Method for producing cell particles with anti-tumor activity

Abstract

A method for producing cell particles having anti-tumor activity comprises isolating cell particles derived from a cell population having a phenotype attributable to human adipose tissue-derived pericytes (AD-PCs) expressing anti-tumor TRAIL.

Description

Technical Field

[0001] The present invention relates to a method for producing cell particles having anti-tumor activity in cell therapy, which method can be carried out in vitro. Background Art

[0002] It is well known that adult progenitor cells, including a population of cells with a phenotype attributed to human adipose tissue-derived pericytes (hereafter referred to as AD-PCs), can be used as carriers for carrying bioactive molecules.

[0003] In particular, AD-PCs can carry so-called "death ligands", i.e., a family of molecules belonging to the tumor necrosis factor superfamily.

[0004] Among them, tumor necrosis factor-related apoptosis-inducing ligand (hereinafter referred to as TRAIL) molecules can induce cell death in diseased tissues, so the possible uses of TRAIL molecules in certain biomedical fields are particularly interesting.

[0005] From European Patent EP2424979 a method for producing a medicament for treating tumors is known, which method comprises preparing a retroviral vector encoding a soluble variant of the TRAIL molecule and stably transfecting adipose pericytes (AD-PC).

[0006] AD-PCs carrying TRAIL have shown cytotoxic activity against certain tumors in vitro and in vivo and are therefore of particular interest in cancer therapy.

[0007] Furthermore, the term "cellular granules" refers to products of cellular activity consisting of particles that are spontaneously released outside the cell.

[0008] These particles are defined by a closed circular membrane consisting entirely or partially of phospholipids. In addition, these particles are characterized by a diameter size between 40 nm and 5000 nm.

[0009] Cellular particles may originate from protrusions of the plasma membrane that subsequently detach from the plasma membrane.

[0010] Alternatively, cellular particles can originate from intracellular organelles called endosomes and are subsequently released outside the cell by fusion of the endosomal membrane with the plasma membrane.

[0011] Alternatively, during apoptosis, cellular granules may originate from protrusions of the plasma membrane.

[0012] It is known that cell particles may include molecules derived from the cell producing the cell particles in the lumen or membrane of the particles that make up the cell particles.

[0013] These molecules are then transported outside the cell along with cellular granules.

[0014] However, this prior art suffers from several problems.

[0015] The first is that the safety of inoculating living cells carrying bioactive molecules in vivo for therapeutic purposes is still under debate, as the fate of the inoculated cells cannot be controlled in advance, i.e., the behavior of the cells after inoculation is unknown. The main uncertainty concerns the potential for uncontrolled expansion of the inoculated cells, which could lead to damage to healthy organs and altered physiological functions.

[0016] A second problem is that inoculation of an organism with living cells carrying bioactive molecules for therapeutic purposes can trigger rejection reactions in the recipient organism, rendering the treatment itself ineffective.

[0017] A third problem is that the use of living cells carrying the bioactive molecules that must be injected requires the production of large numbers of cells and is therefore costly. Summary of the invention

[0018] It is an object of the present invention to improve the prior art.

[0019] Another object of the present invention is to provide a method for producing cell particles derived from TRAIL-producing AD-PC (AD-PC-TRAIL) for anti-tumor purposes.

[0020] Another object of the present invention is to provide a method for producing cell particles derived from AD-PC-TRAIL without affecting the anti-tumor effect of TRAIL.

[0021] Another object of the present invention is to provide a method for producing cell particles derived from AD-PC-TRAIL which reduces the simultaneous administration of AD-PC-TRAIL, thereby limiting every undesirable side effect on the organism due to AD-PC itself.

[0022] Another object of the present invention is to provide a method for producing cell particles derived from AD-PC-TRAIL, which method reduces the concurrent administration of AD-PC-TRAIL, thereby making the administration of TRAIL substantially economical.

[0023] According to one aspect, the present invention provides a method for producing cell particles having anti-tumor activity according to the features of claim 1 .

[0024] According to another aspect of the present invention, a cell particle is provided according to the features of claim 5. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Other features and advantages of the present invention will become more apparent from the following description of an embodiment of a method for producing cell particles having anti-tumor activity, given as a non-limiting example with reference to the accompanying drawings, in which:

[0026] Figure 1 A dot plot is shown for a class of calibration beads with known diameters between 500 nm and 3000 nm analyzed by flow cytometry. The horizontal line "A" separates the background noise of the instrument (below the line) from the dots representing the acquired data (above the line).

