Compounds with anti-metastatic effects

Abstract

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

Description

[Technical Field]

[0001] The invention finds application in the medical field, in particular in tumor therapy through stimulation of the immune system. [Background technology]

[0002] Several small molecules, both natural and synthetic, have been used in tumor therapy. These molecules exert their activity against tumor cells by inhibiting specific signaling pathways or by exhibiting cytotoxic activity (e.g., mitotic inhibitors, microtubule formation inhibitors, DNA repair system inhibitors). However, all of these small molecules are not specific to tumor cells and have high systemic toxicity, which causes severe side effects and leads to severe disease with poor prognosis in patients. In recent decades, to mitigate or eliminate this problem, the use of monoclonal antibodies directed against specific receptors on tumor cells has been introduced into the therapy of cancer patients. Although this approach has met with some success, issues regarding therapy specificity remain. In fact, the targets of monoclonal antibodies used in tumor therapy are present not only in tumor cells but also in other healthy tissues and organs. Therefore, in this case, the therapy also causes serious side effects in patients. Nevertheless, over the past decade, a new generation of monoclonal antibodies conjugated to cytotoxic molecules has been produced with the aim of increasing their therapeutic efficacy, but at the same time, nonspecific damage to non-diseased tissues in patients has also increased. Recently, a new immunotherapy-based approach to tumor treatment has been introduced. In particular, a patient's T lymphocytes are collected and genetically modified to enhance their ability to specifically recognize and kill tumor cells. This strategy is known as CAR-T (chimeric antigen receptor T cell) therapy. However, this therapy is very expensive, heavily dependent on the characteristics of the facility where it is performed, and, last but not least, its success strictly depends on the characteristics of the treated patient. CAR-T therapy also exhibits significant side effects because the target molecules recognized by genetically modified T lymphocytes are not present exclusively on tumor cells. Recently, a new strategy, still in its infancy, has been proposed to stimulate macrophages, a specific cell population of the immune system. The goal is to equip macrophages with antitumor properties so that they can specifically activate other components of the immune system against abnormal cells.

[0003] International patent application WO 2018 / 069831 describes the use of the β subunit of the follicle-stimulating hormone receptor and recombinant subunits for treating and diagnosing tumors. This document does not describe the use of the subunits for treating and / or preventing metastases. Summary of the Invention

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

[0005] In particular, it has surprisingly been found that such compounds can be used to treat and / or prevent the formation of metastases arising from primary tumors. [Problem to be solved by the invention]

[0006] A first object of the present invention is represented by the pharmaceutical use of compounds for treating and / or preventing tumor metastasis.

[0007] In a preferred embodiment, such a compound for treating and / or preventing tumor metastasis is recombinant (ABRβ).

[0008] In a particular embodiment of the present invention, the pharmaceutical uses described herein for treating and / or preventing tumor metastasis are carried out by stimulation of the immune system.

[0009] In another aspect of the present invention, the pharmaceutical use of the compounds described herein for treating and / or preventing tumor metastasis results from the reprogramming of tumor-associated macrophages M2 (TAM-M2) into tumor-associated macrophages M1 (TAM-M1).

[0010] In a second object, the present invention describes pharmaceutical formulations containing said compounds.

[0011] Of further object, methods for treating and / or preventing tumor metastasis are described, comprising administering to a subject in need thereof a pharmaceutically effective amount of a compound of the present invention. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows the effect of ABRβ1 on macrophage IL10 production: anti-tumor macrophages M1, pro-tumor macrophages M2, untreated NT, and lipopolysaccharide LPS. [Figure 2] Figure 2 shows the effect of ABRβ1 on macrophage IL12 production: antitumor macrophage M1, tumor-promoting macrophage M2, untreated NT, and lipopolysaccharide LPS. [Figure 3] FIG. 3 shows the effect of ABRβ1 on survival in a syngeneic mouse model of melanoma.

