Methods for characterizing products for cell therapy

Abstract

The present invention relates to a method for determining the efficacy of a cell therapy or immunotherapy wherein the method comprises: a. Providing a first set of micropores, each micropore in the first set of micropores comprising effector cells and target cells; b, optionally, providing a second group of micropores, each micropore in the second group of micropores comprising a target cell; c, a micropore containing a single effector cell and n target cells is selected, and n is between 1 and 50, preferably between 1 and 20; d, keeping the culture of the cells for a time t; e, determining the number of dead target cells in each selected micropore containing a single effector cell; and f, calculating an efficacy score consisting of a weighted average of the number of target cells killed by each effector cell.

Description

Background Art

[0001] To characterize and understand the mode of action of cell therapies, identify optimal effector cells, improve the manufacturing process to maximize the efficacy of cell therapies by ensuring minimal depletion of the cytotoxic capacity of immune cells, or for product release, commonly used assays are cytotoxicity and cytokine release assays, which reveal the interaction of immune cells and target cells.

[0002] Currently available methods cannot verify the functional heterogeneity of cell therapy products, nor can they determine whether and how long cells involved in killing target cells maintain cytotoxic capacity to kill other target cells (known as serial killing signatures). In fact, bulk assays are used, from which information is obtained by averaging the analyzed cell populations, but it is impossible to track the cytotoxicity of each individual effector cell or to visualize the progressive activities performed by each effector cell. The flow cytometry assays widely used in this context also define subpopulations based only on phenotypic markers rather than on function.

[0003] There remains a pressing need for methods that can characterize cell therapy products using single-cell-specific information to both observe functional heterogeneity, i.e., how many immune cells exhibit cytotoxic activity against target cells, and to assess how many immune cells are able to maintain cytotoxic activity after killing one or more target cells, thus becoming “serial killer” cells, or whether immune cells are exhausted in their cytotoxic activity after one or more killing events.

[0004] Since there are also multiple approaches to enhance the activity of immune cells against specific targets, there remains a pressing need to compare the effectiveness of these different approaches in conferring a certain degree of potency and depletion of immune cells during cell therapy improvement and production.

[0005] Finally, given that cytotoxicity assays used in cell therapy rely on the ability to maintain target cell viability (typically for 4 to 72 hours), it is critical to maintain ideal conditions and minimize assay noise due to unnecessary and irrelevant target cell death, while ensuring that effector cells are maintained in a local environment where they can exert cytotoxic activity against target cells with minimal alteration of the surrounding environment.

[0006] Microfluidic devices are the most advanced tools available that allow large-scale high-content analysis with single-cell resolution. Although nanoscale volumes offer unique advantages, such as the ability to co-localize a single effector cell with multiple target cells and assay cytokines secreted by a single cell, or to subsequently perform multiple assays on the same cell over an extended period of time, it requires the highest level of control over the local environment to minimize target cell death due to factors unrelated to effector-target cell interactions. Summary of the invention

[0007] The object of the present invention is to provide a method suitable for cell-mediated cytotoxicity assays.

[0008] In one embodiment, the method is performed in an open microwell microfluidic device, which enables the automatic and rapid exchange of liquids into the wells without displacing the loaded suspended cells. This capability enables the automation of the preparation and execution of assays in wells previously loaded with samples, and the supply of nutrients to the microwells at desired time intervals, thereby achieving periodic perfusion of culture media. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 : Illustration of an inverted open microporous system used in the method of the present invention, perspective view (A), vertical cross-sectional view (B) and top view (C).

[0010] Figure 2 : Workflow diagram showing (A) delivering cells to microwells; (B) whole-body staining of cells; (C) perfusing channels with medium supplemented with propidium iodide (PI); (D) incubation without perfusion. Steps (C) and (D) can be repeated multiple times to produce periodic perfusion at specific time points.

