PEPTIDE USEFUL FOR TRANSPORTING MOLECULES ACROSS CELLULAR BARRIERS

Abstract

The present invention relates to a novel peptide and its use as a vector for the transport of molecules after conjugation across cellular barriers, in particular the blood-brain barrier (BBB) ​​for the diagnosis, prognosis or treatment of pathologies of the central nervous system (CNS). Figure 7

Description

Title of the invention: PEPTIDE USEFUL FOR THE TRANSPORT OF MOLECULES THROUGH CELLULAR BARRIERS Technical field

[0001] The present invention relates to a novel peptide and its use as a vector for the transport of molecules of interest after their fusion with said peptide, thus forming a fusion protein (hereinafter referred to as conjugated peptide or peptide conjugate) across cellular barriers, in particular the blood-brain barrier (BBB) ​​for the diagnosis, prognosis or treatment of pathologies of the central nervous system (CNS), for example cerebrovascular accidents (CVA), tauopathies, more particularly Alzheimer's disease or Parkinson's disease, etc.

[0002] In the description below, references in brackets ([ ]) refer to the list of references at the end of the text. State of the art

[0003] The brain barriers protect our central nervous system (composed of the brain and spinal column) by controlling oxygen, nutrients and blocking the entry of substances or microorganisms that are harmful to the brain. There are 3 barriers between the blood and the central nervous system (CNS): the blood-brain barrier (BBB), the blood-cerebrospinal fluid barrier or choroid plexus (CP) and the arachnoid barrier which serves as an interface between the brain and the cerebrospinal fluid (Abbott et al., Neurobiol. Dis., 37: 13-25, 2010) [1].

[0004] The BBB concentrates most of the crossing efforts since it is the major interface between the blood and the CNS. However, the BBB is the main obstacle to the development of new diagnostic or therapeutic molecules targeting the brain. Indeed, only 2% of the molecules developed cross it (Pardridge, Molecular Interventions, 3(2): 90-105, 2003) [2] and these are small molecules (<600 Da) (Pardridge, Expert Opinion on Drug delivery, DOI: 10.1517 / 17425247.2016.1171315, 2006) [3],

[0005] The BBB is composed of small capillaries specific to the brain, surrounded by endothelial cells forming the endothelium, a key element of this barrier.

[0006] The total surface area of ​​this endothelium represents 12 to 20 m2, or kilometers of interface between the capillaries and the brain (De Boer and Gaillard, Clin. Phar-macokinet., 46(7): 553-576, 2007) [4]. But crossing this interface is very controlled and selective. Indeed, the endothelial cells are closely attached by tight junctions, thus forming a veritable fortress between the capillaries and the brain. Tight junctions are made up of adhesion proteins capable of selecting the molecules that can or cannot cross this endothelium. This is called paracellular transport, which is opposed to transcellular transport, where the passage is made by crossing the membrane of the endothelial cells (Patel and Patel, CNS Drugs, DOI: 10.1007 / / s40263-016-0405-9, 2017) [5],

[0007] For this second type of transport, crossing the endothelium can be done by passive diffusion for small non-polar molecules, liquids and alcohols or via a transporter present on the surface of the cells as for glucose or amino acids. But when the size of the molecules is too large for the use of a transporter, the passage of the membrane is then done by transcytosis. Some proteins such as insulin or transferrin bind to receptors present on the membrane of endothelial cells and which mediate transcytosis. For others devoid of receptors, transcytosis can only take place if these molecules meet the conditions necessary for their adsorption such as the presence of a patch of positive charges on their surface (Patel and Patel, 2017) [5].

[0008] Once this endothelium has been crossed, the molecules collide, to a lesser extent, with other types of cells constituting the BBB. Indeed, the endothelial cells are partially surrounded by pericytes, contractile cells participating in the structure of the endothelium and capillaries and playing a role in the vasodilation or vasoconstriction of the latter. Communication between the capillaries, the pericytes and the endothelial cells is achieved thanks to the basement membrane. Not being a completely enclosed layer, the molecules escape it more easily.

[0009] Finally, astrocytes, which act as mediators between capillaries and neurons, participate in the formation of tight junctions with pericytes, and help maintain the structure of the BBB and capillaries, constitute the last obstacle. But just like pericytes, their assembly is not continuous, and their passage is therefore probably not as complex as that of the endothelium.

