host cell comprising one or several nucleic acids of the invention and/or one or several vectors of the invention.
• host cell also encompasses 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 may be prokaryotic (e.g., a bacterium) or eukaryotic (e.g., yeast, plant, insect or mammalian cell). Specific illustrative examples of such cells include E. coli strains, CHO cells, Saccharomyces strains, plant cells, sf9 insect cells etc.
• VHH molecules of the invention can bind to TfR and thus target/deliver molecules to TfR-expressing cells or organs.
• binding is preferably specific, so that binding to TfR occurs with higher affinity than binding to any other antigen in the same species.
• Preferred VHH molecules of the invention bind human TfRl and a murine or rat TfR. More preferably, the VHH molecules bind the human and murine receptors with a substantially similar affinity.
• the invention thus relates to methods of targeting/delivering a compound to/through a TfR-expressing cell or organ, comprising coupling said compound to at least one VHH of the invention.
• the invention further relates to the use of a VHH such as defined above, as a vector for the transport of a compound to/through a TfR-expressing cell or organ.
• the invention also relates to the use of a VHH such as defined above for preparing a drug capable of crossing the BBB.
• the invention also relates to a method for enabling or improving the passage of a molecule across the BBB, comprising the coupling of the molecule to a VHH of the invention.
• the VHH of the invention may be used to transport or deliver any compound, such as small drugs, proteins, polypeptides, peptides, amino acids, lipids, nucleic acids, viruses, liposomes, etc.
• the invention also relates to a pharmaceutical composition characterized in that it comprises at least one VHH or VHH-drug conjugate such as defined above and one or more pharmaceutically acceptable excipients.
• the invention also relates to a diagnostic composition characterized in that it comprises a VHH or VHH-diagnostic or medical imaging agent conjugate compound such as defined above.
• the conjugate can be used in the form of any pharmaceutically acceptable salt.
• pharmaceutically acceptable salts refers to, for example and in a non- restrictive way, pharmaceutically acceptable base or acid addition salts, hydrates, esters, solvates, precursors, metabolites or stereoisomers, said vectors or conjugates loaded with at least one substance of interest.
• salts refers to nontoxic salts, which can be generally prepared by reacting a free base with a suitable organic or inorganic acid. These salts preserve the biological effectiveness and the properties of free bases.
• Representative examples of such salts include water-soluble and water-insoluble salts such as acetates, N-methylglucamine ammonium, amsonates (4,4-diaminostilbene- 2,2’-disulphonates), benzenesulphonates, benzonates, bicarbonates, bisulphates, bitartrates, borates, hydrobromides, bromides, buryrates, camsylates, carbonates, hydrochlorates, chlorides, citrates, clavulanates, dichlorhydrates, diphosphates, edetates, calcium edetates, edisylates, estolates, esylates, fumarates, gluceptates, gluconates, glutamates, glycolylarsanylate
• compositions of the invention advantageously comprise a pharmaceutically acceptable carrier or excipient.
• the pharmaceutically acceptable carrier can be selected from the carriers classically used according to each mode of administration. According to the mode of administration envisaged, the compounds can be in solid, semi-solid or liquid form.
• the active substance can be combined with: a) diluents, for example lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b) lubricants, for example silica, talc, stearic acid, its magnesium or calcium salt and/or polyethylene glycol; c) binders, for example magnesium and aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethyl cellulose and/or polyvinylpyrrolidone; d) disintegrants, for example starch, agar, alginic acid or its sodium salt, or effervescent mixtures; and/or d) absorbents, dyes, flavoring agents and sweeteners.
• diluents for example lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine
• lubricants for example silica
• the excipients can be, for example, mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate and analogues of pharmaceutical quality.
• the excipient can, for example, be an emulsion or oily suspension, or polyalkylene glycol-based, such as polypropylene glycol.
• Liquid compositions in particular injectables or those included in a soft capsule, can be prepared, for example, by dissolution, dispersion, etc., of the active substance in a pharmaceutically pure solvent such as, for example, water, physiological saline solution, aqueous dextrose, glycerol, ethanol, oil and analogues thereof.
• a pharmaceutically pure solvent such as, for example, water, physiological saline solution, aqueous dextrose, glycerol, ethanol, oil and analogues thereof.
• compositions or conjugates of the invention can be administered by any suitable route and, in a non-restrictive way, by parenteral route, such as, for example, in the form of preparations that can be injected by subcutaneous, intravenous or intramuscular route; by oral route (or per os), such as, for example, in the form of coated or uncoated tablets, gelatin capsules, powders, pellets, suspensions or oral solutions (one such form for oral administration can be either with immediate release or with extended or delayed release); by rectal route such as, for example, in the form of suppositories; by topical route, in particular by transdermal route, such as, for example, in the form of patches, pomades or gels; by intranasal route such as, for example, in aerosol and spray form; by perlingual route; or by intraocular route.
• parenteral route such as, for example, in the form of preparations that can be injected by subcutaneous, intravenous or intramuscular route