[0011] Figure 3 : Cell viability of KG-1 (A) and K562 (B) at 72 hours. Although the data for K562 cells do not show statistical differences between the two conditions, for KG-1, 24-hour regular perfusion can maintain cell viability while limiting changes in the cell surroundings. (C) Kinetics of KG-1 and K562 cell viability and cell counts in a 72-hour time-lapse assay. Data refer to the 24-hour perfusion condition.

[0012] Figure 4: Image sequence of KG-1 cell line showing that cell viability is maintained and CMAC labeling is persistent, and cell number increases over time due to proliferation. PI labeling is not visible as cell death was not observed in this microwell.

[0013] Figure 5 : Cell viability measured after 72 h of culture, with varying reperfusion frequency. Solid line: target cells only. Dashed line: target cells co-cultured with effector cells.

[0014] Figure 6 :(A) Representative images of a set of microwells observed by fluorescence and phase-contrast microscopy. Arrows indicate effector cells, highlighted by Calcein AM staining and CD16 / CD56 markers. Wells containing single effector cells (circled in the figure) were selected for analysis, and the results are shown in the figure. (B) The percentage of effector cells that can kill the specified number of target cells is shown.

[0015] Figure 7 : Representative images of a panel of microwells containing (A) inactive effector cells or (B) active effector cells. Effector cells are highlighted by arrows.

[0016] Figure 8 : Quantification of the potency of effector cells obtained from donors D1 to D6. (A) Effector cells that killed 1 or 2 target cells; (B) Effector cells that killed 3 or more target cells; (C) Effector cell potency derived from the results of panels (A) and (B).

[0017] Fig. 9 : Potency of effector cells from donors D1 to D3.

[0018] Fig.10 : Potency of two different phenotypes of effector cells, (A) obtained from donor DA and (B) obtained from donor DC. DETAILED DESCRIPTION

[0019] definition

[0020] In this context, interference coupling refers to the fit between two elements so that the two elements can be considered connected together. When the two elements (in this case, the tip and the vertical channel) are coupled by interference, the fluid injected into the tip and released in the vertical channel is forced to move within the channel, and the interference coupling prevents the fluid from passing, that is, the interference coupling seals the two elements from each other.

[0021] In the present context, a connector refers to any tubular, cylindrical, more or less conical, converging or diverging element suitable for placing two compartments in fluid connection.

[0022] In this article, re-perfusion refers to replacing the culture medium with fresh culture medium. The fresh culture medium is the same or different from the culture medium already present in the culture. In one embodiment, the culture medium is culture medium. In one embodiment, it is a culture medium comprising one or more drugs and / or one or more dyes and / or one or more labeled antibodies and / or one or more cell viability markers.

[0023] Fluid: Any substance in liquid or gaseous form.

[0024] Biological sample: A sample comprising cells obtained from a microorganism, an animal and / or a human (preferably a human), wherein the sample is preferably selected from the group consisting of a biological fluid or a biopsy sample. The sample comprises suspended cells or the sample is a tissue. In a preferred embodiment, the sample is a blood sample or a bone marrow aspirate. Alternatively, the biological sample consists of cultured cells (e.g., a cell line) or a composition comprising cultured cells and cells from a patient.

[0025] High-content assay: A phenotypic assay performed on cells.

[0026] Time-lapse: An imaging technique that takes a series of pictures of the same area over time.

[0027] In vitro: A test conducted by placing tissue obtained from an organism into an environment outside the organism with minimal changes to natural conditions.

[0028] Long-term means that the culture is maintained for 24 hours or 48 hours, preferably 72 hours.

[0029] Serial killer cells: Immune cells that are able to exert cytotoxic activity against each of two or more target cells, leading to their death.

[0030] In one embodiment, the method is performed in an open micropore microfluidic device described in WO2017 / 216739.

[0031] In one embodiment, the open microwell is an open microwell of about 0.5 nL.

[0032] Advantageously, the open microporous interface provides a source of gas exchange that aids in long-term viability.

[0033] In one embodiment, by providing a kit ( Figure 1 The microfluidic device (1) comprises at least one microchannel (3) and an input region (8), the input region (8) comprising at least one vertical channel (18), the dimensions of the tip and the vertical channel (18) being designed to produce interference coupling therebetween.