[0027] Figure 2 Figure 2 shows a dot plot of isolated cell particles analyzed by flow cytometry. The horizontal line "A" separates the background noise of the instrument (below the line) from the dots representing the acquired data (above the line).

[0028] Figure 3 Figure 2 is a histogram showing the fluorescence of cell pellets isolated from AD-PCs and stained with the carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) molecule.

[0029] In detail, line "B" separates the left field of lower fluorescence intensity, which is comparable to the fluorescence intensity of cell particles not stained with CFDA-SE, from the right field of higher fluorescence intensity.

[0030] Figure 4 Table showing the concentration of TRAIL detected in cell pellets derived from AD-PCs modified with an empty vector and thus not expressing TRAIL (AD-PC-EMPTY) or in AD-PC-TRAIL (expressed in pg / million cells).

[0031] Figure 5 Graphs showing cytotoxicity assays in BXPC3 tumor cells incubated with cell pellets derived from AD-PC-EMPTY or AD-PC-TRAIL.

[0032] Figure 6 Graphs showing cytotoxicity assays in A673 tumor cells incubated with cell particles derived from AD-PC-EMPTY or AD-PC-TRAIL.

[0033] Figure 7 Graph showing Caspase 8 activation assay of A673 cells incubated with AD-PC-TRAIL-derived cell particles compared with control A673 cells. DETAILED DESCRIPTION

[0034] The present invention relates to a method for producing cell particles, in particular cell particles derived from AD-PC-TRAIL, which have been found to have anti-tumor activity.

[0035] according to Figure 1 , which shows flow cytometric analysis based on forward scatter (FSC) and side scatter (SSC) morphological parameters of a sample of calibration beads with known sizes between 500 nm and 3000 nm.

[0036] according to Figure 2 , which shows flow cytometric analysis of cell pellets isolated from AD-PCs according to FSC and SSC morphological parameters.

[0037] according to Figure 3 , which shows fluorescence from cell pellets isolated from AD-PCs after staining with CFDA-SE.

[0038] In particular, 95.9% of the cell pellets tested positive for fluorescence.

[0039] according to Figure 4 Table showing TRAIL concentrations detected by enzyme-linked immunosorbent assay (ELISA) in cell pellets derived from AD-PC-EMPTY or AD-PC-TRAIL.

[0040] In particular, it is noteworthy that no TRAIL released from AD-PC-EMPTY derived cell pellets was detected, whereas 446 pg of TRAIL was measured from cell pellets derived from 1 million AD-PC-TRAIL.

[0041] according to Figure 5 The cytotoxicity test showed that the cell particles derived from AD-PC-TRAIL could produce cytotoxic effects on the BXPC3 tumor cell line.

[0042] In particular, when cells were incubated with AD-PC-TRAIL derived cell pellets, the mortality rate of BXPC3 cells was measured to be 34.6 ± 3.6%, while when cells were incubated with AD-PC-EMPTY derived cell pellets, the mortality rate was 16.5 ± 6.2%. Using the T test statistical test, there was a statistically significant difference between the two groups, p < 0.05 (indicated by * in the figure).

[0043] Data are expressed as mean ± SEM (standard error of the mean), and the same representation is used in other figures below.

[0044] according to Figure 6 The cytotoxicity test showed that the cell particles derived from AD-PC-TRAIL could produce cytotoxic effects on the tumor cell line A673.

[0045] In particular, when cells were incubated with AD-PC-TRAIL-derived cell particles, a mortality rate of 93.1% was detected, whereas when cells were incubated with AD-PC-EMPTY-derived cell particles, a mortality rate of 16.1 ± 16.2% was detected.

[0046] Using T-test statistical test, there was a statistically significant difference between the two groups, p<0.01 (indicated by * in the figure).

[0047] Data are expressed as mean ± SEM.

[0048] according to Figure 7 , which shows the percentage activation of caspase 8 on A673 cells after incubation with AD-PC-TRAIL derived cell pellets relative to a reference control. This treatment has determined that the activation of caspases is 296±5% higher than in the control.

[0049] The difference was statistically significant with T-test, p<0.001 compared to the control. Data are presented as mean ± SEM.

