Patent Application
20240053345
The present invention relates to a polypeptide able to bind selectively to cells expressing proteoglycans comprising glycosaminoglycans as heparin or heparan sulfate. The linkage of said polypeptide to an internalization peptide enables the polypeptide to be internalized.
Homeoproteins (HPs) are autonomous transcription factors that are active during development and in adulthood. These proteins are found ubiquitously in plant and animal cells. HPs have paracrine activities and can travel from cell to cell through unconventional transfer or transduction pathway. Their intracellular activity implies a direct access of the traveling proteins to the cytosol and nucleus of recipient cells. HP atypical paracrine activity relies on common structural features shared by all HPs, and includes independent and distinct secretion and internalization regions. These regions reside in the 60-residue DNA-binding homeodomain (HD) that define the HP family. HDs are organized as three stable helices while the N- and C-terminal ends are variable and mostly unfolded. The third helix is responsible for the internalization activity of the protein while the secretion property requires a motif spanning both second and third helices. Notable is that the internalization region from the drosophila Antennapedia HP sequence is a cationic hexadecapeptide called Penetratin (RQIKIWFQNRRMKWKK). Penetratin is a cell-penetrating peptide (CPP). CPPs are a large family of peptides generally containing 5-30 amino acids and own the characteristic of spontaneously crossing the cell membrane (Derossi, D. & Prochiantz, A. Trojan peptides: the penetratin system for intracellular delivery. Trends Cell Biol. 8, 84-87 (1998)). Their positive charges intrinsically represent the major common properties of CPPs. The first CPPs Penetratin (Pen) and Tat are respectively from Antennapedia (Antp) transcription factor and the trans-activator protein of transcription of human immunodeficiency virus-1 (HIV-1).
CPPs interact with the different biomolecules at the cell surface. Cell-penetrating peptide internalization is strongly dependent on the presence of glycosaminoglycans (GAGs) both in vitro (Bechara et al. (2015) Massive glycosaminoglycan-dependent entry of Trp-containing cell-penetrating peptides induced by exogenous sphingomyelinase or cholesterol depletion. Cell Mol Life Sci 72(4):809-820.) and in vivo (Nakase I, Konishi Y, Ueda M, Saji H, Futaki S (2012) Accumulation of arginine-rich cell-penetrating peptides in tumors and the potential for anticancer drug delivery in vivo. J Control Release 159(2):181-188).
Cell membrane is decorated with diverse proteoglycans consisting of a core protein and a large number of linear polysaccharides, termed glycosaminoglycans (GAGs). GAGs are covalently linked to the core protein and negatively charged due to their sulfated groups and carboxyl groups.
GAGs are composed of different repeating disaccharide units. The disaccharide is composed of uronic acid derivatives (GlcA or IdoA) and an N-acylated or N-sulfated hexosamine.
GAGs are classified into the following four categories based on core disaccharides structures: heparan sulfate (HS), chondroitin sulfate (CS) and dermatan sulfate (DS), keratin sulfate (KS) and hyaluronic acid or hyaluronan (HA).
Heparin and heparan sulfate (HS) are composed of a repetition of glucuronic acid (GlcA) and iduronic acid (IdoA) with N-acetyl, N-sulfate and O-sulfate substitutions. Heparin is the structural analogue of HS but with higher sulfation.
Chondroitin sulfate (CS) is composed of a chain alternating N-acetylgalactosaminesulfate and glucuronic acid (GlcA). Sulfation rate vary within the CS subtype: CS-A and CS-C are mono-sulfated, CS-D and CS-E are di-sulfated. Dermatan sulfate (DS) is one subtype of chondroitin sulfate (CS): CS-B. The difference is the epimerization of the glucuronic acid into iduronic acid in DS.
Keratan sulfate (KS) is composed of a chain alternating N-acetylglucosamine and galactose. Keratan sulfate is the only GAG that does not contain uronic acid.
Hyaluronan (HA) is the only GAG existing as a free status without being linked to a core protein. HA is not sulfated and is composed of a chain alternating glucuronic acid (GlcA) and N-acetylglucosamine.
The content of HS on cells generally ranges from 50%-90% while CS is the second-largest component, the ratio of HS and CS is usually found as 4:1 in the surface of vascular endothelium.