[0010] The search for new molecules targeting a CNS pathology must therefore combine the ability to cross the BBB with biological activity, biodistribution, low toxicity and stability. Few molecules achieve this because research almost systematically focuses on finding a target and a ligand adapted to the problem in a way disconnected from crossing the BBB. To cross this BBB, several strategies are envisaged. They are divided into 2 categories, invasive and non-invasive strategies (Dong, Theranostics, 8(6): 1481-1493, 2018) [6]. Among the promising non-invasive strategies, we find the use of “Trojan horse” molecules using receptors on the surface of the BBB or the use of cell penetrating peptides (CPP) adsorbed by transcytosis (Zhou et al., WIREs Nanomed Nanobiotechnol., 13: el695, 2021) [7].

[0011] Peptides, when combined with molecules, including large molecules such as proteins, are able to increase their brain penetration by increasing their capacity to pass through the BBB. Several peptides have been developed for crossing the BBB, linear or cyclic, between 5 and 50 amino acids. Some use the receptors present at the BBB while others use passive diffusion. The latter, called cell penetrating peptides (CPPs), are generally amphipatic and / or cationic and are not specific to the BBB. They nevertheless increase the passage of molecules through cell membranes and, combined with other strategies to deliver them preferentially to the BBB, can be decisive for the delivery of molecules to brain targets (Oller-Salvia, Chem. Soc. Rev., 45(17): 4690-4707, 2016) [8],

[0012] There remains a great need to identify new drugs to treat CNS pathologies such as, for example, cerebrovascular accidents (CVA), tauopathies, for example, Alzheimer's disease or Parkinson's disease. Therapeutic molecules currently being developed for the brain mostly fail to reach their brain target due to their inability to cross the BBB. Nearly a billion people are affected by a neurological disorder (excluding psychiatric disorders). Many effective in vitro molecules have been found for these pathologies but need a vector to allow them to reach their target. The market for the delivery of therapeutic molecules across the BBB will reach 7.4 billion US dollars in 2028 according to Emergen research in 2021. Description of the invention

[0013] With the aim of making biomolecules cross the BBB for the diagnosis or therapy of brain pathologies, the inventors designed by bioinformatics a new CPP (hereinafter referred to as TB peptide) with a three-dimensional structure forming a stable alpha helix, of small size, with positive charges distributed on the surface and a theoretical isoelectric point greater than 10 (12.70 according to the expasy online tool Protparam) - these criteria being essential for the crossing of the BBB, the stability of the TB peptide and the proteins of interest to which it is attached.

[0014] This TB peptide with amino acid sequence RQRIWFQNRRRSRKIKK (SEQ ID NO: 1), encoded by the nucleotide sequence cgccagcgcatttggtttcagaaccgccgccg-cagccgcaaaattaaaaaa (SEQ ID NO: 2), has demonstrated its ability to transport across the BBB a molecule of interest, namely the nanobody 2C5 (VHH, PM=10-15 kDa) with amino acid sequence QVQLVQSGGGLVQAGGSLRLSCAASGRTFSSDT-LAWFRQAPGKEREFVASISPSGGVTYYEDSVKGRFTISRDNSKNTVLLQMNSL TPEDTAVYYCNRDPKYGNTRYWGQGTQVTVSSAAA (SEQ ID NO: 3) capable of detecting pathological forms (oligomeric form) of the Tau protein (Patent Application FR 3058143) [9], after fusion.

[0015] The resulting 2C5-TB peptide conjugate has the amino acid sequence M QVQLV-OSGGGLVOAGGSLRLSCAASGRTFSSDTLAWFROAPGKEREFVASISPSGGVT YYEDSVKGRFTISRDNSKNTVLLOMNSLTPEDTAVYYCNRDPKYGNTRYWGO GTOVTVSSAAARORIWFONRRRRSRKIKKHHHHHH* (SEQ ID NO: 4), encoded by the ATG nucleotide sequence CAGGTGCAGCTGGTGCAGTCTGGGGGAG-GATTGGTGCAGGCTGGGGGTTCTCTGAGACTCTCCTGTGCAGCCTCTGGAC GCACCTTCAGTAGCGATACCCTGGCGTGGTTCCGCCAGGCGCCAGGGAAG GAGCGTGAGTTTGTAGCGTCTATTAGTCCCAGTGGTGGTGTCACATACTAT GAAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACAGCAAGAA CACGGTGTTGCTGCAAATGAACAGCCTGACACCTGAGGACACGGCCGTCTA TTACTGTAACCGGGACCCCAAGTACGGTAACACTAGATACTGGGGCCAGG GGACCCAGGTCACCGTCTCCTCAGCGGCCGCAcgccagcgcatttggtttcagaaccgccg ccgcagccgcaaaattaaaaaaCATCACCACCATCACCATTAA (SEQ ID NO: 5), where the 2C5 nanobody sequence is the one underlined, the TB peptide sequence is the one in bold and the italicized sequence is a polyhistidine tag comprising at least 6 histidine residues.It should be noted that 3 alanines are added after the 2C5 sequence ending with TVSS and before the Tb peptide sequence starting with RQR, to serve as a linker. However, this linker is not essential for the proper transport of the molecule of interest across the BBB (data not shown).