[0013] Detailed Description of the Invention Regarding the first object of the present invention, the pharmaceutical use of compounds for treating and / or preventing tumor metastasis is described.

[0014] According to a preferred embodiment of the present invention, such a compound is represented by the beta subunit of follicle-stimulating hormone (FSH) (SEQ ID NO: 1) or the beta subunit of recombinant follicle-stimulating hormone (FSH), as described herein, for treating and / or preventing tumor metastasis.

[0015] Recombinant compound (ABRβ) For the purposes of the present invention, the term "ABRβ" refers to the β subunit of human follicle-stimulating hormone (FSH) obtained by use of the biotechnology platform described below.

[0016] "ABRβ1" refers to the specific β subunit of human follicle-stimulating hormone (FSH) obtained by the use of a biotechnology platform for the production of Nicotiana benthamiana in plant cells according to the present invention.

[0017] In particular, such subunits are characterized by an amino acid sequence corresponding to SEQ ID NO:2. TIFF2025539024000001.tif22155

[0018] Biotechnology platforms for preparing the β subunit of human follicle-stimulating hormone (FSH) involve the use of transformed cells.

[0019] According to one aspect of the present invention, such cells may be plant cells.

[0020] According to a preferred embodiment of the present invention, such cells are, for example, Nicotiana benthamiana (ABRβ1) cells.

[0021] In particular, the ABRβ1 recombinant compounds of the present invention are glycosylated at asparagine residues 13 and 30 of the mature protein.

[0022] More particularly, the glycosylation site comprises a branched structure of mannose residues.

[0023] The total number of mannose residues is about 45 to 75, preferably about 50 to 70, more preferably about 58 to 62, where they may be 60 or 61.

[0024] Each glycosylation site contains a branched structure of two N-acetylglucosamine and mannose residues.

[0025] In particular, each branched structure contains 29, 30 or 31 mannose residues.

[0026] Each mannose residue may contain phosphorylations, sulfurylations or methylations.

[0027] Additionally, the polysaccharide moiety can be attached to molecules containing phenolic groups.

[0028] Molecules containing phenolic groups are characteristic of plant cells.

[0029] In particular, such phenolic groups are typical of Nicotiana benthamiana plant cells.

[0030] In particular, a method for preparing an ABRβ1 subunit, comprising modifying the C-terminus of the β subunit (SEQ ID NO: 1) of human follicle-stimulating hormone (FSHβ) with a KDEL sequence and the N-terminus with a 6-histidine tail (His-tag).

[0031] A platform allowing the preparation of the subunits of the invention is described, for example, in International Patent Application WO 2018 / 069831.

[0032] More particularly, the method for preparing recombinant beta subunit follicle-stimulating hormone (FSH) comprises: I) obtaining a suitable vector transformed with a plasmid containing a sequence corresponding to SEQ ID NO: 3; II) transforming a plant cell with the vector of step I; III) selecting transformed plant cells; IV) culturing the stabilizing plant cells; V) preparing a cell extract; purifying the compound; Includes.

[0033] In a preferred embodiment, the vector of step I) is expressed by Agrobacterium tumefaciens.

[0034] The sequence used for transformation corresponds to SEQ ID NO:3: TIFF2025539024000002.tif49155

[0035] For the purposes of the present invention, step II) involves the transformation of Nicotiana benthamiana plant cells.

[0036] In particular, in step II), the transformation is carried out by co-cultivation at about 25° C. in the dark under constant agitation for 48 hours.

[0037] Next, the cells are selected.

[0038] Preferably, in step III) a selective medium is used which comprises 0.9% w / v agar and MS supplemented with antibiotics.

[0039] In a preferred embodiment, carbenicillin and kanamycin, more preferably 250 mg / L carbenicillin and 100 mg / L kanamycin, are used for such purposes.

[0040] In a preferred embodiment of the invention, in step IV), cells of possibly Nicotiana benthamiana are cultivated in suspension.