• oral route or per o
• compositions typically comprise an effective dose of a VHH or conjugate of the invention.
• A“therapeutically effective dose” as described herein refers to the dose that gives a therapeutic effect for a given condition and administration schedule. It is typically the average dose of an active substance to administer to appreciably improve some of the symptoms associated with a disease or a pathological state. For example, in treating a cancer of the brain or of other tissue, a pathology, a lesion or a disorder of the CNS, the dose of an active substance that decreases, prevents, delays, eliminates or stops one of the causes or symptoms of the disease or disorder would be therapeutically effective.
• A“therapeutically effective dose” of an active substance does not necessarily cure a disease or disorder but will provide a treatment for this disease or disorder so that its appearance is delayed, impeded or prevented, or its symptoms are attenuated, or its term is modified or, for example, is less severe, or the recovery of the patient is accelerated.
• the“therapeutically effective dose” for a person in particular will depend on various factors, including the activity/effectiveness of the active substance, its time of administration, its route of administration, its toxicity, its rate of elimination and its metabolism, drug combinations/interactions and the severity of the disease (or disorder) treated on a preventive or curative basis, as well as the age, weight, overall health, sex and/or diet of the patient.
• the conjugates and compositions of the invention can be used for treating, preventing, diagnosing or imaging numerous pathologies, notably pathologies affecting the CNS, infectious pathologies or cancers.
• the VHH of the invention have the capacity to target TfR-expressing cells, particularly cells which exhibit marked expression of said receptor, such as notably cancer cells, nervous or non-nervous tissue and/or to cross cell membranes, notably those of the physiological barriers of the CNS and more particularly the blood-tumor barrier (BTB) of cancerous nervous tissue.
• TfR is enriched in organs such as bone marrow, placenta and in the gastrointestinal tract.
• TfR is also highly expressed in brain endothelial cells but not in endothelial cells lining the vessels in other tissues. TfR expression has been confirmed at the plasma membrane of purified brain microvessels and cultured endothelial cells from rat, mouse, pig and non-human primate.
• the invention relates to the use of pharmaceutical conjugates or compositions as described above for treating or preventing CNS pathologies or disorders, brain tumors or other cancer cells, and bacterial, viral, parasitic or fungal infectious pathologies of the brain or other tissues.
• the invention also relates to a VHH, conjugate, or compositions as described above for use for diagnosing, imaging or treating CNS pathologies or disorders, brain tumors or other cancer cells, and bacterial, viral, parasitic or fungal infectious pathologies of the brain or other tissues.
• the invention also relates to a VHH, conjugate, or compositions as described above for use for treating, imaging and/or diagnosing a brain tumor or other types of cancer.
• the invention to a VHH, conjugate or composition such as defined above for use for treating, imaging and/or diagnosing neurodegenerative pathologies such as, in a non- restrictive manner, Alzheimer’s disease, Parkinson’s disease, stroke, Creutzfeldt- Jakob disease, bovine spongiform encephalopathy, multiple sclerosis, amyotrophic lateral sclerosis, etc.
• the invention also relates to a VHH, conjugate or composition such as defined above for use for treating, imaging and/or diagnosing neurological pathologies such as, in a non-restrictive manner, epilepsy, migraine, encephalitis, CNS pain, etc.
• the invention also relates to a VHH, conjugate or composition such as defined above for use for treating, imaging and/or diagnosing rare diseases such as, in non-restrictive manner lysosomal storage diseases, Farber disease, Fabry disease, Gangliosidosis GM1 and GM2, Gaucher disease, different mucopolysaccharidoses etc.
• rare diseases such as, in non-restrictive manner lysosomal storage diseases, Farber disease, Fabry disease, Gangliosidosis GM1 and GM2, Gaucher disease, different mucopolysaccharidoses etc.
• the invention also relates to a VHH, conjugate or composition such as defined above for use for treating, imaging and/or diagnosing neuropsychiatric pathologies such as, in a non-restrictive manner, depression, autism, anxiety, schizophrenia, etc.
• the invention also relates to a VHH, conjugate or composition such as defined above for use for treating, imaging and/or diagnosing cancers such as, in a non-restrictive manner, glioblastoma, pancreatic cancer, ovarian cancer, hepatocellular cancer, etc.
• the invention also relates to a VHH, conjugate or composition such as defined above, wherein the conjugated agent is a virus or a virus-like particle, such as a recombinant virus.
• the invention may indeed be used to increase brain or cancer or any TfR enriched tissue delivery of recombinant (e.g., replication-defective or attenuated) viruses used in gene therapy, such as adenoviruses, adeno-associated viruses, lentiviruses, retroviruses, etc, or virus-like particles.
• Coupling to a virus or VLP may be performed e.g., by coupling to the capsid protein of the virus.
• the invention also relates to methods for treating any of the above conditions or diseases by administering to a subject in need thereof a VHH, conjugate or composition of the invention.
• the invention also relates to the use of a VHH, conjugate or composition of the invention for the manufacture of a medicament for treating any of the above conditions or diseases.
