EP2780028A1

EP2780028A1 - Use of protein s for treating cancer

Info

Publication number: EP2780028A1
Application number: EP12813101.8A
Authority: EP
Inventors: Omar BENZAKOUR; Arnaud MONVOISIN
Original assignee: Centre National de la Recherche Scientifique CNRS; Universite de Poitiers
Current assignee: Centre National de la Recherche Scientifique CNRS; Universite de Poitiers
Priority date: 2011-11-14
Filing date: 2012-11-12
Publication date: 2014-09-24
Also published as: FR2982490A1; WO2013072836A1

Abstract

The invention relates to an isolated sequence of amino acids, consisting of SEQ ID NO:1, a homologue presenting a homology of at least 80% with said sequence and a similar biological activity, or an active fragment consisting of 280 to 675 contiguous amino acids of said sequence or of said homologue, for use as an antiproliferative agent against cancer cells for preventing and/or reducing and/or treating a cancer.

Description

USE OF PROTEIN S TO TREAT CANCER

The present invention relates to the pharmaceutical field, and more particularly to the field of oncology.

Cancer is a human and animal disease that is mostly fatal, and results in uncontrolled growth of endogenous cells. The term "cancer" is used to refer to the formation of malignant tumors and neoplasms (tumors or carcinomas) or malignant degeneration and disordered maturation of white blood cells (leukemia or blood cancer). Generally, cancer growth involves two stages, namely local tumor growth and cancerous spread. Local tumor growth consists of the appearance of a tumor clone, from a "transformed" cell, under the action of carcinogenic initiators and promoters. Cell proliferation results in the constitution of the tumor. The cancerous spread from the initial tumor can be achieved either by regional spread or by development of secondary tumors called metastases. This spread is the gravity of cancer, because it is better to treat the primary tumor than metastases.

In an attempt to prevent and / or defeat cancer, various techniques such as surgery, radiotherapy including brachytherapy, chemotherapy including hormone therapy, immunotherapy and more recently anti-angiogenic therapy have been developed.

However, despite recent advances, these techniques are not all effective, especially against metastases or tumors resistant to treatment with cytotoxic agents, and unfortunately, frequently cause intolerance on the part of patients, as well as side effects such as bone marrow depression, vomiting, nausea, alopecia, asthenia, loss of appetite, anemia, thrombocytopenia, or non-preservation of healthy tissue.

Thus, for obvious reasons, there is a continuing need to identify new, more potent anti-cancer active agents, which selectively act on cancer cells to the exclusion of healthy cells, which are non-toxic, and which result in less, if not very few side effects.

The object of the present invention is to satisfy these needs.

According to one of these first aspects, the present invention relates to an isolated amino acid sequence, consisting of SEQ ID NO: 1, a homolog having a homology of at least 80% with said sequence and a similar biological activity, or a fragment active consisting of 280 to 675 contiguous amino acids of said sequence or homologue, for use as an antiproliferative agent of cancer cells to prevent and / or reduce and / or treat cancer.

Unexpectedly, the inventors have observed that an amino acid sequence consisting of SEQ ID NO: 1, in particular the S protein (S for Seattle), and more particularly the human protein S, has particularly effective antiproliferative properties. with regard to cancer cells. Thus, the protein S may prove to be a therapeutic asset of particular interest for the prevention, reduction or treatment of cancer.

Protein S is a vitamin K-dependent protein that is known to be involved in blood clotting.

Protein S, and its structural homolog Gas6 (growth arrest-specific gene 6), with which it shares 44% of sequence homology, are TAM tyrosine kinase receptor ligands whose members are Axl (also called Ufo or Ark), Tyro3 (also named Rse, Brt or Sky), and Mer (also named c-Eyk) (Stitt et al., Cell, 1995, 80: 661-670). Protein S has growth factor activity for smooth muscle cells (Gasic et al., Proc Natl Acad Sci USA, 1992, 89: 2317-20), and is involved in the regulation of phagocytosis of apoptotic cells (Benzakour et al., Sci Med (Paris), 2007, 23: 826-833, Rankin et al, Cancer Res., 2010, 70: 7570-7579). It has been observed that the S protein and the Tyro-3 receptor are expressed together in lung carcinoma tissues (Wimmel et al., Cancer, 1999, 86: 43-49).

Several studies have shown the existence of a relationship between TAM receptor activation and tumor and / or cancer progression (Linger et al, Adv Cancer Res, 2008, 100: 35-83, He et al, Mol. Carcinog., 2010, 49: 882-891, Ye et al, Oncogene, 2010, 29: 5254-5264).

KR20090090944 proposes the implementation of activators of the LIGHT gene (TNFSF14) selected from ligands of the Axl receptor to sensitize the tumor cells to NK (Natural Killer) cells.

Finally, US Pat. No. 6,593,291 describes the ability of protein S to inhibit the proliferation of HUVEC (Human Umbilical Vascular Endothelial Cell) cells.

However, until now it has never been observed that the S protein may prove to be particularly effective for directly inhibiting the proliferation of cancer cells, regardless of its anti-angiogenic effect. For the purposes of the invention, the term "antiproliferative agent of cancer cells" an agent capable of reducing and / or blocking the proliferation of cancer cells by direct action on these cells, without affecting the growth or proliferation of healthy cells.

By "direct action" is meant an action on a constituent element of the cancer cell or an element of one of its metabolic pathways. Such an effect opposes an effect obtained by action on the environment of the cell, such as an anti-angiogenic effect, which is described as an indirect action. The therapeutic effect obtained by an active ingredient of the invention can therefore be exerted independently of a possible anti-angiogenic action.

In the present invention, the term "antiproliferative" with respect to a given agent qualifies the property of the latter to exert an inhibitory action directly on the cells whose proliferation is to be stopped. Such a property is therefore necessarily independent of any action on cells other than those whose proliferation is to be stopped. Thus, the antiproliferative effect with respect to the cancer cells according to the invention is, for example, independent of an inhibition of the angiogenesis or the activation of immune cells.