[0041] In another preferred embodiment, the culture comprises an initial inoculum of plant cells, perhaps of Nicotiana benthamiana, equal to 10% of the final culture volume.

[0042] The cell culture is incubated in MS medium (Murashige 1962) supplemented with sucrose, naphthalene-acetic acid (NAA) and kinetin for 15 days at a temperature of 24 / 27°C and with aeration maintained at 50-100 mbar.

[0043] Additionally, subcultures are set up every 7 days, transferring an aliquot of the cell suspension into fresh medium.

[0044] The cells are incubated under agitation in the dark at a constant temperature of 25°C.

[0045] According to the present invention, step V) involves the use of an extraction buffer containing 50 mM NaHPO, 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, supplemented with 1% (w / v) XAD-4 and 1% (w / v) polyvinylpolypyrrolidone (PVPP).

[0046] Ammonium sulfate is then added to the extract until a saturation concentration of 70% is obtained and incubated at 4° C. for 1 hour under constant agitation.

[0047] The precipitate is then collected by centrifugation and resuspended in IMAC buffer.

[0048] The preparation is centrifuged and filtered.

[0049] The solution thus obtained is purified in step VI) through a column passage.

[0050] In particular, the solution is loaded onto an IMAC chromatography column.

[0051] Preferably, a Ni Sepharose 6 FF column is used.

[0052] The fractions of interest are then collected and loaded onto a desalting column.

[0053] Preferably, a Sephadex G-25 Medium column is used.

[0054] The fractions of interest are then collected and loaded onto an ion exchange chromatography column.

[0055] Preferably, an SP Sepharose HP column is used.

[0056] During the purification, the absorbance is monitored at 280 and 254 nm.

[0057] As mentioned above, the ABRβ1 subunit is obtained by biotechnology (recombinantly) from Nicotiana benthamiana plant cell cultures in suspension.

[0058] According to another embodiment, the ABRβ subunit of the present invention can be obtained by biotechnology in other cells, such as mammalian, yeast, bacterial or other plant cells.

[0059] In particular, among plant cells, cells of carrot (Daucus carota), rice (Oryza sativa), soybean (Glycine max), corn, etc. can be used.

[0060] For the purposes of the present invention, the term "treatment" of tumor metastasis refers to a therapeutic protocol in a patient with a primary tumor with or without the presence of metastases.

[0061] In a preferred embodiment of the invention, such primary tumors are represented by melanoma, breast tumor, ovarian tumor, sarcoma, pancreatic tumor, kidney tumor, stomach tumor, lung tumor, neuroblastoma (also at pediatric age).

[0062] For the purposes of the present invention, the term "tumor metastasis" refers to a tumor, especially a solid secondary tumor arising from a primary tumor.

[0063] In a preferred embodiment of the present invention, such metastases are metastases arising from melanoma.

[0064] In a further preferred embodiment of the present invention, the metastasis is lung metastasis, peritoneal metastasis, brain metastasis, kidney metastasis, or liver metastasis.

[0065] In a further aspect, the present invention describes the pharmaceutical use of compounds, possibly recombinant (ABRβ), to prevent metastasis.

[0066] Thus, the compounds described by this invention are anti-metastatic.

[0067] This means that the recombinant compound of the present invention (ABRβ) can not only regress metastases already formed by the primary tumor (therapeutic effect) in patients with a primary tumor, but also prevent their formation.

[0068] According to a preferred embodiment of the present invention, the pharmaceutical use of the compounds of the present invention for treating and / or preventing tumor metastasis is described, wherein such treatment and / or prevention is achieved by stimulation of the immune system.

[0069] In another aspect of the present invention, a pharmaceutical use for treating and / or preventing tumor metastasis is described, wherein tumor-associated macrophages M2 (TAM-M2) are reprogrammed into tumor-associated macrophages M1 (TAM-M1).