• TfR expression at the BBB was analyzed cell membrane expression profile of the TfR in brain endothelium of various species.
• the kit ProteoExtract Subcellular Proteome Extraction Kit (Calbiochem, La Jolla, CA, USA) was used to prepare membrane extracts of digested or non-digested brain microvessels (BMVs) and of primocultures of brain microvascular endothelial cells (BMEC) from rat, mouse, pig and non-human primate (NHP; rhesus monkey) ( Figure 1).
• Membrane extracts were quantified using the BioRad DC Protein Assay (Bio-Rad, Hercules, CA, USA) following manufacturer’s instructions. Membrane proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 4-12% polyacrylamide gels, and transferred onto nitrocellulose membranes (ThermoFisher Scientific). Proteins were probed with a primary antibody against TfR (Genetex GTX102596; 1/1000), followed by an HRP-conjugated donkey anti-rabbit IgG secondary antibody (Jackson ImmunoResearch) diluted 1/10000. Finally, proteins were detected using chemiluminescence.
• TfR is expressed in digested and non-digested brain microvessels from rat, mouse, pig and non-human primate. TfR is also expressed in brain endothelial cells from mouse rat and pig (note that only 1 mg of membrane proteins was loaded on SDS-PAGE for brain microvascular endothelial cells versus 10 mg or 5 mg for brain microvessels). TfR expression is enhanced in digested NHP brain microvessels.
• TfR-binding VHHs The prerequisite to the identification and characterization of TfR-binding VHHs was the establishment in eukaryotic cells (Chinese hamster ovary cells, CHO) of stable cell lines expressing hTfR and mTfR, constitutively and at high rates. These cell lines were then used i) for the identification and characterization of agents binding to the receptor expressed at the cell surface, in its native configuration; and ii) to test whether the receptor could internalize such agents by endocytosis.
• eukaryotic cells Choinese hamster ovary cells, CHO
• the cDNA coding for the hTfR was cloned using sequence information available in databases (accession number: NM_003234.3).
• the primers necessary for cDNA amplification by RT-PCR were selected (see table below), comprising at their end (in bold type) the restriction sites (EcoRI and Sail) necessary for cloning in the pEGFP-Cl expression vector (Clontech) ( Figure 2-A).
• RNA prepared from human brain was used for RT-PCR amplification of the cDNA fragment coding for hTfR.
• the PCR product was digested by EcoRI-Sall restriction enzymes, and ligated in the pEGFP-Cl expression vector (Clontech), digested by the same restriction enzymes.
• this vector enables the expression, under control of the CMV promoter, of the hTfR fused to EGFP at its N-Terminal end, i.e., at the end of its intracellular domain.
• transforming competent E. coli DH5a bacteria After transforming competent E. coli DH5a bacteria, obtaining isolated colonies and preparing plasmid DNA, both strands of the construct were fully sequenced for verification.
• Plasmid coding for the mTfR fused to EGFP was purchased from GeneCopoeia (plasmid reference: EX-Mm05845-M29).
• Transient transfections in CHO-K1 cells were carried out and used to select stable transfectants by limit dilution and resistance to antibiotic (G418). These cell lines were amplified while maintaining selective pressure.
• Membrane expression of the receptors of the expected size was checked by western blot on cell membranes extracted with ProteoExtract Subcellular Proteome Extraction Kit. Antibodies were directed either against GFP or against the TfR. Proteins corresponding to the combined sizes of EGFP and h/mTfR (170 kDa), were recognized by an anti-GFP antibody and by an anti-TfR antibody ( Figure 2-C). A CHO K1 wild type (WT) cell line was used as negative control and antibodies detected no proteins.
• a llama (. Lama glama ) was immunized subcutaneously 4 times with membrane preparations from CHO stable cell lines expressing the human and murine receptors of interest.
• VHH library construction was performed as previously described (Alvarez- Rueda et al., 2007, Behar et al., 2009). Briefly, mRNAs coding for VHH were amplified by RT-PCR from the total RNAs of peripheral blood mononuclear cells isolated by ficoll gradient, and cloned into the pHENl phagemid. Reiterative selections enabled the isolation of phages presenting VHH exhibiting strong affinity for the TfR expressed at the cell surface.
• VHHs with improved binding to both the murine and the human cell lines, cell penetration and transport properties were obtained.
• Illustrative VHH are VHH A, VHH B, VHH C, VHH D (see also the list of sequences). These VHHs do not bind to cells of the control CHO cell line.
• TfR-binding VHH with appropriate, improved binding properties were generated by site-directed mutagenesis. More particularly, site directed mutagenesis was performed to introduce single alanine substitutions into the VHH A complementarity-determining regions (CDR) 1, 2 and 3, giving rise to the VHH Al to A9. VHH Al and A2 were mutated in the CDR1, VHH A3 and A4 were mutated in the CDR2 and VHH A5 to A9 were mutated in the CDR3. Furthermore, single site directed mutagenesis was also performed by substituting some CDR amino acids by structurally-close amino acids. VHH A10-A19 were obtained, wherein VHH A10 and Al l were mutated in the CDR1, VHH A12 to A14 were mutated in the CDR2, and VHH A15 to A19 were mutated in the CDR3.
• humanized TfR-binding VHH were generated, to improve in vivo efficacy by, e.g., avoiding immunogenicity, and were designated VHH A20-A25.
• VHH molecules were produced, to facilitate purification and/or coupling.
• VHHs of the invention Binding and endocytosis of purified VHHs of the invention to confirm the ability of selected VHH molecules to bind the TfR, and to be endocytosed, immunocytochemical experiments involving the incubation of VHHs on living CHO cell lines expressing the 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), were performed and observed with a confocal microscope. The results obtained with VHH A are shown as an example.