For the purposes of the invention, the term "prevent" means reducing the risk of occurrence or slowing the occurrence of a given phenomenon, namely in the present invention, a cancer or a metastasis.

In another aspect, the present invention provides an isolated nucleic acid sequence encoding an amino acid sequence of the invention for use as an antiproliferative therapeutic agent of cancer cells to prevent and / or reduce and / or treat a Cancer.

In yet another aspect, the present invention relates to an expression vector comprising a nucleic acid sequence of the invention for use as an antiproliferative therapeutic agent of cancer cells for preventing and / or reducing and / or treating cancer.

According to another aspect, the present invention relates to a pharmaceutical composition comprising, in a pharmaceutically acceptable medium, at least one amino acid sequence or at least one nucleic acid sequence of the invention, or at least one vector of Expression of the invention as an antiproliferative agent of cancer cells for preventing and / or reducing and / or treating cancer

In yet another aspect, the present invention relates to a combination product comprising at least one amino acid sequence of the invention, or at least one a nucleic acid sequence of the invention, or at least one expression vector of the invention as antiproliferative agent of cancer cells, and at least one chemotherapeutic agent or at least one immunotherapeutic agent, for simultaneous use , separate or sequential to prevent and / or reduce and / or treat cancer.

Also disclosed is a therapeutic treatment method comprising administering at least one amino acid sequence of the invention, or at least one nucleic acid sequence of the invention, or at least one expression vector of the invention, as antiproliferative therapeutic agent of cancer cells, for preventing and / or reducing and / or treating cancer.

The anticoagulant properties of protein S are only manifested by the action of activated protein C. Its administration to an individual in the absence of active coagulation process does not affect the hemostatic balance. Moreover, as shown by the examples illustrating the invention, the implementation of the protein S as an antiproliferative agent of cancer cells is advantageously devoid of toxicity and side effects. Human protein S does not cause bone marrow depression, gastrointestinal symptoms, hair loss, standard chemotherapy features.

Since the antiproliferative properties of the protein S with respect to cancer cells are independent of an anti-angiogenic effect, the present invention makes it possible to propose a novel treatment for tumors or cancers that are insensitive or have acquired resistance to cancer. anti-angiogenic agents.

Advantageously, the present invention makes it possible to propose a new anticancer treatment with little or no side effects and less toxicity with regard to the treatments usually employed.

Advantageously, the present invention may be particularly useful with regard to tumors or cancers whose growth and / or dissemination does not involve angiogenesis.

Advantageously, the present invention makes it possible to increase the survival of patients presenting one or more metastases, and more particularly one or more lung metastases.

Protein S

An isolated amino acid sequence suitable for the invention may be represented by the sequence SEQ ID NO: 1. Also, an isolated amino acid sequence suitable for the invention may be a homologue of the sequence SEQ ID NO: 1 having a homology of at least 80% with this sequence and a similar biological activity.

According to another variant embodiment, an isolated amino acid sequence suitable for the invention may be an active fragment consisting of 290 to 670, preferably 300 to 660 amino acids, or even 310 to 650 or 320 to 640 amino acids. and preferably from 330 to 630 contiguous amino acids of said sequence or said homologue.

The term "homologue" of an amino acid sequence of the invention is intended to denote any amino acid sequence having a sequence identity of at least 80%, preferably at least 85%, or even at least 87% o, still at least 90% o, still at least 93% o, or even at least 97% o, and still more preferably at least 99% o, and still more more preferably 100% with said sequence, and a similar biological activity or of the same nature.

According to an alternative embodiment, a sequence homology may be more particularly determined with regard to the region including amino acids 299 to 666 of SEQ ID NO: 1. This region is presumed to be the domain through which the S protein interacts with and activates. TAM receivers (Tyro-3, Axl, and Mer).

When the sequence homology is determined with regard to the region including amino acids 299 to 666 of SEQ ID NO: 1, this can be at least 87% o, preferably at least 90% o, or even at least less than 93% o, or at least 95% o, or at least 97% o, even 99% o, and preferably 100% o.

Sequence homology can be determined by any method known to those skilled in the art, such as visual comparison, or by any computer tool generally used in the field, such as BLAST programs available at www.ncbi.nlm. .nih.gov and used with the default settings.

A homologue suitable for the invention may be a homologue of human protein S.

A homologue of an amino acid sequence of the invention may result from modifications resulting from mutation or variation in an amino acid sequence according to the invention, originating either from the deletion or the insertion of one or several amino acids, either the substitution of one or more amino acids or alternatively splicing. Many of these changes can be combined.

Advantageously, a homologue of an amino acid sequence of the invention may comprise conservative substitutions with respect to this amino acid sequence. By way of example of mutations which may be considered in the present invention, it may be mentioned, in a non-exhaustive manner, the replacement of one or more amino acid residues by amino acid residues having a subscript. similar hydropathic without substantially affecting the biological properties of the polypeptide. The hydropathic index is an index attributed to amino acids as a function of their hydrophobicity and charge (Kyte et al (1982) J. Mol Biol., 157: 105).

Among the homologues of the sequence SEQ ID NO: 1 that are suitable for the invention, mention may be made of the macaque protein S (Macaca mulatta, accession number NP 001038191.1), zebrafish (Danio rerio, accession number NP 001070791.1), xenopus (Xenopus (Silurana) tropicalis, accession number NP 001 1 19539.1), rat (Norwegian rats, accession number NP 1 12348.2), mouse (Mus musculus, accession number NP 035303.1 ), cattle (Bos taurus, accession number NP 776863.1, chimpanzee (Pan troglodytes, accession number XP 003309948.1), orangutan (Pongo abelii, accession number XP 002813442.1), turkey ( Meleagris gallopavo, accession number XP 003202865.1), dog (Canis lupus familiaris accession number XP 535708.3), mandarin diamond (Taeniopygia guttata accession number XP 002190185.1), African savannah elephant (Loxodonta africana, accession number XP 003410240.1), opossum (Monodelphis domestica, accession number export XP 001380313.2), green anole (Anolis carolinensis, accession number XP 003219160.1), nile tilapia (Oreochromis niloticus, accession number XP 003445823.1), marmoset (Callithrix jacchus, accession number XP 002761331.1 ), hen (Gallus gallus, accession number XP 0416641.2), horse (Equus caballus, accession number XP 001503040.2), gibbon (Nomascus leucogenys, accession number XP 003261735.1), chimpanzee (Pan troglodytes , accession number XP 516603.3), wild rabbit (Oryctolagus cuniculus accession number XP 002716802.1), panda (Ailuropoda melanoleuca accession number XP 002929359.1), and guinea pig (Cavia porcellus number of accession XP 003469383.1).

