PEPTIDE USEFUL FOR THE TRANSPORT OF MOLECULES THROUGH CELL BARRIERS

Abstract

A new peptide and its use as a vector for the transport of molecules after conjugation through cellular barriers for the diagnosis, prognosis or treatment of pathologies of the central nervous system (CNS).

Claims

1. A peptide comprising a peptide of amino acid sequence SEQ ID NO 1.

2. A peptide conjugate comprising the peptide according to claim 1, wherein the peptide is covalently linked to a molecule of interest, directly or via a linker.

3. The peptide conjugate according to claim 2, wherein the linker is composed of 1 to 6 amino acids.

4. The peptide conjugate according to claim 2, wherein the molecule of interest is chosen from the group consisting of an antibody, a DNA, an RNA, a peptide, and a drug.

5. The peptide conjugate according to claim 4, wherein the molecule of interest is a nanobody.

6. The peptide conjugate according to claim 5, wherein the nanobody is an anti-Tau nanobody that comprises the amino acid sequence SEQ ID NO: 3.

7. The peptide conjugate according to claim 6 comprising the amino acid sequence SEQ ID NO: 4.

8-11. (canceled)

12. A nucleic acid sequence encoding the peptide according to claim 1, wherein the peptide is covalently linked to a molecule of interest, directly or via a linker.

13. An expression vector comprising a nucleic acid sequence as defined in claim 12.

14. (canceled)

15. A method comprising diagnosing, prognosing, or treating a pathology of the central nervous system (CNS) with the peptide conjugate according to claim 2.

16. The method according to claim 15, wherein the CNS pathology is chosen from the group consisting of brain tumors, cerebrovascular accidents (CVA), Huntington's disease, multiple sclerosis and tauopathies.

17. The method according to claim 16, wherein the tauopathies are chosen from the group consisting of Parkinson's disease, Pick's disease, and Alzheimer's disease.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0030] FIG. 1 represents the three-dimensional structure of the TB peptide.

[0031] FIG. 2 represents the confirmation of the alpha helix of the TB peptide by the PepFold and heliquest programs.

[0032] FIG. 3 represents the conjugates comprising the TB or BIP peptide fused to the 2C5 nanobody.

[0033] FIG. 4 represents the cellular model of the BBB.

[0034] FIG. 5 represents the effect of the nanobodies and conjugates (2C5, 2C5-TB and 2C5-BIP) on the sucrose permeability of the endothelial cells.

[0035] FIG. 6 represents the sucrose clearance curves through the endothelial cells exposed to the nanobodies and conjugates (2C5, 2C5-TB and 2C5-BIP).

[0036] FIG. 7 represents the permeability coefficient in L/h/cm.sup.2 of the nanobodies and conjugates (2C5, 2C5-TB and 2C5-BIP).

[0037] FIG. 8 represents the data of the HoxA-13 peptides and penetratin obtained by the heliquest program.

[0038] FIG. 9 represents the body temperature variation curves of mice after their intravenous injection of the different peptides (NT, TB, TAT-NT and TB-NT).

EXAMPLES

Example 1: Design of a New CPP: THT TB Peptide

Protein Sequence of the TB Peptide

[0039] The sequence of the TB peptide was modeled in 3D using the COOT program to form a stable alpha helix with many positive charges distributed on the surface, with a theoretical isoelectric point (pl) close to 13 (12.70 after protparam) in order to subsequently lower the pl of the molecules of interest which are fused to it.

[0040] The TB peptide was then synthesized by gene synthesis and produced in bacterial system or by peptide synthesis using a synthesizer.

Resulting Three-Dimensional Structure (FIG. 1)

[0041] The alpha helix of the TB peptide was confirmed by the PepFold and heliquest programs (FIG. 2).

[0042] The TB peptide was designed in silico so as to have positive charges better distributed on its surface, better solubility in aqueous solvents and a more structurally stable -helix. All of these properties allow the TB peptide, as well as all its conjugates, to have an increased solubility in the aqueous solvents, for use in vivo. Ultimately, TB and its various conjugates have a greater capacity to cross the BBB than for any other known CPP.

