Compound with anti-metastasis effect

Abstract

The present invention relates to the medical use of compounds in the treatment of tumor metastasis.

Description

Technical Field

[0001] The present invention can be applied to the medical field, especially for treating tumors by stimulating the immune system. Background Art

[0002] Small molecules from several natural and synthetic sources have been used to treat tumors. These molecules act on tumor cells by inhibiting specific signaling pathways or showing cytotoxic activity (mitotic inhibitors, microtubule formation inhibitors, DNA repair system inhibitors, etc.). However, all these small molecules are not specific to tumor cells and have high systemic toxicity, causing severe side effects, leading to serious diseases in patients and poor prognosis. In recent decades, to minimize or eliminate this problem, the use of monoclonal antibodies targeting specific receptors on tumor cells has been introduced in the treatment of cancer patients. Although this approach has achieved some success, problems related to therapy specificity still exist. In fact, the targets of monoclonal antibodies used for tumor treatment are present not only in tumor cells but also in other healthy body tissues / organs. Therefore, in this case, the therapy also causes severe side effects to patients. Nevertheless, in the past decade, to improve its therapeutic effect, a new generation of monoclonal antibodies related to cytotoxic molecules has continued to be produced, while at the same time, the non-specific damage to non-diseased tissues of patients has also increased. Recently, a new tumor treatment method based on immunotherapy has been introduced. In particular, the patient's T lymphocytes are collected and genetically modified to enhance their ability to specifically recognize and kill tumor cells. This strategy is called CAR-T (chimeric antigen receptor T cells). However, this therapy is very expensive, largely depends on the characteristics of the facilities implementing the therapy, and last but not least, its success strictly depends on the characteristics of the patients being treated. CAR-T therapy also shows non-negligible side effects because the target molecules recognized by genetically modified T lymphocytes are not only present on tumor cells. Recently, still in its embryonic stage, a new strategy has been proposed that can stimulate macrophages (a special type of immune system cell). The goal is to endow macrophages with anti-tumor characteristics and enable them to specifically activate other components of the immune system to fight against abnormal cells.

[0003] International Patent Application WO 2018 / 069831 describes the use of the β subunit of the follicle-stimulating hormone receptor and the recombinant subunit for the treatment and diagnosis of tumors; this document does not describe the use of the subunit for the treatment and / or prevention of metastasis. Summary of the Invention

[0004] The present invention is based on the surprising discovery of a compound capable of reprogramming macrophages in an anti-tumor sense.

[0005] In particular, it has surprisingly been found that this compound can be used for the treatment and / or prevention of the formation of metastases caused by primary tumors.

[0006] Object of the Invention

[0007] A first object of the present invention is represented by the medical use of a compound for the treatment and / or prevention of tumor metastases.

[0008] In a preferred aspect, this compound for the treatment and / or prevention of tumor metastases is recombinant (ABRβ).

[0009] In a particular aspect of the present invention, the described medical use for the treatment and / or prevention of tumor metastases occurs by stimulating the immune system.

[0010] In another aspect of the present invention, the described medical use for the treatment and / or prevention of tumor metastases occurs because tumor-associated macrophages M2 (TAM-M2) are reprogrammed into tumor-associated macrophages M1 (TAM-M1).

[0011] In a second object, the present invention describes a pharmaceutical preparation comprising said compound.

[0012] In another object, a method for the treatment and / or prevention of tumor metastases is described, comprising administering a pharmaceutically effective amount of the compound of the present invention to a subject in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Shows the effect of ABRβ1l on macrophage IL10 production: anti-tumor macrophage M1, pro-tumor macrophage M2, untreated NT, lipopolysaccharide LPS.

[0014] Figure 2 Shows the effect of ABRβ1 on macrophage IL12 production: anti-tumor macrophage M1, pro-tumor macrophage M2, untreated NT, lipopolysaccharide LPS.

[0015] Figure 3 Shows the effect of ABRβ1 on the survival rate of a syngeneic mouse model of melanoma. DETAILED DESCRIPTION

[0016] According to the first object of the present invention, the medical use of a compound for the treatment and / or prevention of tumor metastases is described.

[0017] According to a preferred aspect of the present invention, this compound is represented by the β subunit of follicle-stimulating hormone (FSH) (SEQ.ID NO:1) or is the β subunit of recombinant follicle-stimulating hormone (FSH) described herein for the treatment and / or prevention of tumor metastases.