Among the homologues of the sequence SEQ ID NO: 1 that is more particularly suitable for the invention, mention may be made of the NP macrophage protein NP 001038191.1, 1 of the NP 001070791.1 zebrafish, the NP 001 1 19539.1 xenopus, and the NP 1 rat. 12348.2, the NP 035303.1 mouse and the NP 776863.1 cattle. These six homologs exhibited sequence homology with SEQ ID NO: 1, respectively, of 93%, 94%, 97%, 98%, 99% and 100% with this sequence. An amino acid sequence, or a homologue thereof, contemplated by the present invention may be an amino acid sequence having undergone one or more post-translational processing (s).

By "post-translational processing (s)" is intended to encompass all the modifications that an amino acid sequence is likely to undergo at the end of its synthesis in a cell, such as, for example, a or phosphorylation (s), thiolation (s), acetylation (s), glycosylation (s), lipidation (s), such as farnesylation or palmitoylation.

By "similar biological activity or of the same nature" with respect to an amino acid sequence according to the invention is meant the antiproliferative properties of the S protein with respect to the cancer cells. These properties can in particular be evaluated by means of the experimental protocols described in the examples.

In particular, the antiproliferative properties of an amino acid sequence of the invention, or a homologue, can be determined on tumor cells cultured under appropriate conditions in vitro. The determination of suitable in vitro culture conditions for cancer cells is within the knowledge of those skilled in the art. The tumor cells may be derived from primary cultures of tumors or cell lines. The effect, resulting from the contacting of an amino acid sequence of the invention with tumor cells, on the proliferation of the latter can be compared to an effect obtained under negative control conditions, in which the cancer cells are maintained in their culture medium without a test compound or in the presence of an amino acid sequence having less than 70% homology with SEQ ID NO: 1 and having no effect with regard to the proliferation of cells tumor, and optionally to an effect obtained under positive control conditions, wherein the tumor cells are in contact with an amino acid sequence SEQ ID NO: 1.

Within the meaning of the invention, the term "fragment of an amino acid sequence" any portion of an amino acid sequence according to the invention consisting of 290 to 670, preferably 300 to 660 amino acids , or 310 to 650 or 320 to 640, and preferably 330 to 630 consecutive amino acids of said sequence, and having a biological activity of the same kind. More preferably, a fragment suitable for the invention may be derived from the sequence SEQ ID NO: 1.

According to one embodiment, a fragment of protein S which is more particularly suitable for the invention may be wholly or partly derived from the region of the protein of S binding to the TAM receptors. More preferably, a fragment suitable for the invention may be an amino acid sequence derived from a region defined by amino acids 250 to 675, preferably 260 to 670, or even 270 to 665, or 280 to 660, even 290 to 650 or 300 to 640, with regard to the numbering of the amino acid sequence SEQ ID NO: 1, or from any equivalent region derived from a homologue of SEQ ID NO: 1.

The biological activity of a fragment of an amino acid sequence of the invention can be determined as indicated above with respect to an amino acid sequence of the invention or a homologue thereof. this.

According to a preferred embodiment, an amino acid sequence that is suitable for the invention may be a human protein S. This protein has been described by Lundwall et al. (Proc Natl Acad Sci, 1986, 83: 6716-6720). Preferably, an amino acid sequence that is suitable for the invention may be represented by the sequence SEQ ID NO: 1. This sequence has the accession number NCBI NP 000304.2.

Human protein S is a secreted glycoprotein, and exhibits anticoagulant factor activity. It is naturally present in the blood and acts as a cofactor for activated protein C (APC). Its anticoagulant activity being dependent on activated protein C (APC), its injection, even at very high doses, does not lead to an inhibition of blood coagulation.

Human protein S is naturally expressed by a wide variety of tissues and cell types such as the liver, brain, heart, ovaries, placenta, spleen, kidneys, endothelial cells, megakaryocytes, osteoblasts, nervous system cells, smooth vascular cells and Leydig cells.

Human protein S has a half-life in the human body of about 48 hours, advantageously conferring on it a better bioavailability and an ability to exert a long-term effect, necessary for an effective anticancer treatment.

Protein S is an endogenous protein and can be readily purified from blood by conventional precipitation-based and chromatographic purification methods and the implementation of which has been published (Dahlback, Biochem J., 1983, 209 : 837-846).

Purified human S protein can also be obtained commercially from various companies such as Merck, Calbiochem, Biogenic, Anaria or Haematologic Technologies.

The cDNA sequence as well as the corresponding amino acid sequence of human protein S are known to those skilled in the art (Lundwall et al., Proc Natl Acad Sci. USA, 1986, 83: 6716-6720). Thus, human protein S can be obtained in very large quantities and with a high degree of purity by transfection of its cDNA into eukaryotic cells. The techniques for transfection and expression of nucleic acid sequences in host cells, as well as purification of the proteins obtained are known to those skilled in the art which can refer for example to the manual "Molecular Cloning: A Laboratory Manual "- Sambrook et al., Ed. 2001, Cold Spring Harbor Laboratory, New York.

Thus, according to one embodiment, the present invention also relates to a nucleic acid sequence encoding an amino acid sequence of the invention, a homologue or a fragment thereof, for use as an antiproliferative agent of cells. cancerous to prevent and / or reduce and / or treat cancer.

For the purposes of the present invention, the term "nucleic acid sequence fragment" means a nucleic acid sequence consisting of 780 to 1980, or even 900 to 1950, or even 930 to 1920, or even 960 to 1890. , and more preferably in 990 to 1860 consecutive base pairs of said sequence, and encoding an amino acid sequence having a biological activity of the same nature as the amino acid sequence encoded by said sequence.