[0043] In this sense, by comparing the properties of the TB peptide to those of two other known CPPs, the HoxA-13 peptide and penetratin, we see that the TB peptide has negative hydrophobicity unlike HoxA-13 and penetratin (FIG. 8). This difference gives the TB peptide a solubility in the aqueous solvents, unlike HoxA-13 and penetratin, allowing it to cross the BBB, particularly when it is linked to a molecule of interest, such as the nanobody (2C5) or neurotensin (NT).

Example 2: Fusion of the TB Peptide to a Nanobody

Fusion of the TB Peptide to a Nanobody that does not Pass the BBB

[0044] To evaluate the diffusion capacity through a differentiated cellular BBB model, the TB peptide was fused to a nanobody (2C5) not passing this model but whose target is cerebral (International application WO 2018/078140) [11].

[0045] The conjugation of the TB peptide to the 2C5 nanobody was carried out by gene fusion such that the resulting protein is composed, from the N-terminus to the C-terminus, of the 2C5 nanobody, of a linker composed of 3 alanines, of the peptide TB and a 6 histidine tag. A similar fusion with a promising CCP according to Cho, 2017 [9], the BIP peptide, was also carried out (FIG. 3).

[0046] The corresponding genes were inserted into the pET15b plasmid using the NcoI/BamHI restriction enzymes (data not represented).

Production of 2C5 Nanobodies, and 2C5-TB and 2C5-BIP Peptide Conjugates

[0047] The plasmids encoding the 3 proteins (2C5, 2C5-TB and 2C5-BIP) were inserted into E. coli Shuffle bacteria (Biolabs) according to the supplier's protocol.

[0048] From a colony resulting from the transformation, a preculture was carried out in a volume of 25 mL of LB containing 100 g/mL of ampicillin. This preculture was incubated overnight at 37 C. with stirring at 190 rpm.

[0049] The following day a 1 L culture was carried out by diluting the 25 mL of preculture in LB containing 100 g/mL of ampicillin. The preculture was incubated at 37 C. with stirring at 190 rpm and, when the optical density measured at 600 nm reached 0.8, 1 mM of IPTG was added to induce the T7 promoter of pET15b plasmid. The culture was incubated for an additional 3 hours under the same conditions.

[0050] The bacteria were collected after 3 hours of induction, by centrifugation for 40 min at 9500 G. The supernatant was eliminated and the bacterial pellet was resuspended in 40 mL of lysis buffer (50 mM Tris HCl pH9 for 2C5-TB and pH8 for 2C5 and 2C5-BIP, 250 mMNaCl, 30 mM imidazole, 1 mg/ml lysozyme, 2 tablets of complete EDTA free antiprotease (Roche) and 1 L of benzonase). The suspension was placed at 4 C. for 1 hour with gentle stirring to allow lysis under the action of lysozyme. An additional sonication step was then carried out and the soluble fraction containing the nanobodies or conjugates was collected by centrifugation for 40 min at 9500 G.

[0051] The nanobodies having a poly-histidine tail, the nanobodies and conjugates were purified on a Ni-NTA agarose affinity resin (Qiagen). The soluble fraction was loaded into a gravity column containing 1 mL of Ni-NTA resin pre-equilibrated in TpA (50 mM Tris HCl pH9 for 2C5-TB and pH8 for 2C5 and 2C5-BIP, 250 mM NaCl, 30 mM Imidazole). Then the resin was washed using 150 mL of TpA and the nanobodies and conjugates were eluted by addition of 250 mM of imidazole in TpA.

[0052] Fractions of 1 mL were collected and those containing the nanobodies or conjugates after analysis on a 18% SDS gel were combined and injected onto a Superdex 75 10/300 column for a second purification step on a Biorad NGC FPLC system, in PBS buffer.

[0053] The fractions containing the nanobodies or conjugates, more than 95% pure, were combined and collected at 1 mg/ml for the 2C5 nanobodies and the 2C5-BIP conjugates and at 0.8 mg/ml for the 2C5-TB conjugates.

Evaluation of the Passage of the Nanobodies and Peptide Conjugates Through a Differentiated Cellular BBB Model (FIG. 4)

[0054] Cerebral microvessel endothelial cells prepared from 5-week-old male rats were seeded on microporous filters in order to: [0055] evaluate the toxicity of the nanobodies and conjugates during their transfer by kinetic measurement of the sucrose transfer. [0056] evaluate the permeability of the nanobodies and conjugates in the blood/brain direction.