[0016] This TB peptide therefore represents an effective vector capable of transporting through cellular barriers, in particular the BBB, molecules of interest for diagnostic, prognostic and therapeutic purposes of CNS pathologies which for the most part fail to reach their cerebral target due to their inability to cross the BBB alone, as in the case of the 2C5 nanobody.

[0017] The subject of the present invention is therefore a peptide comprising or consisting of a peptide of amino acid sequence SEQ ID NO 1.

[0018] Said peptide can be synthesized by any methods known to those skilled in the art, for example using a peptide synthesizer or by gene synthesis, insertion of the latter into an expression vector and expression in a bacterial, cellular or acellular system.

[0019] The present invention also relates to a peptide conjugate comprising or consisting of a peptide according to the invention, where said peptide is covalently linked to a molecule of interest, directly or via a linker.

[0020] According to a particular embodiment of the present invention, said linker, when present, is chosen from the group consisting of peptides, polymers or chemical agents enabling cross-linking. Preferably, said linker, when present, is composed of 1 to 6 amino acids, preferably 3 amino acids, preferentially 3 alanines.

[0021] According to a particular embodiment of the present invention, the molecule of interest is chosen from the group consisting of any chemical or biological molecule of diagnostic, prognostic or therapeutic interest. For example, it may be an antibody (e.g. a nanobody), a DNA, an RNA, a peptide (e.g. analgesic, anesthetic, e.g. neurotensin), a drug (e.g. inhibitors of BACE1 (beta-site APP cleaving enzyme 1) or Gamma-secretases). Preferably, the nanobody is an anti-Tau nanobody capable of binding early pathological forms of the human Tau protein while being devoid of the ability to cross the BBB, preferably a Tau nanobody of amino acid sequence SEQ ID NO: 3.

[0022] According to a particular embodiment of the present invention, the peptide conjugate comprises or consists of the sequence SEQ ID NO: 4.

[0023] The present invention also relates to a peptide conjugate according to the present invention for use as a medicament. In particular, the peptide conjugate is useful in the diagnosis, prognosis, or treatment of a pathology of the central nervous system (CNS).

[0024] According to a particular embodiment of the present invention, the CNS pathology is selected from the group consisting of brain tumors, cerebrovascular accidents (CVA), multiple sclerosis, Huntington's disease and tauopathies. For example, tauopathy is selected from the group consisting of Parkinson's disease, Alzheimer's disease, Pick's disease.

[0025] The present invention also relates to a nucleic acid sequence coding for a peptide or a peptide conjugate according to the present invention, preferably the nucleic acid sequence comprises or consists of the sequence SEQ ID NO: 2 or the sequence SEQ ID NO: 5, respectively.

[0026] The present invention also relates to an expression vector comprising a nucleic acid sequence according to the present invention, for example the plasmid pET15b. BRIEF DESCRIPTION OF THE FIGURES

[0027] [Fig. 1] represents the three-dimensional structure of the TB peptide.

[0028] [Fig.2] represents the confirmation of the alpha helix of the TB peptide by the PepFold and heliquest programs.

[0029] [Fig.3] represents the conjugates comprising the TB or BIP peptide fused to the 2C5 nanobody.

[0030] [Fig.4] represents the cellular model of the BBB.

[0031] [Fig.5] represents the effect of nanobodies and conjugates (2C5, 2C5-TB and 2C5-BIP) on the sucrose permeability of endothelial cells.

[0032] [Fig.6] represents the sucrose clearance curves through endothelial cells exposed to nanobodies and conjugates (2C5, 2C5-TB and 2C5-BIP).

[0033] [Fig.7] represents the permeability coefficient in pL / h / cm2 of the nanobodies and conjugates (2C5, 2C5-TB and 2C5-BIP). EXAMPLES

[0034] EXAMPLE 1: DESIGN OF A NEW CCP: THE TB PEPTIDE Protein sequence of the TB peptide

[0035] The TB peptide sequence 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 (pi) close to 13 (12.70 according to protparam) in order to subsequently lower the pi of the molecules of interest fused to it.