For the purposes of the present invention, the term "nucleic acid sequence analogue" means a nucleic acid sequence having a sequence identity of at least 65%, preferably at least 80%, and more. preferably 100% with said sequence, and coding for an amino acid sequence having a biological activity of the same nature as the amino acid sequence encoded by said sequence.

By "analog of a nucleic acid sequence" is meant a nucleic acid sequence, possibly resulting from the degeneracy of the nucleic acid code, and coding for an amino acid sequence according to the invention, in particular as defined above.

A nucleic acid sequence suitable for the invention may in particular be a sequence consisting of SEQ ID NO: 2, a homologue or a fragment thereof. More preferably still, a nucleic acid sequence that is suitable for the invention may be the sequence SEQ ID NO: 2 coding for the human protein S. This sequence has accession number NCBI NM_000313.3.

According to yet another embodiment, an expression vector may be implemented to introduce a nucleic acid sequence of the invention into a host cell to produce in vitro a recombinant S protein, as indicated previously, or in a cancer cell or in a cell healthy environment of a cell cancer patient of an individual suffering from cancer or metastases. This last implementation advantageously allows the in situ production of the anticancer active ingredient, and an improvement in its bioavailability.

An expression vector suitable for the invention may comprise a transcription cassette comprising a transcription initiation agent, a nucleic acid sequence of the invention and a transcription termination agent, all of which is operably linked. The transcription cassette can be introduced into a variety of expression vectors, such as a plasmid, a retrovirus, e.g. a lentivirus, or an adenovirus. An expression vector may allow stable or transient expression of the amino acid sequence of interest, usually for a period of at least one day, more generally for several days, or even weeks.

Administration and targeting of an expression vector of the invention to specific cells of an individual suffering from cancer is within the skill of those skilled in the art. The targeting of an expression vector of the invention to cancer cells to be treated may be carried out for example by means of an antibody directed specifically towards an antigen specific for or overexpressed by cancer cells, such as Mage, Bage or Her2-neu, or by means of a ligand of a receptor specific for or overexpressed by cancer cells, such as the folic acid receptor. Pharmaceutical composition

According to another of its aspects, the invention relates to a pharmaceutical composition comprising, in a pharmaceutically acceptable medium, at least one amino acid sequence of the invention, or at least one nucleic acid sequence of the invention, or at least one expression vector of the invention, as antiproliferative agent of cancer cells for preventing and / or reducing and / or treating cancer.

Also, the invention relates to the use of at least one amino acid sequence of the invention, or at least one nucleic acid sequence of the invention, or at least one expression vector of the invention. as an antiproliferative agent of cancer cells for preparing a pharmaceutical composition for preventing and / or reducing and / or treating cancer.

Thus, is also considered by the invention, a sequence of amino acids or nucleic acids, or an expression vector according to the invention as a medicament for preventing or inhibiting the proliferation of cancer cells to prevent and / or reduce and / or treat cancer. An amino acid or nucleic acid sequence, or an expression vector according to the invention, or a drug or a pharmaceutical composition of the invention may be formulated with any pharmaceutically acceptable medium or vehicle, and can be formulated in any solid, semi-solid, liquid or gaseous form, such as a tablet, capsule, capsule, powder, granule, emulsion, suspension, gel, microsphere , or an inhaled form.

According to one embodiment, an amino acid or nucleic acid sequence, or an expression vector in accordance with the invention, or a pharmaceutical composition of the invention may be formulated for the oral route, in the form of a tablet, capsule or capsule, with extended or controlled release, of a pill, a powder, a solution, a suspension, a syrup or emulsion.

According to another embodiment, an amino acid or nucleic acid sequence, or an expression vector in accordance with the invention, or a pharmaceutical composition of the invention may be prepared in injectable form. An amino acid or nucleic acid sequence of the invention may be formulated with different carriers, such as a liposome or a transfection polymer.

A pharmaceutical composition of the invention may preferably comprise a sequence of amino acids or nucleic acids suspended in a pharmaceutically acceptable vehicle, for example an aqueous vehicle. Different aqueous vehicles may be used, for example water, saline buffer solution, 0.4% or 0.3% glycine solution, or a solution of hyaluronic acid.

A pharmaceutical composition can be sterilized by any known conventional method, such as filtration. The resulting aqueous solution may be packaged for use as it is, or lyophilized. A lyophilized preparation can be combined with a sterile solution before use.

A pharmaceutical composition of the invention may comprise any pharmaceutically acceptable excipient required to approximate physiological conditions, such as buffering agents or agents for adjusting pH or isotonicity, wetting agents. Such preparations may also include antioxidants, preservatives, adjuvants, and optionally other therapeutic agents.

A pharmaceutical composition of the invention naturally comprises an amino acid or nucleic acid sequence of the invention in an effective amount. An effective amount of an amino acid or nucleic acid sequence of the invention is an amount which, alone or in combination with subsequent doses, induces the desired response, namely an antiproliferative effect on cells. cancerous. An effective amount may depend on a varied set of parameters, such as the route of administration, single or multiple dose administration, patient related parameters including age, fitness, height, weight and the pathological condition. These parameters and their influence are well known to those skilled in the art and can be determined by any known method.

An amino acid or nucleic acid sequence of the invention may be administered in an amount of from 0.1 mg / kg to 15 mg / kg, preferably from 1 mg / kg to 5 mg / kg, and even more preferably ranging from 2 mg / kg to 3 mg / kg body weight.

A pharmaceutical composition that is suitable for the invention may comprise at least one amino acid or nucleic acid sequence of the invention, or at least one expression vector according to the invention in an amount ranging from 0.1 mg / kg at 15 mg / kg, preferably from 1 mg / kg to 5 mg / kg, and even more preferably from 2 mg / kg to 3 mg / kg of body weight.

An amino acid or nucleic acid sequence, or an expression vector according to the invention, or a pharmaceutical composition may be administered by any suitable route, such as the oral, buccal, sub-lingual, rectal, parenteral, intraperitoneal, intradermal, transdermal, intratracheal, topical, or ophthalmic.