Experimental Conditions

[0057] The endothelial cells were seeded on 28 Transwell Polycarbonate filters (porosity 0.4 m and diameter 12 mm), and cultured in differentiation medium. [0058] The measurement of toxicity (study A) or permeability (study B) of the nanobodies and conjugates was carried out in HBSS Ca/Mg medium supplemented with 0.1% BSA. The transfer volumes were respectively 280 and 1200 l for the donor luminal compartment and the acceptor basolateral compartment. [0059] The nanobodies and conjugates were diluted in the same HBSS Ca/Mg0.1% BSA buffer. [0060] The transfer was initiated by adding the nanobodies or conjugates to the upper compartment and transferring the filter to a well containing HBSS Ca/Mg0.1% BSA. [0061] The integrity of control monolayers or those exposed to nanobodies or conjugates (study A) was evaluated by measuring the sucrose permeability through a kinetic analysis over 2 hours (comparable to the time used to estimate the permeability of the nanobodies and conjugates). [0062] The luminal and abluminal media collected for study B were analyzed by ELISA test. [0063] The groups are as follows:

TABLE-US-00003 TABLE 1 Study A. Toxicity of B. Permeability of the nanobodies the nanobodies Groups/number of fibers and conjugates and conjugates Luminal sampling 50 l/120 min 20 l/8 min vol/time 250 l/120 min Abluminal sampling 900 l/15, 30, 45, 250 l/60 min vol/time 60, 90, 120 min total vol/120 min

[0064] For study B, the medium collected at 60 min basolaterally was replaced by the same volume of fresh medium.

Results of Study A:

[0065] The sucrose permeability of the endothelial cells reflecting the integrity of the tight junctions was not modified in the presence of the nanobodies and conjugates (FIG. 5). The clearance curves averaged for the 4 filters were linear for the four filter groups (FIG. 6).

[0066] These results demonstrated that the three nanobodies and conjugates (2C5, 2C5-TB and 2C5-BIP) tested do not have an acute deleterious effect on the permeability of the endothelial cells to sucrose. They therefore validate the possible differences observed within the values of the permeability coefficients for the 3 nanobodies and conjugates tested.

Results of Study B:

[0067] The samples collected at 60 and 120 min were analyzed by ELISA test to evaluate the quantity of nanobodies and conjugates which passed during study B. To be able to analyze the different samples by ELISA, it was necessary to first purify them to remove BSA limiting adhesion to 96-well plates of ELISA test. Thanks to the polyhistidine label present on the 3 nanobodies and conjugates, purifications on a 96-well plate coated with Nickel-NTA were carried out.

[0068] The purifications were carried out as follows: [0069] Incubation of the samples for 1 hour at 4 C. on the Ni-NTA plate. [0070] Elimination of unretained fractions by pipetting. [0071] 3 washes of 200 L in PBS+30 mM imidazole buffer. [0072] Incubation for 30 min with 200 L of PBS+300 mM imidazole elution solution. [0073] Transfer of the elutions to 96-well plates for ELISA tests.

[0074] In parallel, concentration ranges of the different nanobodies and conjugates were carried out and incubated on the same 96-well plates as the previous elutions, overnight at 4 C. The following day, the non-adsorbed fractions were removed by inversion and blocking was carried out with a solution of PBS+1% BSA for 1 hour at 23 C. A solution of PBS+1% BSA containing anti-histidine and HRP-coupled antibodies replaced the blocking solution and an incubation for 1 hour at 23 C. was carried out to allow the antibodies to link to the nanobodies and conjugates present. 3 washes in PBS+0.1% tween were then carried out to eliminate the elements not retained and a TMB solution was added to reveal the ELISA tests by colorimetry.

[0075] An absorbance reading was carried out at 450 nm after stopping the colorimetric reaction using a 1N HCl solution.

[0076] The concentration ranges carried out made it possible to trace the absorbance curves as a function of the concentration of nanobodies and conjugates and to determine the limit of linearity as well as the coefficient making it possible to pass from the absorbances to the concentrations in the samples taken during the study B. To be able to compare the quantities of nanobodies and conjugates passed, the concentrations of the wells were then converted into quantities of nanobodies and conjugates passed per hour and per cm.sup.2 of model. In order to compare these values to the references (a dextran of 15000 Da and sucrose), the diffusion of the nanobodies was finally expressed as a permeability coefficient, i.e. in L/h/cm.sup.2 (FIG. 7).