• the VHH binds to the CHO-hTfR-EGFP ( Figure 3-B) and CHO- mTfR-EGFP ( Figure 3 -A) cell lines and is incorporated by endocytosis to accumulate in the cells as shown using triton permeabilization, which is not the case for the control VHH (VHH Z) ( Figure 3-C, D).
• VHHs with affinity for the TfR were tested using flow cytometry, and apparent affinities (K d app ) were determined. All experiments were performed in 96 well plates using 2-3 x 10 5 cells/well, at 4 °C with shaking. CHO cell lines expressing the TfR fused to EGFP or CHO WT cells were saturated with PBS/BSA 2% solution during 30 min to avoid nonspecific binding, followed by incubation with purified VHHs at concentrations ranging from 2 mM to 1 pM for 1 hr.
• VHH Z There was no nonspecific labelling in the control conditions where cells were incubated with control VHH (VHH Z). All tested VHHs induced a concentration-dependent shift of the signal, confirming binding to the receptor of interest ( Figure 4- A). No labeling of the CHO WT control cells was detected with all the tested VHHs (not shown).
• the VHH K d app were calculated using GraphPad Prism software ( Figure 4-B). K d app were in the same range for all VHH, ranging from 7.5 nM (VHH B) to 56 nM (VHH D) on mTfR, and from 1.6 nM (VHH B) to 2.7 nM (VHH A) on hTfR.
• TfR-binding VHHs were used as tracers (Figure 5-B) and competitors (Figure 5-C). In all conditions, there was no competition between VHHs and the ligand Tf, suggesting than VHHs bind to TfR on an epitope different than that of Tf.
• VHH A1-A19 for the TfR were tested using flow cytometry, and apparent affinities (K d app ) were determined. All experiments were performed in 96 well plates using 2 x 10 5 cells/well, at 4 °C with shaking. CHO cell lines stably expressing the hTfR or the mTfR fused to EGFP or CHO WT cells were saturated with PBS/BSA 2% solution during 30 min to avoid non-specific binding, followed by incubation with purified VHHs at concentrations ranging from 50 mM to 5 pM for 1 hr.
• VHH A1-A19 all induced a concentration-dependent shift of the signal on both cell lines (with the exception of VHH A12) confirming their efficient binding to the receptor of interest ( Figures 11; 12). While VHH A, VHH A1 to A4, and VHH A10 to A15, showed similar B max (plateau of the curve) on both hTfR and mTfR expressing cell lines, VHH A6 to A9 and VHH A16 to A19 showed slight to drastic lower B max on both cell lines, as well as slight to strong curve shift. Only VHH A12 showed a lower B max and a strong curve shift on the hTfR expressing cell line compared to the other VHH Ax. No labeling of the CHO WT control cells was detected with all the tested VHHs (not shown).
• VHH K d app were calculated using GraphPad Prism software ( Figure 11-B; 12-B). Regarding the binding to the human TfR, VHH A, A1 to A4, A6, A9 to A11, and A13 to A 17, all showed similar K d app of about 3-4 nM. Conversely, VHH A5, A8, A18 and 19 showed slightly lower affinities of 9.2 to 25 nM, while VHH A7 and A12 showed drastically lower affinities of 255 nM and 363 nM, respectively.
• VHH A and A9 showed similar K d app of about 50 nM. All other VHH Ax showed slightly lower affinities of 131 to 259 nM, with the exception of VHH A5, A8 and A18 that showed significantly lower affinities of 604 nM, 427 nM and 416 nM, respectively.
• VHH-Fc fusion molecules Binding and endocytosis of purified VHH-Fc fusion molecules with affinity for TfR and affinity determination.
• Anti-TfR VHH molecules of the invention were fused to an IgG Fc fragment.
• DNA fragments encoding the VHHs (with no tag) were amplified by PCR and cloned into the pINFUSE-IgGl-Fc2 vector (InvivoGen) in order to encode a human IgGl-Fc fragment encompassing in its N-ter or in its C-ter the VHHs.
• Fusion proteins were prepared using the Expi293 Expression System according to the manufacturer’s instructions (Life Technologies). Seventy -two hrs post transfection, supernatants were recovered and purified using Protein A GraviTrap columns (GE Healthcare). The purified fusion proteins were quantified using an in- house anti-Fc ELISA.
• VHH-Fc and Fc-VHH fusion proteins with an affinity for the TfR were tested in flow cytometry experiments, and apparent affinity (K d app ) were determined. All experiments were performed in 96 well plates using 2-3 x 10 5 cells/well, at 4 °C with shaking. CHO cell lines expressing the receptors of interest fused to EGFP or CHO WT cells were saturated with PBS/BSA 2%, followed by an incubation with purified VHH-Fcs or Fc-VHHs at concentrations ranging from 350 nM to 0,03 pM for 1 hr. After washes, cells were incubated for 1 hr with an Alexa647- conjugated anti-hFc antibody (Jackson ImmunoResearch).
• BMEC rat or mouse brain microvascular endothelial cells
• rat or mouse astrocytes to set up the co-culture model.
• This type of in vitro BBB model is used to evaluate the passive passage or active transport of numerous molecules, notably pharmacological agents, across BMEC and thus, by extrapolation, their capacity to reach CNS tissue in vivo.
• the different models developed to date (bovine, porcine, murine, human) have ultrastructural properties characteristic of the brain endothelium, notably tight junctions, absence of fenestrations, low permeability to hydrophilic molecules and high electrical resistance.
• the in vitro rat BBB model brings into play a co-culture of BMEC and astrocytes (Molino et al., 2014, J. Vis. Exp. 88, e51278).
• membrane inserts Prior to cell culture, membrane inserts (Coming, Transwell 1.0 mm porosity, for 96-well or 12-well plates) were treated on the upper part with collagen type IV and fibronectin in order to enable optimal adhesion of BMEC and to create the conditions of a basal lamina.