An amino acid or nucleic acid sequence or an expression vector according to the invention, or a pharmaceutical composition of the invention may preferably be administered by injection, such as an intravenous route, or by a mucosal route, or a combination of these pathways.

Injection administration may be effected, for example, intraperitoneally, intradermally, subcutaneously, intravenously or intramuscularly.

Any mucosal route may also be implemented, such as the genitourinary, anorectal, respiratory, oral-nasal, sublingual, or a combination thereof.

Also disclosed is a therapeutic treatment method comprising administering at least one amino acid sequence of the invention, or at least one nucleic acid sequence of the invention, or at least one expression vector of the invention, as antiproliferative therapeutic agent of cancer cells, for preventing and / or reducing and / or treating cancer. The implementation of a therapeutic treatment method of the invention makes it possible to observe a reduction, or even a disappearance, of the tumor (s) and / or the metastasis (s). Combination with other therapies

According to another aspect of the invention, an amino acid or nucleic acid sequence of the invention may be used alone or in combination with other treatments such as surgery, radiotherapy, chemotherapy, and the like. immunotherapy.

Thus, the present invention also relates to a combination product comprising at least one amino acid or nucleic acid sequence of the invention or at least one expression vector of the invention, as an antiproliferative agent for cancer cells, and least one chemotherapy agent or at least one immunotherapy agent, for simultaneous, separate or sequential use to prevent and / or reduce and / or treat cancer.

A chemotherapeutic agent that is suitable for the invention may be selected from cytostatic agents, antimetabolites, DNA intercalators, topoisomerase I and II inhibiting compounds, tubulin inhibitors, alkylating agents, neocarcinostatin, calichéamicine. , dynemicine or aspiramicin A, ribosome inhibitor compounds, tyrosine phosphokinase inhibitors, cell differentiation inducing compounds, or histone deacetylase inhibitors.

Even more particularly, a chemotherapeutic agent that is suitable for the invention may be chosen from cytostatics and antimetabolites, such as 5-fluorouracil, 5-fluoro-cytidine, 5-fluorouridine, cytosine arabinoside or methotrexate. among intercalating substances of DNA, such as doxorubicin, daunomycin, idarubicin, epirubicin or mitoxantrone, among topoisomerase I and II inhibiting compounds, such as camptothecin, etoposide or m -AMSA, among tubulin inhibitors, such as vincristine, vinblastine, vindesine, taxol, nocodazole or colchicine, among alkylating agents, such as cyclophosphamide, mitomycin C, rachelmycin, cisplatin, phosphoramide mustard gas, melphalan, bleomycin, N-bis (2-chloroethyl) -4-hydroxyaniline, or among neocarcinostatin, calichéamicine, dynémicine or espéramicine A, or among ribosin inhibiting compounds omega, such as verrucarin A, among tyrosine phosphokinase inhibitors, such as quercetin, genistein, erbstatin, tyrphostin or rohitukin and its derivatives, among compounds inducing cell differentiation, such as retinoic acid; , butyric aid, esters of phorbol or aclacinomycin, among histone deacetylase inhibitors, such as CI-994 or MS275.

An amino acid or nucleic acid sequence of the invention, or an expression vector of the invention may also be used in combination with a radiotherapy treatment.

A radiotherapy treatment that may be suitable for the invention may be external radiotherapy using an external source of X-rays, brachytherapy consisting in bringing the radioactive source into contact with the tumor, or vector-borne metabolic radiotherapy. These methods are known to those skilled in the art.

An immunotherapeutic treatment that is suitable for the invention may comprise the administration of at least one immunotherapy agent chosen from cytokines, such as interleukin-2, cells of the immune system, in particular the immune cells of the patient, which have been activated. and reinjected to the latter, or antibodies directed against cancer cells, such as anti-Her2-neu or anti-CD20 antibodies.

Cancer

An amino acid or nucleic acid sequence of the invention, or an expression vector of the invention, or a pharmaceutical composition of the invention prove to be particularly advantageous for reducing or inhibiting the proliferation of cancer cells. .

As shown by the examples, an amino acid sequence of the invention inhibits or significantly reduces cancer cell proliferation, tumor growth, and metastasis development.

An amino acid or nucleic acid sequence of the invention, or an expression vector of the invention, are advantageously used as an antiproliferative agent for cancer cells expressing at least one receptor of the TAM family. (Tyro-3, Axl or Sea).

According to another embodiment, an amino acid or nucleic acid sequence of the invention, or an expression vector of the invention, can be used as an antiproliferative agent of cancer cells overexpressing at the same time. least one receiver of the TAM family.

For "overexpression" of a receptor, is meant, for the purposes of the invention, to designate a level of expression of this receptor greater than that observed on average in non-cancerous cells. The expression level of the receptor can be increased by approximately more than 10 time, preferably about more than 100-fold, and more preferably about 1000-fold more than the average expression level in a non-cancer cell. A non-cancer cell that can be used as a reference for determining an average level of expression may be a non-cancer cell derived from the same type of tissue or organ from which the cancer cell is derived. The reference organ or tissue may be from the same individual as that with cancer or metastases, or from a separate individual presumed to have no cancer or metastasis.

It is possible to determine the expression of at least one receptor of the TAM receptor family in cancer cells by conventional molecular biology tests such as immunolistochemistry, in situ hybridization and Reverse Transcriptase Polymerase Chain Reaction. quantitative (RT-qPCR).

According to another embodiment, an amino acid or nucleic acid sequence of the invention, or an expression vector of the invention, can be used as an antiproliferative agent for cancer cells. to prevent, reduce or treat cancer having a natural or acquired resistance to chemotherapeutic or immunotherapeutic treatment, such as those usually employed.

According to another embodiment, an amino acid or nucleic acid sequence of the invention, or an expression vector of the invention can be used as antiproliferative agent of cancer cells to prevent, reduce or treat a metastasis, and in particular a metastasis whose cells express or overexpress at least one receptor of the TAM receptor family.