[0077] These results demonstrated the ability of the 2C5-TB conjugate to pass through the BBB. Thus the TB peptide has demonstrated its capacity as a vector to transport through the BBB a molecule of interest that does not pass the BBB when administered alone.

[0078] Furthermore, comparative studies, in which the 2C5 nanobody was fused to HoxA-13 and penetratin, confirmed the unique ability of the TB peptide to be a transport vector through the BBB, of a molecule of interest such as the 2C5 nanobody. Indeed, the peptide conjugates 2C5-penetratin and 2C5-HoxA-13 could not be completely solubilized during the lysis step of the bacteria used for its production (method identical to that of the 2C5-TB conjugate), thus revealing an inability (or at least a very low capacity) to cross the BBB. Therefore, these studies have confirmed that among these three cell penetrating peptides (CPP), only the TB peptide of the invention allows, when conjugated to the 2C5 nanobody, the transport of this molecule of interest through the BBB.

Example 3: Fusion of the TB Peptide to a Neurotensin

Fusion of the TB Peptide to a Neuropeptide that does not Pass the BBB

Sequences of the Different Peptides Used

TABLE-US-00004 NT: RRPYIL TB: RQRIWFQNRRRSRKIKK TAT-NT: GRKKRRQRRRPQRRPYIL TB-NT: RQRIWFQNRRRSRKIKKRRPYIL

Method

[0079] The neurotensin (NT) is a neuropeptide that causes hypothermia when present in the brain but is unable to reach the brain from the periphery on its own. This peptide, if fused to TB, can thus serve as a reporter for crossing the BBB. For this, the TB, NT and TB-NT peptides were synthesized using a peptide synthesizer and the dose of 2.5 mmol/kg was injected intravenously into mice (n=4 to 6). Body temperature was monitored using Anipill capsules previously implanted intraperitoneally.

[0080] These data were studied in comparison to those carried out with the reference cell penetration peptide TAT, also fused to NT neurotensin. The resulting TAT-NT conjugate was used as a positive control, in order to compare the capacity of TB to make NT cross the BBB, with regard to that of the TAT peptide. The TAT peptide, originating from the human immunodeficiency virus (HIV), was shown in 1999 to be capable of transporting a conjugate through the BBB [10].

[0081] The graph resulting from this study (FIG. 9) shows the variations in body temperature of the mice after their intravenous injection of the different peptides. The monitoring was carried out over 90 minutes.

Results

[0082] Mice that received NT alone do not show any variation in temperature in the minutes following the injection and confirms the absence of passage from the blood to the brain of this peptide alone. In other words, this curve confirms the inability of NT to cross the BBB alone.

[0083] Mice that received TB alone showed no signs of hypothermia.

[0084] Mice that received NT fused to TAT showed hypothermia of approximately 1 degree Celsius, observed after 30 minutes following injection. This confirms the ability of the TAT peptide to transport a conjugate through the BBB.

[0085] Nevertheless, mice that received NT fused to TB showed greater hypothermia of approximately 3 degrees Celsius, observed after 35 minutes following injection. Thus, this reflects a capacity of the TB peptide to make a molecule of interest such as neurotensin cross the BBB which is significantly greater than that of the TAT peptide.

[0086] All animals returned to a similar body temperature after 2 hours and 30 minutes.

LIST OF REFERENCES

[0087] 1. Abbott et al., Neurobiol. Dis., 37: 13-25, 2010 [0088] 2. Pardridge, Molecular Interventions, 3(2): 90-105, 2003 [0089] 3. Pardridge, Expert Opinion on Drug delivery, DOI: 10.1517/17425247.2016.1171315, 2006 [0090] 4. De Boer and Gaillard, Clin. Pharmacokinet., 46(7): 553-576, 2007 [0091] 5. Patel and Patel, CNS Drugs, DOI: 10.1007//s40263-016-0405-9, 2017 [0092] 6. Dong, Theranostics, 8(6): 1481-1493, 2018 [0093] 7. Zhou et al., WIREs Nanomed Nanobiotechnol., 13: e1695, 2021 [0094] 8. Oller-Salvia, Chem. Soc. Rev., 45(17): 4690-4707, 2016 [0095] 9. Patent Application FR 3058143 [0096] 10. Schwarze et al., 1999