[0034] The tip is selected from among commercially available tips, the tip comprising at least a proximal portion intended to cooperate with a fluid dispensing system and an open, tapered distal portion.

[0035] Preferably, the distal portion of the tip and the vertical channel (18) are made of plastic and make the system sufficiently elastic to ensure sealing, avoiding the use of gaskets. In a particularly preferred embodiment, the system geometry described below ensures that the contact between the vertical channel (18) and the tip does not occur at a single point, but is distributed over a surface portion, thereby further ensuring an effective seal. This condition is advantageously verified when the half-opening angles of the terminal portion of the tip and the vertical channel (18) do not differ much, preferably less than 10°. Even more preferably, the vertical channel (18) is a cylinder, optionally slightly tapering downwards.

[0036] A method for loading / unloading fluid in a microfluidic device (1) contained in a kit comprises the following steps: a) providing a kit according to the present specification; b) optionally, loading the fluid into the tip; c) positioning the tip above the input area (8) and inserting it until reaching an interference coupling position between the distal area of ​​the tip and the vertical channel (18) in the input area (8); and d) releasing the fluid contained in the tip in the microfluidic device (1) through the input area (8), or, alternatively, aspirating the fluid already contained in the microfluidic device with the same tip.

[0037] The present invention also describes a microfluidic device, which is an inverted open microwell system, which includes an array of open microwells (2), at least one microchannel (3), at least one input port (8) for reagents and / or for one or more biological samples, and at least one output port (10) for reagents and / or for one or more biological samples, wherein the input port and the output port are microfluidically connected to one or more of the microchannels (3), wherein the microchannel (3) has a cross-sectional area of ​​micrometer size and provides fluid to the microwells (2); wherein, in one embodiment, the inverted open microwell system is inserted into an automated management system, which includes the following features: an incubator with controlled temperature, humidity and CO2, a fluid distribution system, phase contrast and fluorescence image acquisition.

[0038] The automated management system is achieved by assembling components known in the art, such as an incubator with controlled temperature, humidity and CO2, a microplate pipetting system, a fluorescence and phase contrast microscope lens connected to an image acquisition camera (such as a CMOS or CCD camera), wherein all or part of the components are managed by software known to those skilled in the art through the hardware connected thereto.

[0039] In a particularly preferred embodiment, each microchannel (3) is associated with an input port (8) and an output port (10).

[0040] In a preferred embodiment, the microfluidic device (1) further comprises a reservoir, wherein the reservoir is at least one reservoir for reagents and at least one reservoir for one or more biological samples. The reservoir is selected from the group consisting of a plate, one or more multi-well plates (e.g., 96-well plates), and Eppendorf tubes. The reservoirs can be 2, or 4, 8, 16, 24, 48, 96, 384.

[0041] In one embodiment, the method according to the present invention comprises: providing at least two cell populations, wherein at least a first cell population comprises effector cells grown in suspension and at least a second cell population comprises target cells; co-culturing the cells for at least 24 h, such as 48 h or 72 h, and regularly re-perfusing the culture, wherein the re-perfusion is performed regularly over a period of time between 1 hour and 30 hours, such as once every two hours, or once every 12 hours, or once every 24 hours; and assessing cell viability.

[0042] In one embodiment, the method is performed in a microfluidic system.

[0043] In one embodiment, the method is performed in an inverted open microwell system (1), which comprises an array of open microwells (2), at least one microchannel (3), at least one input port (8) for reagents and / or for one or more biological samples, and at least one output port (10) for reagents and / or for one or more biological samples, wherein the input port and the output port are microfluidically connected to one or more of the microchannels (3), wherein the microchannel (3) has a cross-sectional area of ​​micrometer size and provides fluid to the microwells (2).

[0044] In one embodiment, the microfluidic device comprises 16 microchannels (3).

[0045] In one embodiment, 1200 open microwells (2) are connected to each of the microchannels (3).