[0050] Example 1

[0051] Isolate the cell pellet generated by AD-PCs.

[0052] The method used to isolate the cell pellet generated by AD-PCs is as follows.

[0053] The cultured AD-PCs that had reached about 80% confluence were washed with PBS (manufactured by Biochrom GmbH), and the medium was replaced with αMEM medium (manufactured by ThermoFisher Scientific Inc.) containing 1% L-glutamine (manufactured by Lonza) and 1% penicillin / streptomycin (10000 U / mL penicillin, 10 mg / mL streptomycin in 0.9% NaCl, manufactured by PAA Laboratories).

[0054] AD-PCs were then cultured in an incubator for 48 hours before supernatant collection.

[0055] Supernatants collected from cell cultures of AD-PC-TRAIL and AD-PC-EMPTY were subjected to differential centrifugation: initial centrifugation at 2,000 g for 20 min at +4°C, followed by gravity filtration of the supernatant through a 0.8 μm filter and further centrifugation at 20,000 g for 40 min at +4°C.

[0056] At the end of these stages, the pellet containing the cell pellet is resuspended in the most appropriate culture medium for subsequent applications.

[0057] Visualize the cell pellet in flow cytometer.

[0058] Before display on a flow cytometer (BD FACS ARIA III, manufactured by Becton Dickinson), the cell pellet obtained by the aforementioned separation method was resuspended in PBS containing 0.1% BSA (bovine serum albumin, manufactured by Sigma) and stained with 1 μL of CFDA-SE (CellTrace CFSE Cell Proliferation Kit, manufactured by Invitrogen).

[0059] Optimal parameters for displaying FSC and SSC of MVs were set using microbeads of known size (Megamix, produced by Biocytex).

[0060] Results of Example 1

[0061] Cell pellets from media conditioned with AD-PC-EMPTY and AD-PC-TRAIL were separated by two separate centrifugation passes filtered through 0.8 μm filters and visualized on a flow cytometer.

[0062] As shown in the size comparison with Megamix beads of known size (see Figure 1 and Figure 2 ), the method can separate a class of particles with a size between 40nm and 5000nm, especially between about 500nm and 3000nm.

[0063] Staining with CFDA-SE allows for better visualization of MVs in flow cytometry (see Figure 3 ).

[0064] This is a dye that is able to permeate into the cytoplasm where it is converted by intracellular esterases into a succinimidyl ester compound of carboxyfluorescein (CFSE), which remains inside the cell where it binds to intracellular proteins and emits a luminescent fluorescent signal that can be detected.

[0065] Due to this property, CFDA-SE can distinguish closed structures covered by a membrane (in this case, cell particles) from cell debris.

[0066] Example 2

[0067] The amount of TRAIL present in the cell pellet was quantified.

[0068] The cell pellets derived from AD-PC-EMPTY and AD-PC-TRAIL were subjected to ELISA test (Quantikine ELISA, manufactured by R&D Systems) to determine the TRAIL concentration.

[0069] Specifically, according to the manufacturer's protocol, the cell pellet isolated using the isolation protocol in the aforementioned Example 1 was resuspended in PBS containing 0.1% BSA and lysed with an appropriate lysis buffer (Lysis Buffer, manufactured by R&D Systems) before being tested.

[0070] Results of Example 2

[0071] After inducing the particles to lyse themselves, the TRAIL content in the cell pellets derived from AD-PC-EMPTY and AD-PC-TRAIL was quantified using an ELISA test. The results showed no presence of TRAIL in the AD-PC-EMPTY derived samples, while a concentration of 446 pg was detected in the cell pellets derived from 1 million AD-PC-TRAIL (see Figure 4 ).

[0072] It is known that the soluble TRAIL molecules produced by AD-PC-TRAIL lack the structural elements necessary for the insertion of the granule-forming particles into the membrane. Therefore, the detected TRAIL molecules are initially located in the lumen of the granule-forming particles.

[0073] Example 3

[0074] Cytotoxicity assays were performed on the cell pellets on tumor cell lines.

[0075] Using the MTS assay ( The antitumor ability of cell pellets from AD-PC-EMPTY and AD-PC-TRAIL against tumor cell lines A673 and BXPC3 was tested using the AQueous One Solution cell proliferation assay (manufactured by Promega).