[0070] In a second object, the present invention describes pharmaceutical formulations containing one of the compounds described herein.

[0071] In particular, the formulation is administered intravenously.

[0072] According to certain aspects of the present invention, such formulations may comprise a compound of the present invention, possibly in recombinant form, and one or more pharmaceutically acceptable carriers and / or excipients.

[0073] In a preferred embodiment of the present invention, such compounds may optionally be conjugated to a suitable molecule having therapeutic activity.

[0074] Such molecules may be selected from groups of molecules used to treat particular forms of tumors.

[0075] More particularly, such preparations are formulated for intravenous administration.

[0076] In a further object, the present invention describes a method for treating and / or preventing tumor metastasis, comprising administering a pharmaceutically effective amount of a compound of the present invention to a subject in need thereof.

[0077] In particular, such methods involve reprogramming tumor-associated M2 macrophages (TAM-M2) to M1 tumor-associated macrophages (TAM-M1).

[0078] In one embodiment of the present invention, the production of IL12 is induced in M2-type macrophages.

[0079] This induction is greater than that of IL12 production in M1 macrophages. [Example]

[0080] Example 1 Effect of ABRβ1 on IL10 production in macrophages Isolation of human monocytes To prepare monocytes from human peripheral blood, collect 2–4 bags of fresh plasma (buffy coat) and transfer them to a 50 ml sterile tube. Centrifuge at 3000 × g for 15 minutes at room temperature to remove residual platelets and combine all supernatants. Place 15 ml of Ficoll solution (1.077 g / ml) in the bottom of a 50 ml falcon and prepare the falcon at room temperature. For each falcon, gently attach 30–35 ml of buffy coat to the Ficoll solution to create the first density gradient. Centrifuge at 400 × g for 30 minutes at room temperature, decelerating very slowly to prevent remixing. Collect the white ring of peripheral blood mononuclear cells (PBMCs) that forms between the two phases of each 50 ml falcon and transfer it to a new sterile container. At this point, add excess PBS-EDTA (1 mM) to the collected cells and centrifuge at 300 × g for 10 minutes at room temperature. Remove the supernatant and repeat the procedure to wash the collected cells. At this point, the washed cell pellet is resuspended in phenol red-free RPMI-1640 medium supplemented with 10% complement-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. 23 ml of this solution is transferred to a 50 ml tube, and 27 ml of RPMI-1640 supplemented with phenol red and complement-inactivated FCS (finally, 10%) is added. All operations are performed at room temperature. 25 ml of the freshly prepared solution is placed in the 50 ml tube, and the PBMCs are deposited on top of this solution very slowly to avoid remixing. At this point, centrifuge at 550 x g for 30 minutes at room temperature, decelerating very slowly. The white ring of monocytes located between the two layers of different densities was gently collected and transferred to a new 50 ml tube. PBS-EDTA (1 mM) was added and the mixture was centrifuged at 400 × g for 10 minutes at room temperature. The supernatant was removed and the cell pellet was resuspended in RPMI-1640 medium supplemented with decomplemented FCS and phenol red to a final concentration of 10%.

[0081] In vitro culture and differentiation of human monocytes First, after isolation from human plasma, the concentration of monocytes in the resuspension must be determined. To do so, the resuspended cells are counted using the vital dye trypan blue. Next, the monocytes are seeded onto culture dishes in 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 differentiation is induced. To this end, 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 (antitumor), while M-CSF differentiates monocytes into M2-type macrophages (tumor-promoting).

[0082] Figure 1 shows that M2 macrophages (differentiated in vitro) produce IL-10 when appropriately stimulated with lipopolysaccharide (LPS), unlike M1 macrophages. Treatment with the compound ABRβ1 induces a phenotypic change in M2 macrophages, where they lose the ability to produce IL-10 upon stimulation, a characteristic more typical of M1 macrophages.