The potential of CPPs for therapeutic molecules delivery into cells has been emphasized following the natural CPPs discovery. The lack of selectivity of the CPP-based delivery system between healthy cells and cancer cells causes side effects which is one of the important obstacles that hamper their biomedical application (Lindberg, S., Copolovici, D. M. & Langel, Ü. Therapeutic delivery opportunities, obstacles and applications for cell-penetrating peptides. Ther. Deliv. 2, 71 82 (2011)).
Regarding HPs, it was reported that the homoprotein Otx2 (orthodenticle homolog 2) internalization is restricted in vivo to specific neurons of the brain in mouse. Secreted Otx2 accumulates in parvalbumin neurons through interactions with surrounding perineural nets enriched in disulfated chondroitin sulfate (CS) GAGs, and regulates plasticity in the developing mouse visual cortex (WO2010081975). The region responsible for the addressing of Otx2 to its target cells is constituted by a peptide sequence of 15 amino acids.
Considering these drawbacks, the Inventors have searched for other HPs comprising a CPP, which could selectively target specific cells.
The Inventors have discovered a new region of Engrailed-2 (En2) that may selectively bind some cancer cells. This region has sequence similarities with nuclear localization signals (NLS) but, in contrast to Otx2, the newly discovered region is highly enriched in basic aminoacids and amazingly this NLS region of En2 is not the homologue of the Otx2 sequence.
En2 is a transcription factor that plays a role in the patterning of metazoan embryos. En2 regulates in particular the formation of boundaries during development of the brain in vertebrates. En2 does not accumulate in parvalbumin neurons and the region preceding the homeodomain strongly differs from that of Otx2, suggesting a different GAG selectivity.
The third helix of the homeodomain of En2 is a CPP, and its sequence is similar to Penetratin (Sgadó, P.; Genovesi, S.; Kalinovsky, A.; Zunino, G.; Macchi, F.; Allegra, M.; Murenu, E.; Provenzano, G.; Tripathi, P. P.; Casarosa, S.; Joyner, A. L.; Bozzi, Y. Loss of GABAergic Neurons in the Hippocampus and Cerebral Cortex of Engrailed-2 Null Mutant Mice: Implications for Autism Spectrum Disorders. Experimental Neurology 2013, 247, 496-505. https://doi.org/10.1016/j.expneurol.2013.01.021.).
Interestingly, the Inventors have discovered that the NLS region or cell recognition peptide of En2 links specifically to cells expressing heparin or heparan sulfate, especially some cancer cells.
They have further shown that this region associated with a third helix of a CPP is also able to address to specific cells any cargos linked to this chimeric polypeptide.
The present invention relates to an isolated polypeptide defined by the sequence: RK-XXX-KK-XXXXXX-KR, in which X is an amino acid, each X being independent from each other, and wherein X is selected from the group arginine or histidine or lysine or asparagine or glutamine or tryptophan or serine or cysteine or threonine or methionine or proline, or aspartate or glutamate.
According to the preferred embodiment the sequence RK-XXX-KK-XXXXXX-KR, in which X is an amino acid, each X being independent from each other, and wherein X is selected from the group arginine or histidine or lysine or asparagine or glutamine or tryptophan or serine or cysteine or threonine or methionine or proline or aspartate or glutamate, and wherein at least three X are proline.
According to a preferred embodiment, the isolated polypeptide of the invention is: RKP-XX-KK-X-P-XXXX-KR P.
This isolated polypeptide according to the invention is a cell recognition polypeptide binding selectively heparin or heparan sulfate glycosaminoglycans.
The present invention further relates to the isolated polypeptide defined by the sequence:
“None amino acid” means that the polypeptide of SEQ ID No. 1 does not comprise any amino acid on the defined position; for example, if X1 represents none amino acid, then the SEQ ID No. 1 does not comprise any amino acid at the 1st position and begins by the X2 amino acid.
In a preferred embodiment,
Preferably, the present invention relates to an isolated polypeptide defined by the sequence:
In a more preferred embodiment,
According to one preferred embodiment of the invention, the cell recognition polypeptide is characterized by the sequence RSRKPKKKNPNKEDKRPR (SEQ ID No. 2). The SEQ ID No. 2 is the sequence of the NLS of Ent.
The present invention also relates to a conjugated polypeptide comprising the polypeptide of SEQ ID No. 1 or 2 and a biomarker conjugated at its N-terminal end or its C-terminal end or on a lateral position linked to an amino acid.