[0018] Recombinant compound (ABRβ)

[0019] For the purposes of the present invention, the term "ABRβ" refers to the β subunit of human follicle-stimulating hormone (FSH) obtainable by using the biotechnology platform described below.

[0020] "ABRβ1" refers to the specific β subunit of human follicle-stimulating hormone (FSH) obtained according to the present invention by using a biotechnology platform for producing Nicotiana benthamiana in plant cells.

[0021] In particular, this subunit is characterized by an amino acid sequence corresponding to SEQ.ID NO:2.

[0022]

[0023] The biotechnology platform for preparing the β subunit of human follicle-stimulating hormone (FSH) involves the use of transformed cells.

[0024] According to an aspect of the present invention, such cells can be plant cells.

[0025] According to a preferred aspect of the present invention, such cells are, for example, Nicotiana benthamiana (ABRβ1) cells.

[0026] In particular, the ABRβ1 recombinant compound of the present invention is glycosylated at asparagine residues 13 and 30 of the mature protein.

[0027] More specifically, the glycosylation sites include a branched structure of mannose residues.

[0028] The total number of mannose residues is about 45 - 75, preferably about 50 - 70, and even more preferably about 58 - 62, where they can be 60 or 61.

[0029] Each glycosylation site includes a branched structure of two N-acetylglucosamine residues and mannose residues.

[0030] In particular, each branched structure includes 29, 30, or 31 mannose residues.

[0031] Each mannose residue can include phosphorylation or sulfation or methylation.

[0032] In addition, the polysaccharide moiety can be linked to a molecule containing a phenolic group.

[0033] The molecule containing a phenolic group is characteristic of plant cells.

[0034] In particular, these phenolic groups are typical features of Nicotiana benthamiana plant cells.

[0035] In particular, the method for preparing the ABRβ1 subunit comprises modifying the C-terminus of the β-subunit of human follicle-stimulating hormone (FSHβ) (SEQ.ID NO:1) with a KDEL sequence and modifying the N-terminus with a 6-histidine tail (His-tag).

[0036] For example, the platform that allows the preparation of the subunits of the present invention is described in international patent application WO 2018 / 069831.

[0037] More specifically, the method for preparing the recombinant form of the β-subunit follicle-stimulating hormone (FSH) comprises the following steps:

[0038] I) Obtaining a suitable vector transformed with a plasmid containing the sequence corresponding to SEQ.ID NO.3;

[0039] II) Transforming plant cells with the vector of step I);

[0040] III) Selecting the transformed plant cells;

[0041] IV) Culturing the stable plant cells;

[0042] V) Preparing a cell extract;

[0043] VI) Purifying the compound.

[0044] In a preferred aspect, the vector of step I) is represented as Agrobacterium tumefaciens.

[0045] The sequence for transformation corresponds to SEQ.ID NO.3:

[0046]

[0047] For the purposes of the present invention, step II) comprises transforming Nicotiana benthamiana plant cells.

[0048] In particular, in step II), the transformation is a co-culture carried out in the dark at about 25 °C and with continuous stirring for 48 hours.

[0049] The cells are then screened.

[0050] Preferably, in step III), a selective medium is used, comprising: MS supplemented with 0.9% w / v agar and antibiotics.

[0051] In a preferred aspect, carbenicillin and kanamycin are used for this purpose, more preferably 250 mg / L carbenicillin and 100 mg / L kanamycin.

[0052] In a preferred aspect of the present invention, in step IV), cells that may be Nicotiana benthamiana are cultured in suspension.

[0053] In another preferred aspect, the culture includes an initial inoculation of plant cells, which may be plant cells of Nicotiana benthamiana, equivalent to 10% of the final culture volume.

[0054] The cell culture is incubated in MS medium (Murashige 1962) supplemented with sucrose, naphthalene acetic acid (NAA), and kinetin at a temperature of 24 - 27 °C for a period of 15 days, and maintained with aeration at 50 - 100 mbar.

[0055] In addition, subcultures are established every 7 days by transferring an aliquot of the cell suspension to fresh medium.

[0056] The cells are incubated with stirring, in the dark, and at a constant temperature of 25 °C.