[0046] In one embodiment, the inverted open microwell system is operated by an automated management system that includes the following features: a temperature, humidity and CO2 controlled incubator, a fluid distribution system, phase contrast and fluorescence image acquisition.

[0047] In one embodiment, the method comprises: providing an inverted open microwell system (1), the inverted open microwell system (1) comprising an open microwell array (2), at least one microchannel (3), at least one input port (8) for reagents and / or for one or more biological samples, and at least one output port (10) for reagents and / or for one or more biological samples, wherein the input port and the output port are microfluidically connected to one or more of the microchannels (3), wherein the microchannels (3) have a cross-sectional area of ​​micrometer size and provide fluid to the microwells (2); providing an automated management system for the inverted open microwell system, the automated management system comprising: The automated management system comprises the following features: an incubator with controlled temperature, humidity and CO2, a fluid distribution system, phase contrast and fluorescence image acquisition; placing the inverted open microwell system (1) in the automated management system; loading reagents through one or more of the input ports (8), wherein the reagents include: filling buffer and / or washing solution and / or one or more drugs and / or one or more dyes and / or one or more labeled antibodies and / or one or more cell viability markers; loading at least two cell populations; staining the cells with one or more dyes and / or one or more labeled antibodies and / or one or more cell viability markers ( Figure 2 B); at time point T0, acquiring images from one or more of the microwells (2); regularly refilling the culture with fresh culture medium, which may optionally include one or more drugs and / or one or more dyes, and / or one or more labeled antibodies and / or one or more cell viability markers ( Figure 2 C to Figure 2 D); and at time point T1, acquiring an image from one or more of said microwells (2), wherein the steps of loading and acquiring images are repeated over time.

[0048] In one embodiment, the at least two cell populations are present in an amount of about 1.2×10 5 The effector cell population was loaded at a concentration of 3.3 × 10 cells / mL and 6 The target cell population was loaded at a concentration of cells / mL ( Figure 2 A).

[0049] In one embodiment, one of the at least two cell populations is an effector cell population.

[0050] In one embodiment, a single effector cell is loaded into at least 400 microwells out of the 1200 microwells (2) connected to each microchannel (3).

[0051] In one embodiment, one of the at least two cell populations is KG-1, a human macrophage cell line.

[0052] In one embodiment, one of the at least two cell populations is K562, a human lymphoblastoid cell line.

[0053] In one embodiment, the effector cells are NK lymphocytes.

[0054] In one embodiment, the effector cells are T lymphocytes.

[0055] In one embodiment, the T cells or NK cells are genetically modified.

[0056] The effector cells are co-cultured with target cells.

[0057] The target cell is a primary cell obtained from a subject, or a cell line.

[0058] In one embodiment, said target cell expresses an antigen that is recognized by said effector cell.

[0059] In one embodiment, the assay and time lapse is performed for a maximum of 72 hours.

[0060] In one embodiment, the marker is used for cell detection, and the marker is 7-amino-4-chloromethylcoumarin (CellTracker TM Blue CMAC).

[0061] In one embodiment, the marker is used to assess cell death and the marker is propidium iodide (PI).

[0062] In one embodiment, the marker is Calcein AM.

[0063] In one embodiment, a homogenous co-culture is selected, i.e., a microwell is selected that contains a single effector cell and a number of target cells within a defined range. Figure 6 A, A microwell containing a single effector cell NK was selected, highlighted by Calcein AM staining. In the microwell, the dead target cells stained with P1 were counted. This allowed defining the percentage of effector cells that were able to kill 0, 1, 2, 3, 4 or more than 5 target cells ( Figure 6B) From this profile, a so-called potency score can be calculated, which consists of a weighted average of the number of target cells killed by each effector cell, by first subtracting from the count the average number of dead target cells in microwells that did not contain any effector cells.

[0064]

[0065] where tk i represents the number of target cells killed by effector cell i, normalized to the number of spontaneous deaths of the same target cells in the absence of effector cells, and N represents the total number of effector cells.