• Primary cultures of mixed astrocytes were established from neonatal rat cerebral cortex. Briefly, meninges were removed and the cortical pieces were mechanically, then enzymatically dissociated in a trypsin solution.
• Dissociated cells were seeded into cell culture flasks in glial cell media (GCM) containing DMEM supplemented with 10% fetal bovine serum then frozen in liquid nitrogen for later use.
• GCM glial cell media
• Primary cultures of BMEC were prepared from 5-6 weeks old Wistar rats. Briefly, the cortical pieces were mechanically then enzymatically dissociated in a collagenase/dispase solution. The digested tissues were separated by a density-dependent centrifugation in 25% bovine serum albumin.
• microvessels pellet were seeded on culture flask, pre-coated with collagen type IV and fibronectin, in endothelial cell media (ECM) containing DMEM/F12 supplemented with 20% bovine platelet poor plasma derived serum and basic fibroblast growth factor (bFGF) 2 ng/ml.
• ECM endothelial cell media
• bFGF basic fibroblast growth factor
• VHH-Fc The binding/uptake at the BBB of inventive VHHs conjugated to the human Fc fragment of an IgGl antibody (VHH-Fc) was verified on the in vitro rat model described above ( Figure 9).
• VHH A-Fc or VHH B-Fc were co-incubated with Tf- Alexa647 for 2 hrs on live rBMEC monolayers at 37°C ( Figure 9A).
• the cell monolayer was washed extensively and fixed with PFA 4%.
• the cell monolayer was permeabilized with a solution of 0.1% triton X-100.
• VHH-Fcs were detected using immunostaining with an antibody against the human Fc fragment. Then confocal microscopy was used to assess the co-localization between fluorescence signal of VHH A-Fc or VHH B-Fc with Tf-A647 ( Figure 9A).
• VHH A-Fc and VHH B-Fc were readily endocytosed and co-localized almost perfectly with Tf-Alexa647.
• VHH- Fcs were incubated at 10 nM in the luminal compartment of the culture system for 24 hrs to 72 hrs ( Figure 9-C, D).
• filter inserts, containing rBMEC monolayers were placed in 96-well plates containing fresh transport buffer (75 m ⁇ in the luminal and 250 m ⁇ in the abluminal compartments).
• VHH-Fcs were co-incubated with lucifer yellow (LY), a small fluorescent molecule that does not cross the BBB.
• LY lucifer yellow
• LY accumulated in the abluminal compartment was quantified by fluorescence spectrophotometry and results were expressed as endothelial surface permeability (or Pe) in 10 -3 cm/min.
• the in vitro barrier was considered“permeable” or“open” if the Pe value of LY was greater than 0.6x10 -3 cm/min.
• Transendothelial electrical resistance (TEER) measured with an ohmmeter and expressed in ohm. cm 2 , also makes it possible to measure BBB integrity in vitro during tests of passage across the BBB.
• the quality threshold value is set at >400 ohm.
• VHH B-Fc and VHH A-Fc conjugates show higher transport than VHH Z-Fc (negative control), around 10-fold at 24 hrs and 5-fold at 72 hrs. This transport reached an apparent saturation between 24 hrs and 72 hrs, further suggesting the involvement of a specific and saturable receptor mediated process (Figure 9-D).
• VHH-Fc conjugates of the invention to target organs enriched with receptors of interest in vivo .
• conjugates VHH A-Fc, VHH A-Fc-Agly and VHH Z-Fc were injected into tail vein at 5 mg/kg and the mice were perfused with saline at different times. Plasmas and brains were collected. Brains were processed by the capillary depletion method to isolate brain parenchyma from capillary. The amount of VHH-Fc in plasma, brain parenchyma and microvessels was measured using an in- house anti-Fc ELISA. Results are presented as concentrations (nM), or by organ-to- plasma ratio (Figure 10).
• TfR-binding conjugates VHH A-Fc and VHH A-Fc-Agly exhibit a significant brain targeting at 2 hrs pi, with concentrations of 0.25 and 0.32 nM in 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).
• Figure 10-D When looking at parenchyma-to-plasma ratios, a clear advantage is confirmed, especially at 24 hrs pi where VHH A-Fc-Agly is still measurable in brain parenchyma whereas there is only 8 nM present in plasma.
• VHH A-Fc and VHH A-Fc-Ag In microvessels, VHH A-Fc and VHH A-Fc-Agly accumulate significantly more than VHH Z-Fc at 2 hrs pi, with concentrations 9 and 5 times higher, respectively. Moreover, VHH A-Fc concentration in microvessels is still 3 times higher than VHH Z-Fc at 24 hrs pi ( Figure 10-C). These results were confirmed when looking at microvessel -to-plasma ratios ( Figure 10-E). These results demonstrate that TfR-targeting VHH of the invention can be used to effectively deliver or to improve pharmacokinetic properties of agents, notably protein cargos.
• Anti-TfR VHH A, Al, A5, A6, A7 and A8 of the invention were fused to the mouse IgGl 13C3 monoclonal antibody, with high specific affinity for the protofibril form of b-amyloid peptide (W02009/065054).
• a DNA fragment encoding the selected VHH was synthetized and cloned into the 13C3 heavy chain (HC) vector in order to encode the 13C3-HC- VHH conjugate containing, in its C-ter, the selected VHH sequence fused to the antibody heavy chain C-ter amino acid residue.
• the DNA fragment encoding the selected VHH was cloned into the 13C3 light chain (LC) vector in order to encode the 13C3 LC conjugate containing in its C-ter the selected VHH sequence fused to the antibody light chain C-ter amino acid residue.
• Fusion proteins were produced using the Expi293TM Expression System according to the manufacturer’s instructions (Life Technologies). Seventy -two hrs post transfection, supernatants were recovered and purified using HiTrap® Protein G High Performance columns (GE Healthcare). The purified fusion proteins were quantified using 280 nm absorbance measurement.
• amino acid sequence of a 13C3-HC-VHHA conjugate is provided as SEQ ID NO: 93 :