[0057] According to the present invention, step V) includes using an extraction buffer that contains: 50 mM Na2HPO4, 150 mM NaCl, 20 mM citric acid, 40 mM ascorbic acid, 5 mM EDTA, 1 mM PMSF, 0.05% (v / v) Tween - 20, pH 6.5, and adding 1% (w / v) XAD - 4 and 1% (w / v) polyvinylpyrrolidone (PVPP).

[0058] Then, ammonium sulfate is added to the extract until a saturation concentration of 70% is obtained, and incubated at 4 °C with continuous stirring for 1 hour.

[0059] Then the precipitate is recovered by centrifugation and resuspended in IMAC buffer.

[0060] The preparation is centrifuged and filtered.

[0061] In step VI), the solution thus obtained is purified by column passage.

[0062] In particular, the solution is loaded onto an IMAC chromatography column.

[0063] Preferably, a Ni Sepharose 6FF column is used.

[0064] Then the fractions of interest are collected and loaded onto a desalting column.

[0065] Preferably, a Sephadex G-25 medium column is used.

[0066] Then, the fractions of interest are collected and loaded onto an ion exchange chromatography column.

[0067] Preferably, an SP Sepharose HP column is used.

[0068] During the purification process, the absorbance is monitored at 280 nm and 254 nm.

[0069] As described above, the ABRβ1 subunit is obtained by biotechnology (recombinant) from Nicotiana benthamiana plant cell cultures in suspension.

[0070] According to an alternative aspect, the ABRβ subunit of the present invention can be obtained by biotechnology in other cells, such as in mammalian, yeast, bacterial, or other plant cells.

[0071] In particular, cells of Daucus carota, Oryza sativa, Glycine max, maize, etc. can be used in plant cells.

[0072] For the purposes of the present invention, the term "treatment" of tumor metastasis refers to a treatment regimen for patients with primary tumors with or without metastases.

[0073] In a preferred aspect of the present invention, such primary tumors are represented by melanoma, breast tumors, ovarian tumors, sarcomas, pancreatic tumors, kidney tumors, gastric tumors, lung tumors, neuroblastomas (even in childhood).

[0074] For the purposes of the present invention, the term "tumor metastasis" refers to tumors, particularly solid, secondary tumors originating from a primary tumor.

[0075] In a preferred aspect of the present invention, such metastases are metastases originating from melanoma.

[0076] In an even more preferred aspect of the present invention, the metastases are lung metastases, peritoneal metastases, brain metastases, kidney metastases, liver metastases.

[0077] In another aspect, the present invention describes the medical use of a potentially recombinant compound (ABRβ) for preventing metastasis.

[0078] Therefore, the compounds described in the present invention have anti-metastatic properties.

[0079] This means that, in patients suffering from a primary tumor, the recombinant compound (ABRβ) of the present invention can not only cause the regression of metastases that have already formed in the primary tumor (therapeutic effect), but also prevent their formation.

[0080] According to a preferred aspect of the present invention, the medical use of the compounds of the present invention for the treatment and / or prevention of tumor metastasis is described, wherein such treatment and / or prevention occurs by stimulating the immune system.

[0081] In another aspect of the present invention, the medical use for the treatment and / or prevention of tumor metastasis is described, wherein tumor-associated macrophages M2 (TAM-M2) are reprogrammed into tumor-associated macrophages M1 (TAM-M1).

[0082] In a second object, the present invention describes a pharmaceutical preparation comprising one of the described compounds.

[0083] In particular, the preparation is administered intravenously.

[0084] According to a specific aspect of the present invention, such preparations may comprise the compounds of the present invention (possibly in recombinant form) and one or more pharmaceutically acceptable carriers and / or excipients.

[0085] In a preferred aspect of the present invention, such compounds may optionally be conjugated with a suitable molecule having therapeutic activity.

[0086] These molecules may be selected from the group of molecules used for the treatment of specific forms of tumors.

[0087] More specifically, such preparations are formulated for intravenous administration.

[0088] In another object, the present invention describes a method for the treatment and / or prevention of tumor metastasis, comprising administering to a subject in need a pharmaceutically effective amount of a compound of the present invention.

[0089] In particular, this method comprises reprogramming tumor-associated M2 macrophages (TAM-M2) into M1 tumor-associated macrophages (TAM-M1).

[0090] In one aspect of the present invention, the production of IL12 is induced in M2-type macrophages.