[0036] The TB peptide was then synthesized by gene synthesis and produced in a bacterial system or by peptide synthesis using a synthesizer. Resulting three-dimensional structure (Fig. 1)

[0037] The alpha helix of the TB peptide was confirmed by the PepFold and heliquest programs ([Fig.2]). EXAMPLE 2: FUSION OF THE TB PEPTIDE TO A NANOBODY

[0038] Fusion of the TB peptide to a nanobody that does not pass the BBB

[0039] To assess the diffusion capacity across a cellular BBB model dif fertilized, the TB peptide was fused to a nanobody (2C5) which did not pass this model but whose target is cerebral (International application WO 2018 / 078140)

[11] .

[0040] Conjugation of the TB peptide to the 2C5 nanobody was achieved by gene fusion such that the resulting protein consists, from the N-terminus to the C-terminus, of the 2C5 nanobody, a linker composed of 3 alanines, the TB peptide and a tag of 6 histidines. A similar fusion with a promising CCP from Cho, 2017 [9], the BIP peptide, was also achieved ([Fig.3]).

[0041] The corresponding genes were inserted into the plasmid pET15b using the restriction enzymes NcoI / BamHI (data not shown).

[0042] Production of 2C5 nanobodies and 2C5-TB and 2C5-BIP peptide conjugates

[0043] The plasmids coding for the 3 proteins (2C5, 2C5-TB and 2C5-BIP) were inserted into E. coli Shuffle bacteria (Biolabs) according to the supplier's protocol.

[0044] From a colony resulting from the transformation, a preculture was carried out in a volume of 25 mL of LB containing 100 pg / mL of ampicillin. This preculture was incubated overnight at 37°C with shaking at 190 rpm.

[0045] The following day a 1 L culture was carried out by diluting the 25 mL of preculture in LB containing 100 pg / mL of ampicillin. The preculture was incubated at 37°C with 190 rpm shaking and when the optical density measured at 600 nm reached 0.8, 1 mM of IPTG was added to induce the T7 promoter of plasmid pET15b. The culture was incubated for an additional 3 hours under the same conditions.

[0046] The bacteria were collected at the end of the 3 hours of induction, by centrifugation for 40 min at 9500 G. The supernatant was removed 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 of lysozyme, 2 antiprotease pellets supplemented with EDTA free (Roche) and 1 μL 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.

[0047] 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 with 150 mL of TpA and the nanobodies and conjugates were eluted by adding 250 mM imidazole in TpA.

[0048] Fractions of 1 mL were collected and those containing the nanobodies or conjugates after analysis on 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.

[0049] The fractions containing the nanobodies or conjugates, pure at more than 95%, 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.

[0050] Evaluation of the passage of nanobodies and peptide conjugates through a differentiated cellular BHE model ([Fig.4D

[0051] Cerebral microvessel endothelial cells prepared from 5-week-old male rats were seeded onto microporous filters in order to:

[0052] - evaluate the toxicity of nanobodies and conjugates during their transfer by measuring the kinetics of sucrose transfer. - evaluate the permeability of nanobodies and conjugates in the blood / brain direction.

[0053] Experimental conditions:

[0054] - Endothelial cells were seeded onto 28 Transwell Poly-carbonate filters (porosity 0.4 pm and diameter 12 mm), and cultured in dif reference. - 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 280 and 1200 μl for the donor luminal compartment and the acceptor basolateral compartment, respectively. - The nanobodies and conjugates were diluted in the same HBSS Ca / Mg-0.1% BSA buffer. - Transfer was initiated by adding the nanobodies or conjugates to the upper compartment and transferring the filter to a well containing HBSS Ca / Mg-0.1% BSA. - The integrity of control monolayers or monolayers exposed to nanobodies or conjugates (study A) was assessed by measuring sucrose permeability through a kinetic analysis over 2 hours (comparable to the time used to estimate the permeability of nanobodies and conjugates). - Luminal and abluminal media collected for study B were analyzed by ELISA test. - The groups are as follows:

[0055] [Tableauxl] Study A. Toxicity of nanobodies and conjugates A. Permeability of nanobodies and conjugates Groups / number of fibers Luminal sampling vol / time 50 pl / 120 min 20 pl / 8 min 250 pl / 120 min Abluminal sampling vol / time 900 pl / 15, 30, 45, 60, 90, 120 min 250 pl / 60 min total vol / 120 min

[0056] For study B, the medium collected at 60 min basolaterally was replaced with the same volume of fresh medium.

[0057] Results of study A:

[0058] The sucrose permeability of endothelial cells reflecting the integrity of tight junctions was not modified in the presence of nanobodies and conjugates ([Fig.5]). The clearance curves averaged for the 4 filters were linear for the four groups of filters ([Fig.6]).