[0066] Therefore, the present invention also relates to a method for determining the efficacy of cell therapy (or immunotherapy), the method comprising: a. providing a first group of microwells, each microwell in the first group of microwells containing effector cells and target cells; b. providing a second group of microwells, each microwell in the second group of microwells containing target cells; c. selecting microwells containing a single effector cell and n target cells, wherein n is between 1 and 50, preferably between 1 and 20; d. keeping the cells in culture for a time t; e. determining the number of dead target cells in each microwell selected to contain a single effector cell; and f. calculating a potency score consisting of a weighted average of the number of target cells killed by each effector cell, comprising first subtracting the average number of dead target cells in the second group of microwells from the average number of target cells killed by each effector cell.

[0067] In one embodiment, in step c, microwells are selected that contain target cells in an amount between 5 and 15, or between 7 and 10, or between 10 and 15. Advantageously, selecting microwells that contain target cells in an amount within a narrow range allows for more uniform co-culture. In addition, microwells that include at least 5 or at least 7 target cells allow for better observation of sequential killing activity.

[0068] In one embodiment, the microwell is an inverted open microwell of a microfluidic device as described above.

[0069] In one embodiment, effector cells are selected only when they are positive for a surface marker, or effector cells are selected only when they are negative for a surface marker, for example selected from the group consisting of CD3, CD4, CD16, CD56, CD45RA, CCR7, NKp30, NKp44, NKp46, PD-1, LAG-3, TIM-3.

[0070] In one embodiment, dead target cells are counted only when they are positive for a surface marker, for example selected from the group consisting of CD38, HLA-DR, CD34, or dead target cells are counted only when they are negative for a surface marker.

[0071] The efficacy score advantageously enables the efficacy of a therapy to be expressed in an absolute manner.

[0072] The potency score defined herein is the average number of target cells that a single effector cell is able to eliminate, this activity having been tested in replicates with well controlled test conditions, ie, a known number of cells in a known volume.

[0073] The advantage of this approach is that different variants of cell therapies can be compared, such as different sources of immune cells (donors or patients), different engineering methods, different production processes, and different processes and methods for selecting certain subpopulations.

[0074] From the data currently available, it can be observed that, for example, CAR-NK therapy can achieve a score > 1 at t = 12 h or t = 24. In another set of experiments, it was observed that unmodified NK cells can usually achieve a score < 1.

[0075] Advantageously, the method according to the invention enables a combined functional and phenotypic characterization.

[0076] For example, in conjunction with cytotoxicity, up to 2 surface markers can be evaluated by observing 4 different colors. In one embodiment, the cytoplasm of the target cell is stained with CMAC (in the blue DAPI channel), and PI (in the yellow TRITC channel) is used as a cell death marker. Therefore, in the green FITC channel and the red CY5 channel, two surface markers can be used. In this embodiment, for example, antibody 1 positive cell populations can be selected, and then continuous killer cells, killer cells and inactive cells can be identified. This makes it possible to observe that the presence of surface markers is correlated with the increase in efficacy scores (in other words, the efficacy scores of the subpopulations defined based on the immunophenotype are higher than the average efficacy scores of the parental group).

[0077] According to the method described herein, even with specific stains for cell detection (e.g., 7-amino-4-chloromethylcoumarin (CellTracker TM Target cell viability can be maintained in the absence of immune effector cells for 72 h in the presence of long-term immunofluorescence staining (ELISA), immunohistochemistry (IHC), and time-lapse death assessments (e.g., propidium iodide (PI)).

[0078] The entire setup was optimized in terms of imaging, fluidics, and staining methods to achieve high enough reagent concentrations to allow cells to be properly detected and sorted without significantly affecting cell viability.

[0079] The methods described herein enable the application of these stains throughout an experiment to support time-lapse analysis.

[0080] In summary, the conditions identified herein ensure that a model consisting of a target cell line, a panel of stains, and cell treatment methods ensures downstream use in potency assays based on cell killing assays.

[0081] Example

[0082] Example 1: Viability analysis of human macrophage cell line KG-1 and human lymphoblastoid cell line K562, loading, treatment Processing and imaging over a time lapse of up to 72 hours.