[0091] This induction is greater than the production of IL12 in M1-type macrophages.

[0092] Example 1

[0093] Effect of ABRβ1 on the production of IL10 in macrophages

[0094] Isolation of Human Monocytes

[0095] To prepare monocytes from human peripheral blood, collect 2 - 4 bags of fresh plasma (buffy coat) and transfer it to a 50 ml sterile tube. Centrifuge at 3000 x g for 15 minutes at room temperature to remove residual platelets and collect all the supernatant. At room temperature, prepare a 50 ml falcon tube with 15 ml of Ficoll solution (1.077 g / ml) at the bottom. For each falcon tube, very gently deposit 30 - 35 ml of the buffy coat on top of the Ficoll solution to form the first density gradient. Centrifuge at 400 x g for 30 minutes at room temperature and decelerate extremely slowly to prevent remixing. Collect the white ring of peripheral blood mononuclear cells (PBMCs) formed between the two phases in each 50 ml falcon tube and transfer it to a new sterile container. At this point, add an excess of PBS - EDTA (1 mM) to the collected cells and centrifuge at 300 x g for 10 minutes at room temperature. Remove the supernatant and repeat the procedure to wash the collected cells. At this time, resuspend the washed cell pellet in RPMI - 1640 medium without phenol red and supplemented with 10% heat - inactivated FCS. Meanwhile, prepare an isotonic Percoll solution for the second density gradient. To do this, mix the solutions in the following ratio: 23.13 ml of Percoll solution (density 1.131 g / ml) and 1.87 ml of 10X PBS. Transfer 23 ml of this solution to a 50 ml test tube and add 27 ml of RPMI - 1640 with phenol red and supplemented with heat - inactivated FCS (final 10%). All operations are carried out at room temperature. Place 25 ml of the newly prepared solution into a 50 ml test tube and very slowly deposit the PBMCs on top of this solution to avoid remixing. At this time, centrifuge at 550 x g for 30 minutes at room temperature and slow down very slowly. Gently collect the white ring of monocytes located between the two phases of different densities and transfer it to a new 50 ml test tube, add PBS - EDTA (1 mM), and centrifuge at 400 x g for 10 minutes at room temperature. Remove the supernatant and resuspend the cell pellet in RPMI - 1640 medium with phenol red and supplemented with heat - inactivated FCS to a final concentration of 10%.

[0096] In Vitro Culture and Differentiation of Human Monocytes

[0097] First, it is necessary to determine the concentration of monocytes in the resuspension solution after isolation from human plasma, and thus count the resuspended cells using the vital dye trypan blue. Then, the monocytes are seeded in culture dishes using a medium consisting of RPMI-1640, 2% AB human serum, and 1% penicillin / streptomycin. The cultures are incubated at 37 °C in a 5% CO incubator for several days before inducing differentiation. For this purpose, the monocyte cultures are incubated for 10 days in the presence of 2 ng / ml GM-CSF or 2 ng / ml M-CSF. GM-CSF differentiates monocytes into M1-type macrophages (anti-tumor), while M-CSF differentiates monocytes into M2-type macrophages (pro-tumor).

[0098] Figure 1 It is shown that, unlike M1-type macrophages, M2-type macrophages (in vitro differentiated) produce IL10 when appropriately stimulated with lipopolysaccharide (LPS). Treatment with compound ABRβ1 leads to a change in the phenotype of macrophage M2, which will lose the ability to produce IL10 if stimulated. This characteristic, instead, is typical of M1-type macrophages.

[0099] Example 2

[0100] Effect of ABRβ1 on IL12 production in human macrophages

[0101] To determine the type of effect of compound ABRβ1 on the phenotype of in vitro differentiated human macrophages, the production of IL12, which characterizes M1-type macrophages, and the production of IL10, which is normally produced by M2-type macrophages, were analyzed. The above-mentioned interleukins were measured in the cell culture medium after different treatments using specific ELISA kits.

[0102] Figure 2 It is shown that M1-type macrophages produce IL12. Compound ABRβ1 shows a moderate effect on the production of IL12 by M1-type macrophages. In contrast, compound ABRβ1 significantly stimulates the production of IL12 in M2-type macrophages, which normally cannot do this. The data clearly show that compound ABRβ1 is able to reprogram M2-type macrophages, especially to endow them with the characteristics of M1-type macrophages.