According to another embodiment, an amino acid or nucleic acid sequence of the invention, or an expression vector of the invention may be used as antiproliferative agent of cancer cells selected from cancerous cells for which expression or overexpression of TAM receptors has been observed, and in particular cancer cells of breast cancer, colorectal cancer, esophageal cancer, stomach cancer, digestive stromal tumors ( GIST), brain tumors (astrocytoma), liver cancer, lung cancer, skin cancer, ovarian cancer, osteosarcoma, pancreatic cancer, kidney cancer, prostate cancer thyroid cancer, endometrial cancer, Kaposi's sarcoma, glioma or leukemia (Verma et al., Mol Cancer Ther., 201 1, Sep 20).

According to another embodiment, the invention may be more particularly suitable for preventing and / or reducing and / or treating a melanoma, or metastases associated with melanoma or other metastatic cancers in the lungs such as breast, colon, prostate, pancreatic, kidney, thyroid, stomach, and ovarian cancers, melanomas , sarcomas, gliomas or leukemias. For the purposes of the present invention, "a" must be understood, unless otherwise indicated, in the sense of "at least one". Also, within the meaning of the invention, the values of the limits of an interval defined by the expression "between ... and ..." are included in the interval.

The examples and figures below are presented solely by way of illustration of the invention.

Legends of the figures

Figure 1: illustrates the inhibition of human protein S-induced (control) human growth in vitro, 1 or 10 μg / ml, applied to skin tumor cells in culture for 24 hours (16F10 B cells) . The proliferation of cells is evaluated by the BrdU incorporation method.

FIG. 2 illustrates the inhibition of tumor growth in vivo by the human protein S, administered intraperitoneally, at 0 (placebo, n = 6) or 2.5 mg / kg (protein S, n = 6), determined in 9-10 week old male C57 / BL / 6 mice injected subcutaneously with 1 x 10 ⁶ of skin cancer cells (melanoma) called B16 / F10 and isolated from spontaneous mouse melanoma (commercial source : American Type Culture Collection / ATCC reference: CRL-6475). The mice were treated for 13 consecutive days, then sacrificed and the tumors were removed and weighed.

Figure 3: shows the in vivo inhibition of metastasis growth by human protein S, administered intraperitoneally, at 0 (placebo, n = 10) or 2.5 mg / kg (protein S, n = 6), determined on 9-10 week old male C57 / BL / 6 mice injected intravenously with 2x10 ⁵ lung cancer cells called LLC (Lewis Lung Carcinoma) initially isolated from mice (Bertram and Janik, Cancer Lett, 1980, 11: 63-73). The mice were treated for 14 consecutive days, then sacrificed, and the lungs were removed and weighed, and the total number of metastatic nodules counted.

Figure 4: Shows the survival rate of 9-10 week old male C57 / BL / 6 mice carrying lung metastases formed by the injection of 2x10 ⁵ lung cancer cells called CLL isolated from mice (Bertram and Janik, Cancer). Lett., 1980, 11: 63-73) induced by human protein S, administered intraperitoneally, at 0 (placebo, n = 5) or 2.5 mg / kg (protein S, n = 5). The chart shows the percentage of live mice for a period of 35 days.

Figure 5: illustrates the safety of human protein S administered intraperitoneally, at 0 (placebo, n = 5) or 2.5 mg / kg (protein S, n = 5), in C57 / BL / 6 mice males 9-10 weeks old. The diagram shows the variation of the weight of the mice (in g) treated for 14 consecutive days.

EXAMPLES

Example 1

In vitro inhibition of tumor growth by human protein S B16 / F10 cutaneous cancer cells (commercial source: American Type Culture Collection / ATCC reference: CRL-6475) were inoculated into 96-well plates at ³ × 10 ³ cells / well / 100 μl of RPMI basal medium supplemented with fetal calf serum at a final concentration of 10%. The cells are cultured in a CO ₂ incubator (5% final) at 37 ° C. 24 hours later, the medium is removed and replaced with serum-free RPMI containing bromodeoxyuridine (BrdU) at a final concentration of 0.1 mg / ml and two concentrations of human protein S (Enzyme Research Laboratories; UK). , namely 1 μg / ml and 10 μg / ml. The cells were incubated for 24 hours and then the percentage of BrdU incorporated into the cancer cells is determined according to the protocol provided by the reseller (Roche Diagnostics France, France, catalog number 11669915001).

As shown in FIG. 1, the levels of BrdU incorporated by the cancer cells treated with 1 μg / ml or 10 μg / ml were reduced, respectively, to approximately 70% and 60% of the control value.

These results show that the treatment of cancer cells with the protein S inhibited their proliferation, and all the more efficiently as the concentration of protein S is high. In addition, the observation of this effect on cells in culture demonstrates that it is obtained independently of a possible inhibitory action of angiogenesis.

Example 2

In vivo inhibition of tumor growth by human protein S Subcutaneous tumors were induced in twelve 9-10 week old male C57 / BL / 6 mice randomized into two groups of six animals (Griswold, Cancer Chemother Rep, 1972, 3, 315-324).

These groups were treated by daily intraperitoneal injection of human protein S at 2.5 mg / kg (protein S), or saline (control) for thirteen consecutive days.

At the end of the treatment, the mice were euthanized by exsanguination after the animals were rendered unconscious by the injection of an intraperitoneal anesthetic (chloral hydrate, 400 mg / kg) according to the European regulation (European Communities council directive of 24 November 1986 - 86/609 / EEC), and the tumors were removed and weighed.

As illustrated in Figure 2, the tumor weight for the "placebo" group is between about 1400 mg and 2300 mg while the tumor weight for the "Protein S" group is between about 700 mg and 1500 mg. Thus, the treatment of mice with the S protein led to a significant decrease in tumor weight of nearly 40%.

Example 3

In vivo inhibition of growth of pulmonary metastases by human protein S

2x10 ⁵ of pulmonary cancer cells called LLCs initially isolated from mice (Bertram and Janik) in 100 μl of phosphate buffered saline (PB S, phosphate buffered saline) were injected intravenously into sixteen male C57 / BL / 6 mice. 9- 10 weeks, randomized into two groups.