[0083] K562 cell line and KG-1 cell line were loaded into multiple microchannels of the microdevice. The experimental workflow steps included:

[0084] ·2×10 6 The cells were delivered into the microwells at a concentration of cells / mL;

[0085] Automatic preparation / dilution of 7pM concentration of cytoplasmic marker CMAC;

[0086] Automated cell staining by injecting CMAC into microfluidic channels;

[0087] Automated immunophenotyping through automated reagent dilution, preparation, and injection;

[0088] Automatically prepare and dilute the cell death marker PI with complete culture medium (RPMI+10% FBS+1% L-Glu+1% Penstrep) at a concentration of 5 mM;

[0089] Automatic cell staining by injecting complete medium supplemented with PI;

[0090] Time-lapse imaging was performed in bright field / phase contrast and 4 fluorescent channels, repeated every 12 h for 72 h.

[0091] Repeat the preparation at a certain time point and regularly perfuse the culture medium with PI to replenish nutrients and restore the fluorescence intensity of the PI signal. Each time it is re-injected, 100 μl is injected into each microchannel at a flow rate of 10 μl / s (rinsing time: 10 seconds). The repeatability of the test is evaluated by repeating the test independently within three days. The cell viability during the duration of time is determined by single cell detection based on Al and quantification of the percentage of cells that take up PI.

[0092] The K562 cell line showed mean 72-hour endpoint viability after normalization to T0: 90.58% ± 6.8% with perfusion every 12 hours, 86.24% ± 7.3% with perfusion every 24 hours, and 87% ± 0.44% with no washout.

[0093] There were no significant differences between the two conditions ( Figure 3 B).

[0094] exist Figure 3 In A, the results for the KG-1 cell line perfused with medium every 12 hours or every 24 hours showed that the average 72-hour endpoint viability was 80.96% ± 8.19% and 87.54% ± 3.36%, respectively, after normalization to T0. For the same cell line, the 24-hour viability without medium perfusion was 96.71% ± 2.36%, indicating optimal health. However, cell viability decreased over time until reaching a viability of 61.11% ± 14.16% at 72 hours, demonstrating the significant advantage of regular medium perfusion.

[0095] There were no statistical differences between the two perfusion strategies tested, so the preferred approach is to flush every 24 h to minimize changes in the environment surrounding the cells.

[0096] In contrast, other microtechniques (Halldorsson et al., Biosensors and Bioelectronics, 2015, 63: 218-231) generally require continuous perfusion to maintain ideal conditions, while the method according to the present invention maintains cell viability by applying short and rapid periodic perfusions. Figure 3 C, these conditions also supported cell growth within the microwells and Figure 4 As shown, the CMAC cell tracker propagates to daughter cells and remains visible for 72 hours.

[0097] exist Figure 4 The results of the co-culture experiments are reported in and compared with the mono-culture experiments.

[0098] Target cells are co-cultured with effector cells (according to the invention) or cultured alone.

[0099] Cell viability was assessed after 72 h of incubation.

[0100] Different microwells were exposed to different reperfusion frequencies, which varied from once per hour to once every 30 hours. The solid line in the figure represents target cells without effector cells. The dotted line represents target cells co-cultured with effector cells.

[0101] In the absence of any reperfusion, a high percentage of cell mortality was observed in both experimental setups, indicating that medium replacement was required to maintain a healthy cell population for up to 72 h.

[0102] Increase the frequency of reperfusion to maintain good cell health. In extreme cases, reperfusion can render the effect of the co-culture meaningless.

[0103] Surprisingly, a "working zone" was demonstrated here, i.e., the optimal reperfusion frequency that maximizes the probability of measuring the effect of effector cells on target cells without being affected by suboptimal culture conditions. The "working zone" is the frequency at which the maximum increment between two experimental settings is observed.

[0104] The viability trends of effector cells cultured alone or in the presence of target cells were comparable to those observed when target cells were grown alone.

[0105] Example 2: Characterization of 6 batches of cell therapy (batches D1 to D6).