[0083] Example 2 Effect of ABRβ1 on IL-12 production in human macrophages To determine the type of effect of compound ABRβ1 on the phenotype of in vitro differentiated human macrophages, we analyzed the production of IL-12, characteristic of M1 macrophages, and IL-10, typically produced by M2 macrophages. The above-mentioned interleukins were measured in the cell culture medium after different treatments using specific ELISA kits.

[0084] Figure 2 shows that M1 macrophages produce IL-12. Compound ABRβ1 has little effect on IL-12 production by M1 macrophages. In contrast, compound ABRβ1 significantly promotes IL-12 production in M2 macrophages, which normally cannot produce IL-12. These data clearly demonstrate that compound ABRβ1 has the ability to reprogram M2 macrophages, specifically to acquire M1 macrophage characteristics.

[0085] Example 3 Effect of ABRβ1 on survival in syngeneic mice bearing metastatic melanoma To evaluate the immunostimulatory and anti-metastatic effects of compound ABRβ1 in vivo, a syngeneic mouse model of melanoma was selected. The immune system of the mice receiving tumor cells was fully functional. The mouse cell line used was B16F10, characterized by high aggressiveness and a high metastatic rate. In this mouse model, death usually occurs due to rapid metastasis at the peritoneal or pulmonary levels. For this purpose, animals were inoculated subcutaneously with B16F10 cells. Two experimental groups were established: one control group (not treated with ABRβ1) and one treated with ABRβ1. The compound was administered intraperitoneally to the animals twice weekly at a dose of 2 mg / kg. Treatment began immediately after tumor cell inoculation. Survival analysis was performed using the Kaplan-Meier model.

[0086] Figure 3 shows that mice inoculated with B16F10 cells did not survive beyond 18 days. In contrast, mice inoculated with tumor cells and treated with compound ABRβ1 showed a high survival rate (80%) of over 18 days. Surprisingly, autopsy analysis of the animals upon sacrifice revealed no metastases at the peritoneal or pulmonary levels. Autopsy analysis of deceased animals not treated with ABRβ1 revealed extensive metastases in the peritoneal cavity and pulmonary levels. This evidence clearly demonstrates that compound ABRβ1 has significant anti-metastatic activity, even in particularly aggressive tumor models such as melanoma.

[0087] From the above description, the advantages offered by the present invention will be apparent to those skilled in the art.

[0088] Of particular importance, the subunits of the invention have been shown to have clear and significant anti-metastatic activity, as demonstrated by highly encouraging data obtained in one of the most aggressive metastatic models, a mouse model of melanoma metastasis.

[0089] Furthermore, the use of the subunit ABRβ1 offers high quality and biological safety thanks to a production method with little risk of contamination with viruses, oncogenes, prions, toxins, or residues of hazardous reagents typically used in the production of therapeutic proteins.

Claims

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

2. The compound for medical use according to the preceding claims, wherein the metastasis arises from a tumor selected from the group comprising melanoma, breast tumor, ovarian tumor, pancreatic tumor, kidney tumor, gastric tumor, lung tumor, sarcoma, neuroblastoma.

3. 10. 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. A compound for medical use according to any one of the preceding claims, wherein the treatment and / or prevention of tumor metastasis is carried out 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. A compound for pharmaceutical use according to any one of the preceding claims, obtained by a process comprising the steps of: I) obtaining a suitable vector transformed with a plasmid containing a sequence corresponding to SEQ ID NO: 3; II) transforming a plant cell with the vector of step I); III) selecting transformed plant cells; IV) culturing stable plant cells in suspension; V) preparing a cell extract; Purifying the compound.

7. A compound for medical use according to the preceding claims, wherein in step II) plant cells of Nicotiana benthamiana are transformed.

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

2.

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

10. 2. The method of treating and / or preventing 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 IL12 production is induced in M2 macrophages.

12. The method according to claim 1 , wherein the induction is higher than IL12 production in M1 macrophages.

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