[0059] 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 endothelial cells. thelials to sucrose. They therefore validate the possible differences observed within the values ​​of the permeability coefficients for the 3 nanobodies and conjugates tested.

[0060] Results of study B:

[0061] The samples collected at 60 and 120 min were analyzed by ELISA test to evaluate the quantity of nanobodies and conjugates that passed during study B. To be able to analyze the different samples by ELISA, it was necessary to first purify them to remove the BSA limiting adhesion to the 96-well plates of the ELISA test. Thanks to the polyhistidine tag present on the 3 nanobodies and conjugates, purifications on 96-well plates coated with Nickel-NTA were carried out.

[0062] The purifications were carried out as follows:

[0063] - Incubation of samples for 1 hour at 4°C on the Ni-NTA plate. - Elimination of “unretained” fractions by pipetting. - 3 washes of 200 pL in PBS buffer + 30 mM imidazole. - Incubation 30 min with 200 pL of PBS elution solution + 300 mM imidazole. - Transfer of eludons onto 96-well plates for ELISA tests.

[0064] In parallel, concentration ranges of the different nanobodies and conjugates were produced and incubated on the same 96-well plates as the previous eludons, overnight at 4°C. The following day, the non-adsorbed fractions were removed by inversion and blocking was carried out with a PBS + 1% BSA solution for 1 hour at 23°C. A PBS + 1% BSA solution containing anti-histidine and HRP-coupled antibodies replaced the blocking solution and a 1-hour incubation at 23°C was carried out to allow the antibodies to bind to the nanobodies and conjugates present. 3 washes in PBS + 0.1% tween were then carried out to remove the unretained elements and a TMB solution was added to reveal the ELISA tests by colorimetry.

[0065] An absorbance reading was taken at 450 nm after stopping the colorimetric reaction using a 1N HCl solution.

[0066] The concentration ranges achieved made it possible to plot 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 allowing the transition from absorbances to concentrations in the samples taken during study B. In order 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 conjugate passed per hour and per cm2 of model. In order to compare these values ​​with the references (a dextran of 15000 Da and sucrose), the diffusion of the nanobodies was finally expressed as a permeability coefficient, i.e. in pL / h / cm2 ([Fig.7]).

[0067] These results demonstrated the ability of the 2C5-TB conjugate to pass through the BBB. Thus, the TB peptide demonstrated its vector capacity to transport through the BBB a molecule of interest that does not pass through the BBB when administered alone. List of references

[0068] 1. Abbott et al., Neurobiol. Dis., 37: 13-25, 2010 2. Pardridge, Molecular Interventions, 3(2): 90-105, 2003 3. Pardridge, Expert Opinion on Drug delivery, DOI: 10.1517 / 17425247.2016.1171315,2006 4. De Boer and Gaillard, Clin. Pharmacokinet., 46(7): 553-576, 2007 5. Patel and Patel, CNS Drugs, DOI: 10.1007 / / s40263-016-0405-9, 2017 6. Dong, Theranostics, 8(6): 1481-1493, 2018 7. Zhou et al., WIREs Nanomed Nanobiotechnol., 13: el695, 2021 8. Oller-Salvia, Chem. Soc. Rev., 45(17): 4690-4707, 2016 9. Patent Application FR 3058143

Claims

Claims

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

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

3. Peptide conjugate according to claim 2, wherein the linker is composed of 1 to 6 amino acids, preferably 3 alanines.

4. A peptide conjugate according to claim 2 or 3, wherein the molecule of interest is selected from the group consisting of an antibody, a DNA, an RNA, a peptide, and a drug.

5. Peptide conjugate according to claim 4, wherein the molecule of interest is a nanobody, preferably an anti-Tau nanobody.

6. A peptide conjugate according to claim 5, wherein the anti-Tau nanobody comprises the amino acid sequence SEQ ID NO:

3.

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

4.

8. A peptide conjugate according to any one of claims 2 to 7, for use as a medicament.

9. A peptide conjugate according to any one of claims 2 to 7, for use in the diagnosis, prognosis or treatment of a central nervous system (CNS) pathology.

10. A peptide conjugate for use according to claim 9, wherein the CNS pathology is selected from the group consisting of brain tumors, strokes, Huntington's disease, multiple sclerosis and tauopathies.

11. A peptide conjugate for use according to claim 10, wherein the tauopathy is selected from the group consisting of Parkinson's disease, Pick's disease, and Alzheimer's disease.

12. A nucleic acid sequence encoding a peptide according to claim 1 or a peptide conjugate according to any one of claims 2 to 7, preferably a nucleic acid sequence comprising the sequence SEQ ID NO: 2 or the sequence SEQ ID NO:

5.

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

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