• the 13C3 fusions K d app were calculated using GraphPad Prism software ( Figure 13- B). Affinities for the hTfR were similar for all fusions, with K d app of about 10-20 nM. Despite different B max , VHH A, A1 and A6 13C3 HC fusions showed similar affinities of 10 to 20 nM, while 13C3-HC-VHH A8 and 13C3-LC-VHH A fusions showed lower affinities of 315 nM and 106 nM, respectively.
• 13C3-HC-VHH A and 13C3-HC-VHH A1 conjugates, or unvectorized 13C3 were injected into C57B16 mice tail vein at the dose of 35 nmoles/kg.
• the mice were perfused with saline solution at different times.
• Brains were collected at 2 hrs and 6 hrs time points post-injection (p.i.).
• Half of mice brains were processed to isolate the capillary network from the brain parenchyma by a capillary depletion method that consists in centrifugation on 20% Dextran solution (Sigma Aldrich) of the resuspended half brain homogenate and recovery of the parenchyma fraction.
• mice brains were directly processed (homogenized and lysed) for total brain quantification.
• the amount of 13C3-HC-VHH conjugate in total brain and brain parenchyma was measured using an in-house qualified Meso Scale Discovery (MSD) direct coating (Abeta) immunoassay. (CV ⁇ 20% and recovery ⁇ 30%). Results are presented as concentrations (nM) ( Figure 14).
• Results show that TfR-binding conjugates 13C3-HC-VHH A and 13C3-HC-VHH A1 exhibited a significant brain uptake advantage at 2 and 6 hrs p.i. by comparison to the control unvectorized 13C3 antibody ( Figure 14-A).
• the total brain concentrations of 13C3-HC-VHH A and 13C3-HC-VHH A1 are 8 and 5-fold more important than that of the unvectorized 13C3 antibody at 6 hrs pi, respectively.
• An anti-GFP siRNA comprising chemical modifications for high resistance to nucleases, namely siGFPstl, was conjugated to a tagged VHH A to generate a VHH A-siGFPstl bioconjugate.
• the same conjugation strategy was used to conjugate siGFPstl to the irrelevant VHH Z as a negative control with the same structure and size as the VHH A- siGFPstl conjugate but with no TfR-targeting capacity.
• the conjugation strategy involved a convergent synthesis with the parallel modification of: i) the VHH to site-specifically introduce an azido-linker; and ii) the siGFPstl to introduce a constrained azido moiety complementary to the azido functional group.
• both functionalized VHH-azide and alkyne-siGFPstl precursors are linked to each other using a copper-free click reaction.
• BTG Bacterial Transglutaminase
• the BTG enzyme catalyzes the formation of an isopeptidic bond between a glutamine residue inserted in a tag sequence specifically recognized by the BTG enzyme (namely a Q-tag) and an amino- functionalized substrate.
• the amino-functionalized substrate introduced was a heterobifunctional linker containing at one end an amino moiety that we proved to be a substrate of the BTG enzyme and at the other end an azido moiety for the conjugation to the siGFPstl through copper-free click chemistry.
• siGFPstl was purchased from Dharmacon with a 3’ amine modification on the sense strand (N6-siGFPstl) to allow its further functionalization by the alkyne moiety required for the click chemistry conjugation with the VHH-azide.
• N6-siGFPstl (leq) was dissolved in a NaB (0.09M; pH 8.5) conjugation buffer to obtain a final concentration between 0.3 and 0.8 mM.
• DBCO-NHS (20eq, DMSO) was then added to this solution. Reaction mixture was stirred for 2 hours at room temperature.
• Alkyne-siGFPstl was purified by precipitation in cold absolute ethanol. Absorbance was read at 260 nm to calculate the amount of purified alkyne-siGFPstl construct and thus the conjugation yield (in the 40-50% range).
• VHH-azide and alkyne-siGFPstl precursors were finally conjugated by a copper- free click chemistry reaction to obtain the final conjugate VHH-siGFPstl .
• VHH-siGFPstl con jugation protocol VHH-siGFPstl con jugation protocol
• VHH-siGFPstl VHH A-siGFPstl and VHH Z-siGFPstl were characterized by analytical SEC-HPLC and agarose-gel electrophoresis to check their identity and purity.
• VHH-siRNA bioconjugate In vitro gene silencing activity of a VHH-siRNA bioconjugate Specific cellular targeting and productive intracellular delivery of therapeutic nucleic acids, especially siRNAs, oligonucleotides remain a major challenge. The structural and physico-chemical features of these molecules, being multiply charged hydrophilic oligomers, prevent them from entering any subcellular compartment if unassisted. VHH of the invention were used to transport a small interfering RNA (siRNA) across cellular membranes to access the cytosol.
• siRNA small interfering RNA
• the apparent hTfR-binding affinity (K d app ) of the VHH A-siGFPstl and VHH B- siGFPstl bioconjugates was evaluated as described in Example VII (Determination of binding affinity of VHH A1-A19) by adding concentrations ranging from 2 mM to 30 pM during 1 hr at 4°C on the same CHO-hTfR-GFP cells. Quantification of the cell-surface bound molecules was performed by anti-6His immunocytochemistry and experimental data were fit with a nonlinear regression using GraphPad Prism® software.
• VHH A-siGFPstl and VHH B-siGFPstl bioconjugates demonstrated concentration-dependent and saturable binding to the cell- surface target hTfR, with K d app values in the same low nanomolar range as unconjugated VHH A and VHH B ( Figure 15 A). No significant binding was observed with the control VHH Z. This, in turn, confirmed that coupling of the VHH A and VHH B to an siRNA does not alter their ability to bind specifically and efficiently to hTfR.
• the intrinsic silencing activity of the VHH-siGFPstl bioconjugate was assessed in living CHO cell lines stably expressing the TfR fused to EGFP (CHO-hTfR-EGFP cells) by transfection of the conjugate at 25 nM using Dharmafect 1 (Dharmacon) for direct delivery into the cytosol.
• the total cellular amount of GFP was quantified 72 hours post-transfection using flow cytometry.