These groups were treated by daily intraperitoneal injection of protein S at 2.5 mg / kg (protein S, n = 6), or saline (control, n = 10) for fourteen consecutive days.

At the end of the treatment, the mice were euthanized by exsanguination after the animals were rendered unconscious by the injection of an intraperitoneal anesthetic (chloral hydrate, 400 mg / kg) according to the European regulation (European Communities council directive of 24 November 1986 - 86/609 / EEC), and the lungs were removed and weighed. The total number of metastatic nodules is counted by hand counting using a binocular microscope. As shown in Figure 3, the number of nodules for the "placebo" group is between about 60 and 220 while the number of nodules for the "protein S" group is between about 40 and 110. These results indicate that the number of nodules for the "placebo" group is between about 60 and 110. Pulmonary metastasis was significantly reduced by treatment with protein S.

Example 4

Survival rate of mouse with lung metastases by human protein S

2x10 ⁵ of lung cancer cells called CLL isolated initially from mice (Bertram and Janik, Cancer Lett, 1980, 11: 63-73) in 100 μl of phosphate buffered saline (PBS, Phosphate-buffered saline) were injected by intravenously in ten male C57 / BL / 6 mice 9-10 weeks, randomized into two groups, each consisting of five mice.

One group was treated by intraperitoneal injection of a saline solution ("placebo") and the other by a solution of human protein S at 2.5 mg / kg ("protein S"). The treatment is administered every two days until the death of the mice.

Figure 4 shows that after a period of 25 days, none of the mice of the "placebo" group survived, whereas after that same period, 40% of the mice that received the human protein S remained alive. And from 26 ^th to 32 ^th day, 20% of mice survived yet. The treatment of mice with lung metastases with human protein S significantly increased their survival in the placebo group.

Example 5

Safety of human protein S

Six 9-10 week old male C57 / BL / 6 mice, randomized into two groups, each consisting of three mice, were treated with daily intraperitoneal administration, saline (placebo) or 2.5 mg / kg human protein S (protein

S), for fourteen consecutive days. During this period the weight of the mice was measured every other day using a scale.

Figure 5 indicates that the weight of the mice varies substantially in the same way between the "protein S" group and the "placebo" group. No signs of weakening of the body, that is, cachexia, have been observed.

At the end of the treatment, the mice were euthanized by exsanguination after the animals were rendered unconscious by injection of anesthetic intraperitoneal (chloral hydrate, 400 mg / kg) according to EU regulations (European Communities Council Directive of 24 November 1986 - 86/609 / EEC), and a visual examination of the abdominal cavity was performed to find the presence of fluid in the abdomen. The absence of observation of inflammatory reaction or ascites in the abdominal cavity indicates a good tolerance of the protein S during the treatment.

The various results obtained above demonstrate that the protein S strongly and reproducibly inhibits proliferation and tumor growth, independently of a possible anti-angiogenic effect. Moreover, the protein S is particularly devoid of toxicity or side effect on cells or healthy organism.

Thus, all of these data make protein S a therapeutic asset of particular interest for the treatment, prevention or reduction of pathologies involving the proliferation of cancer cells, such as lung cancer, melanomas, gliomas, or leukaemias, as well as metastases.

BIBLIOGRAPHY

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SEQUENCE LISTING

SEQ ID NO: 1 (NCBI NP 000304.2)

MRVLGGRCGALLACLLLVLPVSEANFLSKQQASQVLVRKRRANSLLEETKQGNLERECIEELCNKEEARE VFENDPETDYFYPKYLVCLRSFQTGLFTAARQSTNAYPDLRSCVNAIPDQCSPLPCNEDGYMSCKDGKAS FTCTCKPGWQGEKCEFDINECKDPSNINGGCSQICDNTPGSYHCSCKNGFVMLSNKKDCKDVDECSLKPS ICGTAVCKNIPGDFECECPEGYRYNLKSKSCEDIDECSENMCAQLCVNYPGGYTCYCDGKKGFKLAQDQK SCEWSVCLPLNLDTKYELLYLAEQFAGWLYLKFRLPEISRFSAEFDFRTYDSEGVILYAESIDHSAWL LIALRGGKIEVQLKNEHTSKITTGGDVINNGLWNMVSVEELEHSISIKIAKEAVMDINKPGPLFKPENGL LETKVYFAGFPRKVESELIKPINPRLDGCIRSWNLMKQGASGIKEI IQEKQNKHCLVTVEKGSYYPGSGI AQFHIDYNNVSSAEGWHVNVTLNIRPSTGTGVMLALVSGNNTVPFAVSLVDSTSEKSQDILLSVENTVIY RIQALSLCSDQQSHLEFRVNRNNLELSTPLKIETISHEDLQRQLAVLDKAMKAKVATYLGGLPDVPFSAT PVNAFYNGCMEVNINGVQLDLDEAISKHNDIRAHSCPSVWKKTKNS

SEQ ID NO: 2 (NCBI NM 000313.3 Region 342-2372)