In the context of the present invention, the term “biomarker” refers to a detectable and optionally measurable substance in a biological entity such as a patient or isolated organs, tissues or cells.
A “biomarker” denotes any molecule or molecular complex used for labelling purpose, for example, fluorescent biomarker (fluorescein, rhodamine, Cy3, Cy5, nitrobenzoxadiazole (NBD)), radiolabeled biomarker (18F, 68Ga), biotin, organic (micelle, liposome, dendrimer, polymer), inorganic (gold, iron oxide, lanthanide ions, carbon, silica) or composite (core-shell, MOF-metal organic framework) nanoparticles.
More specifically, the conjugated polypeptide of the invention is a diagnostic biomarker both in vivo and in vitro to detect cells expressing heparin or heparan sulfate, especially cancerous cells.
The present invention thus further relates to:
Another object of the invention is an in vitro detection method of cells expressing heparin or heparan sulfate glycosaminoglycans in a biological sample from a patient comprising the steps of:
The detection is measured in vitro.
The detection method is used to detect and to diagnose cancers and the presence of cells expressing heparin or heparan sulfate glycosaminoglycans in the biological sample indicates that the patient has a cancer or is a diagnostic of a cancer.
The general term “biological sample” for the purposes of the present description is a sample of cells from a patient.
According to another object, the present invention relates to a cell penetrating polypeptide, defined by the general formula (I) or (II):
X1X2-RK-X3X4-KK-X5X6X7X8X9X10-KR-X11X12-L-CIP (I)
CIP-L-X1X2-RK-X3X4-KK-X5X6X7X8X9X10-KR-X11X12 (II)
wherein X1 to X12 and their preferred embodiments are the same as those previously described; L is nothing or a spacer and CIP is a cell internalization peptide facilitating the cell penetration of the polypeptide.
The spacer can be a flexible linker with at least three atoms or a conformationally restricted linker with at least three atoms or an aliphatic flexible linker with five atoms or an hydrophilic flexible linker with nine atoms.
In a preferred embodiment, the spacer can be a glycine or a proline or an aminopentanoic acid or polyethylene glycol.
Several cell internalization peptide sequences can be used:
SQIKIWFQNKRAKIKK (SEQ ID No. 3), RQIKIWFQNRRMKWKK (Penetratin, SEQ ID No. 4), RRWWRRWRR (SEQ ID No. 5), RRWRRWWRR (SEQ ID No. 6), RRWWRRWWR (SEQ ID No. 7), RRRRRRRR (SEQ ID No. 8) and YGRKKRRQRRR (SEQ ID No. 9).
CIP sequences which have been described in the prior art (EP1795539, EP2579899) which are incorporated by reference.
According to one specific embodiment of this object, a biomarker, as defined before, can be added to the cell penetrating polypeptide of general formula (I) or (II) in N-terminal end, C-terminal end or on a lateral position linked to an amino acid.
Thanks to the cell recognition peptide, the cell penetrating polypeptide (CPP) can link to cells expressing heparin and heparin sulfate and can enter into cells with its cell internalization sequence (CIP).
Thus, another object of the invention relates to a chimeric polypeptide composed of the cell penetrating polypeptide of general formula (I) or (II) and a biological cargo. The cargo is coupled to the cell-penetrating peptide through an amide, maleimide, disulfide or triazole bond either at the N-terminal or C-terminal end or on an amino acid side chain.
The general term “biological cargo” denotes any molecule or molecular complex, that is desired to target to a target cell. The biological cargos that can be transported by cell penetrating polypeptides in accordance with the invention can be a nucleic acid or a polypeptide. In case of a peptide, a preferred embodiment for the biological cargo is the sequence KRAKLAK (SEQ ID No. 10).
Another object of the present invention is related to a pharmaceutical composition comprising a chimeric polypeptide according to the invention and an acceptable pharmaceutically vehicle.
Another object of the invention relates to the chimeric polypeptide of the invention and said pharmaceutical composition for use as a medicament.
In a specific embodiment, said chimeric polypeptide and said pharmaceutical composition are for use to treat cancer involving cells expressing heparin or heparan sulfate (HS) glycosaminoglycans.