• the results demonstrate that the VHH A- siGFPstl conjugate induced a ca. 85% reduction of GFP protein levels, in the same range than the unconjugated siGFPstl or the control VHH Z-siGFPstl conjugate (Figure 15B).
• VHH A-siGFPstl conjugate was transfected on CHO-hTfR-EGFP cells at concentrations ranging from 10 nM to 1 pM and the total cellular amount of GFP was quantified 120 hours post-transfection using flow cytometry. This resulted in a concentration-dependent reduction of GFP protein levels, with an IC50 of 50.4 pM and a maximum silencing efficiency in this condition of -90.2 % ( Figure 15C).
• VHH A-siGFPstl or the control VHH Z-siGFPstl bioconjugates were incubated on CHO-hTfR-GFP cells at 1 mM during 120 hrs at 37°C to allow free uptake, delivery to the cytosol and gene silencing to take place at the mRNA transcript and protein levels. This led to a significant ca.
• VHH A-siGFPstl bioconjugate allows its delivery into the cytosol in pharmacological amounts, with an IC50 in the same nanomolar range than hTfR-binding affinity of the bioconjugate.
• VHH A-siGFPstl was incubated during 120 hrs at 37°C at the saturating concentration of 30 nM, as defined from the previous experiment, either alone or in the presence of a 100X excess of the free VHHs A, B or Z.
• VHH A-siGFPstl bioconjugate was evaluated using a pulse-chase procedure.
• CHO-hTfR-GFP cells were exposed to VHH A- siGFPstl at concentrations ranging from 300 nM to 1 pM during a short duration (6 hours), followed by chase in ligand-free medium up to a total duration of 120 hrs. This experiment allowed to evaluate the contribution of early cellular uptake to the silencing effect previously observed by continuous incubation during 120 hrs.
• the VHH A-siGFPstl bioconjugate again induced a concentration- dependent reduction of 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 most of the effect previously observed upon 120 hrs continuous incubation was due to productive TfR-mediated uptake within the first 6 hrs.
• This finding is of particular interest since in vivo the plasma pharmacokinetic profile of such bioconjugates generally allows tissue exposure at therapeutic levels during only a few hours when administered by intravenous or subcutaneous bolus injection.
• the TfR-targeting VHH described here hence represents a viable tool for targeted and efficient gene silencing in vivo.
• VHH B ability of the VHH B to trigger hTfR-mediated endocytosis and subsequent gene silencing was evaluated by incubating the VHH B-siGFPstl bioconjugate on CHO- hTfR-GFP cells at 30 nM during 120 hrs.
• the result showed a ca. -60% reduction in GFP levels, similar to that obtained with the VHH A-siGFPstl bioconjugate, confirming that these VHHs display a similar TfR-targeting and intracellular delivery potential (Figure 15H).
• asialoglycoprotein receptor ASGPR
• GalNAc asialoglycoprotein receptor
• the present invention provides a new ligand/receptor system for the targeting and intra-cytoplasmic delivery at nanomolar concentrations of therapeutic nucleic acids, such as siRNAs, into extra-hepatic organs and tissues expressing the TfR.
• VHH-NODAGA conjugates Design of the Q-tagged VHH A
• a DNA fragment encoding VHH A with an AlaLinker, a HisTag, a GlyLinker and a Q-tag (AAA-HisTag-GGG-LQR sequence) introduced at its C-terminal end) was synthetized and cloned into the pHENl vector.
• VHH A-azide Absorbance was read at 280 nm to calculate the amount of purified VHH A-azide construct and thus the conjugation yield (in the 70-80% range).
• Final VHH A-azide was characterized by LCMS analysis to check its identity and the purity.
• VHH A-azide Click chemistry reaction to conjugate VHH A-azide to commercial alkyne-NODAGA VHH A-azide (1 eq.) was allowed to react with the heterobifunctional NODAGA-BCN (5 eq.) (Chematech, Dijon, France) in PBS at room temperature. Reaction was monitored by LCMS. After completion of the reaction, the final conjugate was purified through chromatography on a Protino Ni-ida 1000 packed column according to the manufacturer's instructions to isolate the VHH A-azide from excess of starting material as well as potential by-products. Absorbance was read at 280 nm to calculate the amount of purified VHH A-NODAGA construct and thus the conjugation yield (in the 50-60% range). Final VHH A-NODAGA was characterized by LCMS analysis to check its identity and purity.
• Glioblastoma is the most common primary malignant brain tumor and the U87 cell line, a human primary glioblastoma cell line, is known to express a high TfR levels.
• the radiolabeled VHH A-NODAGA bioconjugate was intravenously administrated to mice previously implanted with glioblastoma cells (xenograft model) and PET-Scan imaging was performed.
• VHH A-NODAGA was radiolabeled using 68Ga chloride.
• Gallium was obtained in 68Ga3+ form using a commercial Ti02-based 68Ge/68Ga generator (Obninsk).
• a radiolabeling reaction was conducted by reacting 60mg of VHH A-NODAGA with 74- 148 MBq (2-4 mCi) of 68Ga in 400 mL of ammonium acetate buffer (1M, pH 6) at
• VHH A-NODAGA and VHH A-68Ga bioconjugates demonstrated concentration-dependent and saturable binding to the cell-surface target receptor hTfR, with K d app values in the same low nanomolar range as the unconjugated VHH A ( Figure 16A). No significant binding was observed with the control VHH Z. This confirmed that coupling of the VHH A to a NODAGA ligand and radiolabeling protocol does not alter its ability to bind specifically to hTfR.
• PET/CT scans were acquired during 2 hrs for 3 mice and at 2 hrs post injection (p.i.) for the 3 other 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%.
• 20 million coincidence events per mouse were acquired for every static PET emission scan (energy window, 400-600 keV; time: 20 minutes for one FOV).
• CT images 35 kVp, exposure time of 350ns and medium zoom