[0103] Example 3

[0104] Effect of ABRβ1 on survival rate in a syngeneic mouse model of metastatic melanoma

[0105] To evaluate the immunostimulatory effect and anti-metastatic activity of the compound ABRβ1 in vivo, a syngeneic mouse melanoma model was selected. The immune system of the mice receiving the tumor cells was perfectly functional, and the mouse cell line used was B16F10, which is characterized by high invasiveness and a high metastasis rate. In this mouse model, animals usually die due to rapid metastases at the peritoneal or pulmonary level. For this purpose, B16F10 cells were subcutaneously inoculated into the animals. Two experimental groups were established: a control group (not receiving ABRβ1 treatment) and a group receiving ABRβ1 treatment. The compound was intraperitoneally administered to the animals twice a week at a dose of 2 mg / kg. Treatment was initiated immediately after inoculating the tumor cells into the animals. The Kaplan-Meier model was used for survival analysis.

[0106] Figure 3 It was shown that the survival time of the mice inoculated with B16F10 cells did not exceed 18 days. In contrast, the mice inoculated with tumor cells and treated with the compound ABRβ1 showed a high survival rate (80%) far beyond 18 days. Surprisingly, after the animals were sacrificed, autopsy analysis showed no metastases at the peritoneal or pulmonary level. Autopsy analysis of the dead animals not receiving ABRβ1 treatment showed extensive metastases at the peritoneal cavity and pulmonary level. This evidence clearly shows that even in a particularly aggressive tumor model such as melanoma, the compound ABRβ1 has significant anti-metastatic activity.

[0107] From the above description, the benefits provided by the present invention will be obvious to those skilled in the art.

[0108] Particularly relevant is that the subunit of the present invention has been shown to have definite and significant anti-metastatic activity, as shown by the high-efficiency data obtained on a metastatic mouse melanoma model (representing one of the most aggressive metastasis models).

[0109] In addition, the use of the subunit ABRβ1 provides high quality and biosafety, as its production process has almost no risk of contamination by viruses, oncogenes, prions, toxins or residues of dangerous reagents commonly used in the production of therapeutic proteins.

Claims

1. A compound for medical use in the treatment and / or prevention of tumor metastasis, having an amino acid sequence corresponding to SEQ. ID NO: 1 or 2.

2. The compound for medical use according to the previous claim, wherein the metastasis originates from a tumor selected from the group consisting of melanoma, breast tumor, ovarian tumor, pancreatic tumor, kidney tumor, stomach tumor, lung tumor, sarcoma, neuroblastoma.

3. The compound for medical use according to any one of the preceding claims, wherein the metastasis is lung metastasis, peritoneal metastasis, brain metastasis, kidney metastasis, liver metastasis.

4. The compound for medical use according to any one of the preceding claims, wherein the treatment and / or prevention of tumor metastasis occurs by stimulating the immune system.

5. The compound for medical use according to any one of the preceding claims, wherein tumor-associated macrophages M2 (TAM-M2) are reprogrammed into tumor-associated macrophages M1 (TAM-M1).

6. The compound for medical use according to any one of the preceding claims, the compound being obtained by a method comprising the following steps: I) Obtaining a suitable vector transformed with a plasmid containing a sequence corresponding to SEQ. ID NO: 3; II) Transforming plant cells with the vector of step I); III) Screening the transformed plant cells; IV) Culturing the stable plant cells in suspension; V) Preparing a cell extract; VI) Purifying the compound.

7. The compound for medical use according to the previous claim, wherein in step II), plant cells of Nicotiana benthamiana are transformed.

8. The compound for medical use according to claim 6 or 7, wherein the compound is represented by a sequence corresponding to SEQ. ID NO:

2.

9. A method for the treatment and / or prevention of tumor metastasis, the method comprising administering to a subject in need a pharmaceutically effective amount of a compound having an amino acid sequence corresponding to SEQ. ID NO: 1 or 2.

10. The method for the treatment and / or prevention of tumor metastasis according to claim 1, wherein tumor-associated M2 macrophages (TAM-M2) are reprogrammed into tumor-associated M1 macrophages (TAM-M1).

11. The method according to claim 9 or 10, wherein the production of IL12 is induced in M2-type macrophages.

12. The method according to the previous claim, wherein this induction is higher than the production of IL12 in M1-type macrophages.

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