[0076] For each sample analyzed, 5,000 tumor cells were seeded into 96-well plates and cultured in a controlled atmosphere incubator (37° C., 5% CO 2 ).

[0077] On the next day, the tumor cell culture medium was replaced with 100 μL DMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin, and the intact cell pellet obtained according to the isolation protocol in Example 1 was resuspended in the culture medium.

[0078] After 24 hours, 20 μL of the solution per MTS was added to each well and the plate was incubated at 37°C for 1 hour before measuring the absorbance at 495 nm, which is proportional to viability and metabolic activity, using a GloMax Discover instrument (manufactured by Promega).

[0079] All experiments included a control for basal mortality consisting of tumor cells exposed to the same medium but without contact with cell pellets.

[0080] To confirm the role of TRAIL in inducing cytotoxicity to A673 cells, the activity of caspase 8 was investigated using the Caspase-Glo 8 Assay Kit (manufactured by Promega).

[0081] Specifically, tumor cells were seeded at a concentration of 5,000 cells per well in white multi-well plates.

[0082] The next day, the medium was replaced with fresh medium containing the intact AD-PC-TRAIL-derived cell pellet isolated as described previously.

[0083] After 18 hours, caspase-8 activity was assessed using the GloMax Discover instrument according to the manufacturer's protocol.

[0084] Results of Example 3

[0085] The induced mortality of BXPC3 and A673 tumor cell lines was examined by cytotoxicity assay with MTS after 24 h incubation with cell pellets derived from AD-PC-EMPTY and AD-PC-TRAIL cells.

[0086] Treatment of BXPC3 cells with AD-PC-TRAIL-derived cell pellets resulted in a mortality rate of 34.6 ± 3.6%, significantly higher than that obtained with AD-PC-EMPTY-derived cell pellets, which showed no relevant cytotoxic effects (see Figure 5 ).

[0087] The mortality of A673 cells induced by AD-PC-TRAIL-derived cell particles was 93.1 ± 9.6%, which was significantly higher than that obtained with AD-PC-EMPTY-derived cell particles, which did not show significant cytotoxic effects (see Figure 6 ).

[0088] The role of TRAIL in inducing tumor cell death was verified by quantifying the percentage of caspase 8 activation after incubation of A673 tumor cells with AD-PC-TRAIL-derived cell pellets for 18 h compared to untreated controls.

[0089] The activation of caspases measured by this treatment was 296 ± 5% higher than that of the control, which supports the cytotoxic effect mediated by TRAIL (see Figure 7 ).

[0090] In practice, it has been found that the invention achieves the intended purposes.

[0091] The invention as conceived is susceptible to modifications and variations, all of which are within the scope of the inventive concept.

[0092] Moreover, all the details may be replaced by other technically equivalent details.

[0093] In practice, any materials, devices and quantities may be used as desired without departing from the scope of protection of the appended claims.

Claims

1. A method for producing cell particles having anti-tumor activity, characterized in that The method comprises separating a cell pellet from a supernatant collected from a culture of a cell population, The cell population is a pericyte phenotype cell population derived from human adipose tissue, wherein cells are stably transfected with a retroviral vector encoding soluble tumor necrosis factor-related apoptosis-inducing ligand, wherein the cells express anti-tumor soluble TRAIL, The cell particles include particles having the following characteristics: - a closed circular membrane formed by a bilayer composed partly or entirely of phospholipids, and - Size between 40nm and 5000nm; The method further comprises initial centrifugation of the separated cell pellet at 2,000 g for 20 minutes at +4°C, followed by gravity filtration of the supernatant through a 0.8 μm filter and further centrifugation at 20,000 g for 40 minutes at +4°C.

2. The method according to claim 1, wherein: The population expressing TRAIL is selected from the group consisting of: autologous cells, allogeneic cells, human cells, and animal cells.

3. The cell particles obtained by the method of claim 1 or 2, characterized in that: The cell particles include particles having a closed circular membrane formed by a bilayer partially or completely composed of phospholipids, wherein the size of the particles is between 40 nm and 5000 nm.

4. The cell particle according to claim 3, wherein The size of the particles is between 500 nm and 3000 nm.

5. The cell particle according to claim 3 or 4, wherein The particles have TRAIL in their lumen and have anticancer activity.

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