The prior art has shown important roles of HS in oncogenic signaling. The deregulations of both HS and heparin sulfate proteoglycan HSPG are involved in solid tumors as well as hematological malignancies among prostate, breast, lung, leukemia, colorectal, pancreatic, ovarian, neuroblastoma, myeloma, carcinoma, gliobastoma, head/neck, melanoma, testicular germ cell, Wilm's tumor, yolk sac tumor, hepablastoma, bladder, sarcoma, endometrial, renal cell, non-Hodgkin's lymphoma. HS and HSPG are considered as good targets to treat cancers (Nagarajan et al., Heparan sulfate and heparin sulfate proteoglycans in cancer initiation and progression, Frontiers in endocrinology, 24 Aug. 2018).
Advantageously, the cell penetrating polypeptide of the invention combines the cellular internalization with the third helix and the GAG selectivity to target cells expressing HS.
The present invention will be better understood with the aid of the additional description which follows, which refers to non-limiting Figures and Examples illustrating the identification of a targeting polypeptide in accordance with the invention and the demonstration of its specificity of addressing.
The chimeric polypeptide P1 have been obtained by synthetizing the third helix 43-58 residues of En2 HD (En2H3) as cell-penetrating part and by picking up a region from the upstream of En2 HD as a putative HS-binding motif (EnHS) to conjugate with En2H3 as represented on
The inventors conjugated CS-binding peptide (OtxCS) with En2H3 to give potentially a CS-binding CPP (P3). Finally, they also synthesized P5 that is the peptide motif in En2 aligned to the CS-binding peptide from Otx2 conjugated to the En2H3.
Peptides listed in Table 1 were incubated separately in the following four ovarian cell types which have different GAGs types and expression levels: wild type CHO-K1 (expressing heparan sulfate (HS), chondroitin sulfate A (CSA) and C (CSC)), CHO-pgs A475 (CHO-745) (genetically modified to express only 5-10% HS and CS), two human ovarian adenocarcinoma cells CaOV-3 (overexpressing HS and CSE) and SKOV-3 (overexpressing CSE). Peptides were incubated with one million cells at 37° C. for 1 h.
Wild type Chinese Hamster Ovary (CHO-K1) cells and GAGs-deficient mutant CHO-745 cells which lack the xylosyltransferase needed for glycosaminoglycan (GAG) synthesis were grown in Dulbecco's modified Eagle's medium F-12 (DMEMF-12) with L-glutamine and 15 mM HEPES. HEK cells and HeLa cells were grown in DMEM with 4.5 g/L D-glucose and pyruvate. Two types of human ovarian cancer cell lines CaOV-3 and SKOV-3 were cultured in DMEM with Glutamax and McCoy's 5A medium, respectively. All complete culture medium was supplemented with 10% fetal bovine serum, penicillin (100,000 IU/L), streptomycin (100 mg/L). Cells were grown in a humidified atmosphere at 37° C. and 5% CO2.
The internalization results are shown in
Comparing the internalization efficacies of the two GAG-binding peptides and the other four CPPs, it is observed that EnHS and OtxCS could not enter efficiently in any of the cell lines (
P1, P3 and P5 are used in this example within the sequences described in the table 1.
Tested Peptides were incubated separately in the following two ovarian cell types: CHO-745 and CaOV-3. The inventors incubated (7.5 μM) peptides with cells lines for 1 h along with exogenous heparin (HI) or chondroitin-4,6-sulfate (CSE) and quantified the internalized peptides by MS.
Cells culture are similar to the example 1.
Desulfation or degradation of cell-surface HS and CS lead to a significant decrease in the internalization of P3 in SKOV-3. In the
P1 and P3 are used in this example within the sequences described in the table 1.
Tested Peptides were incubated separately in the following ovarian cell type: SKOV-3. A pre-treatment with sodium chlorate for 24 h or GAG-specific enzymes for 2 h.
Cells culture are similar to the example 1.
The inventors used different concentrations of chlorate to treat SKOV-3 and analyzed the internalization of P3. The result in
Of outmost interest,
P1 is used in this example within the sequence described in the table 1.
P1 was incubated in the CHO-745 ovarian cell type with different concentrations of heparinase. Cells culture are similar to the example 1.
P1 and P3 are used in this example within the sequences described in the table 1.
P1 and P3 were incubated separately in the CHO-K1 ovarian cell type with different concentrations of heparinase.
Cells culture are similar to the example 1.
The inventors tested cytotoxicity and hemolysis activity of the peptides incubated with CHO-K1 cells at different concentrations. In
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2020/001111 | 12/17/2020 | WO |