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Organic Chemistry (AREA)
• Molecular Biology (AREA)
• Genetics & Genomics (AREA)
• Biomedical Technology (AREA)
• Immunology (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Biotechnology (AREA)
• Medicinal Chemistry (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Biophysics (AREA)
• General Engineering & Computer Science (AREA)
• Physics & Mathematics (AREA)
• Microbiology (AREA)
• Urology & Nephrology (AREA)
• Hematology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Plant Pathology (AREA)
• Virology (AREA)
• Crystallography & Structural Chemistry (AREA)
• Cell Biology (AREA)
• Food Science & Technology (AREA)
• Pathology (AREA)
• Analytical Chemistry (AREA)
• General Physics & Mathematics (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Peptides Or Proteins (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Medicinal Preparation (AREA)

Priority Applications (9)

Applications Claiming Priority (2)

Related Child Applications (2)

Publications (1)

Family

ID=65236972

Family Applications (1)

Country Status (9)

Cited By (20)

Families Citing this family (8)

Citations (10)

Family Cites Families (5)

• 2020
  • 2020-01-08 CA CA3124790A patent/CA3124790A1/en active Pending
  • 2020-01-08 AU AU2020206593A patent/AU2020206593B2/en active Active
  • 2020-01-08 US US17/421,412 patent/US12152317B2/en active Active
  • 2020-01-08 EP EP20700450.8A patent/EP3908608A1/en active Pending
  • 2020-01-08 EA EA202191893A patent/EA202191893A1/ru unknown
  • 2020-01-08 JP JP2021540103A patent/JP7599425B2/ja active Active
  • 2020-01-08 CN CN202080008202.4A patent/CN113474369B/zh active Active
  • 2020-01-08 BR BR112021013559-6A patent/BR112021013559A2/pt unknown
  • 2020-01-08 WO PCT/EP2020/050318 patent/WO2020144233A1/en not_active Ceased

Patent Citations (10)

Non-Patent Citations (13)

Also Published As

Similar Documents

Legal Events