ATGAGGGTCCTGGGTGGGCGCTGCGGGGCGCTGCTGGCGTGTCTCCTCCTAGTGCTTCCCGTCTCAGAGG CAAACTTTTTGTCAAAGCAACAGGCTTCACAAGTCCTGGTTAGGAAGCGTCGTGCAAATTCTTTACTTGA AGAAACCAAACAGGGTAATCTTGAAAGAGAATGCATCGAAGAACTGTGCAATAAAGAAGAAGCCAGGGAG GTCTTTGAAAATGACCCGGAAACGGATTATTTTTATCCAAAATACTTAGTTTGTCTTCGCTCTTTTCAAA CTGGGTTATTCACTGCTGCACGTCAGTCAACTAATGCTTATCCTGACCTAAGAAGCTGTGTCAATGCCAT TCCAGACCAGTGTAGTCCTCTGCCATGCAATGAAGATGGATATATGAGCTGCAAAGATGGAAAAGCTTCT TTTACTTGCACTTGTAAACCAGGTTGGCAAGGAGAAAAGTGTGAATTTGACATAAATGAATGCAAAGATC CCTCAAATATAAATGGAGGTTGCAGTCAAATTTGTGATAATACACCTGGAAGTTACCACTGTTCCTGTAA AAATGGTTTTGTTATGCTTTCAAATAAGAAAGATTGTAAAGATGTGGATGAATGCTCTTTGAAGCCAAGC ATTTGTGGCACAGCTGTGTGCAAGAACATCCCAGGAGATTTTGAATGTGAATGCCCCGAAGGCTACAGAT ATAATCTCAAATCAAAGTCTTGTGAAGATATAGATGAATGCTCTGAGAACATGTGTGCTCAGCTTTGTGT CAATTACCCTGGAGGTTACACTTGCTATTGTGATGGGAAGAAAGGATTCAAACTTGCCCAAGATCAGAAG AGTTGTGAGGTTGTTTCAGTGTGCCTTCCCTTGAACCTTGACACAAAGTATGAATTACTTTACTTGGCGG AGCAGTTTGCAGGGGTTGTTTTATATTTAAAATTTCGTTTGCCAGAAATCAGCAGATTTTCAGCAGAATT TGATTT CCGGACATATGATTCAGAAGGCGTGATACTGTACGCAGAATCTATCGATCACTCAGCGTGGCTC CTGATTGCACTTCGTGGTGGAAAGATTGAAGTTCAGCTTAAGAATGAACATACATCCAAAATCACAACTG GAGGTGATGTTATTAATAATGGTCTATGGAATATGGTGTCTGTGGAAGAATTAGAACATAGTATTAGCAT TAAAATAGCTAAAGAAGCTGTGATGGATATAAATAAACCTGGACCCCTTTTTAAGCCGGAAAATGGATTG CTGGAAACCAAAGTATACTTTGCAGGATTCCCTCGGAAAGTGGAAAGTGAACTCATTAAACCGATTAACC CTCGTCTAGATGGATGTATACGAAGCTGGAATTTGATGAAGCAAGGAGCTTCTGGAATAAAGGAAATTAT TCAAGAAAAACAAAATAAGCATTGCCTGGTTACTGTGGAGAAGGGCTCCTACTATCCTGGTTCTGGAATT GCTCAATTTCACATAGATTATAATAATGTATCCAGTGCTGAGGGTTGGCATGTAAATGTGACCTTGAATA TTCGTCCATCCACGGGCACTGGTGTTATGCTTGCCTTGGTTTCTGGTAACAACACAGTGCCCTTTGCTGT GTCCTTGGTGGACTCCACCTCTGAAAAATCACAGGATATTCTGTTATCTGTTGAAAATACTGTAATATAT CGGATACAGGCCCTAAGTCTATGTTCCGATCAACAATCTCATCTGGAATTTAGAGTCAACAGAAACAATC TGGAGTTGTCGACACCACTTAAAATAGAAACCATCTCCCATGAAGACCTTCAAAGACAACTTGCCGTCTT GGACAAAGCAATGAAAGCAAAAGTGGCCACATACCTGGGTGGCCTTCCAGATGTTCCATTCAGTGCCACA CCAGTGAATGCCTTTTATAATGGCTGCATGGAAGTGAATATTAATGGTGTACAGTTGGATCTGGATGAAG CCATTTCTAAAC ATAATGATATTAGAGCTCACTCATGTCCATCAGTTTGGAAAAAGACAAAGAATTCTTA A

Claims

An isolated amino acid sequence consisting of SEQ ID NO: 1, a homologue thereof having a homology of at least 80% with said sequence and a similar biological activity, or an active fragment consisting of 280 to 675 contiguous amino acids of said sequence or said homologue, for use as an antiproliferative agent of cancer cells for preventing and / or reducing and / or treating cancer.

The sequence for use according to claim 1, said sequence being the amino acid sequence of the human protein S.

3. Sequence for use according to claim 1 or 2, as an antiproliferative agent of cancer cells expressing at least one receptor of the TAM family.

4. Sequence for use according to any one of the preceding claims preventing, reducing or treating cancer having a natural or acquired resistance to chemotherapeutic or immunotherapeutic treatment.

A sequence for use according to any one of the preceding claims for preventing and / or reducing and / or treating a metastasis.

A sequence for use according to any one of the preceding claims, as an antiproliferative agent of cancer cells selected from cancer cells of breast cancer, colorectal cancer, esophageal cancer, stomach cancer, digestive stromal tumors, brain tumors, liver cancer, lung cancer, skin cancer, ovarian cancer, osteosarcoma, pancreatic cancer, kidney cancer, prostate cancer, thyroid cancer, endometrial cancer, and Kaposi's sarcoma.

Sequence for use according to any one of the preceding claims, said sequence being administered in an amount of from 0.1 mg / kg to 15 mg / kg, preferably from 1 mg / kg to 5 mg / kg, and even more preferably ranging from 2 mg / kg to 3 mg / kg body weight.

An isolated nucleic acid sequence encoding an amino acid sequence as defined for use according to claim 1 or 2 for use as an antiproliferative agent of cancer cells for preventing and / or reducing and / or treating cancer .

An expression vector comprising a nucleic acid sequence as defined for use according to claim 8 for use as an antiproliferative agent of cancer cells for preventing and / or reducing and / or treating cancer.

A pharmaceutical composition comprising, in a pharmaceutically acceptable medium, at least one amino acid sequence as defined in claim 1 or 2, or at least one nucleic acid sequence as defined in claim 8, or at least one Expression vector as defined in claim 9 for use as an antiproliferative agent of cancer cells for preventing and / or reducing and / or treating cancer.

A combination product comprising at least one amino acid sequence as defined in claim 1 or 2, or at least one nucleic acid sequence as defined in claim 8, or at least one expression vector such as defined in claim 9, for use as an antiproliferative agent of cancer cells, and at least one chemotherapeutic agent or at least one immunotherapeutic agent, for simultaneous, separate or sequential use to prevent and / or reduce and / or treat a Cancer.