[0106] Three microfluidic devices are provided, namely I, II and III, each of which includes 16 microchannels. Each microfluidic device is loaded with effector cells obtained from a donor, and the cells are CAR-NK. Specifically, 7 microchannels of each device are loaded with effector cells from one donor, and the other 7 microchannels are loaded with effector cells from different donors. The 6 channels loaded with effector cells are also loaded with target cells, and the seventh channel serves as a control channel containing only effector cells. The target cells used are Raji cells, a cell line obtained from human lymphoma. The remaining 2 channels on each device are loaded only with target cells as negative controls.

[0107] The ratio of effector cells to target cells in the co-culture microwells was 1:10. For cell tracking, target cells were stained with CMAC marker. Effector cells could be observed in bright field after evaluating the markers CMAC and Calcein AM, which did not affect cell viability in Raji cells but induced a certain proportion of cell death in CAR-NK.

[0108] The effector cells contained in each well are counted by an automated procedure, and finally a total of 1079 wells containing single effector cells are selected. The 1079 microwells are selected for the next step. Then, the target cells contained in the same well are counted.

[0109] Note that the results are obtained by loading 30 μl of 1.2 × 10 5 The CAR-NK cells were suspended in culture medium at a concentration of 10 cells / mL, and then 30 μl was loaded at 3 × 10 6 In the control case, only 30 μl of 1.2×10 cells / mL of culture medium were inoculated into each microchannel. 5 Cells / mL were suspended in the culture medium of CAR-NK cells (CAR-NK cell viability control) or only 30 μl was inoculated at 3×10 6 cells / mL of medium containing Raji cells suspended in water (negative control).

[0110] Cell death was determined by PI staining after 24 h of co-culture.

[0111] For easy reference, Figure 7 Some images of micropores are shown in Figure 7 In A, a microwell is shown where the CAR-NK cells are inactive because there are no dead target cells. Figure 7 Microwells showing the presence of serial killing CAR-NK cells are shown in B. From left to right, it is noted that there are 1 to more than 5 dead target cells in each well.

[0112] Advantageously, an automated rapid fluid exchange system in the wells allows PI re-labeling every 12 hours to compensate for natural label loss.

[0113] The obtained results are plotted as a graph, such as Figure 8 For each donor, the number of NK cells that killed 1 or 2 target cells is shown ( Figure 8 A), and the number of NK cells that killed 3 or more target cells ( Figure 8 B) Finally, Figure 8 C shows the potency score for each donor as defined according to the present invention. The potency score identifies donor D4 as the most potent donor, being the donor with the highest potency score.

[0114] Example 3: Characterization of 3 batches (Batch D1 to D3) for allogeneic cell therapy.

[0115] NK cells and target cells from 3 different donors were loaded onto the same 16-channel microfluidic device. The target cells used were the K562 cell line. After 24 hours of co-culture incubation, the percentage of NK cells that were able to kill 1 target cell (called killer NK cells) and the percentage of NK cells that were able to kill 2 or more target cells (called serial killer NK cells) were evaluated.

[0116] The results are as follows Fig. 9 , which shows that donor D1 has a higher potency compared to the other two donors tested in the same experiment.

[0117] Example 4: Multiparameter phenotypic / functional analysis.

[0118] NK cells from donor A and donor C were provided and placed in a microfluidic device as described in previous examples.

[0119] The NK cell population is co-cultured with a heterogeneous target cell population expressing phenotype A or phenotype B. The potency of the effector cells is then calculated according to the method of the present invention.

[0120] like Fig.10As shown, for donor C( Fig.10 B), the increase in killing efficacy against cells with phenotype B is obvious.

Claims

1. A method suitable for determining the efficacy of a cell therapy or an immunotherapy, the method comprising: a. providing a first group of microwells, each microwell in the first group of microwells comprising an effector cell and a target cell; b. Optionally, providing a second set of microwells, each microwell in the second set of microwells containing a target cell; c. selecting a microwell containing a single effector cell and n target cells, wherein n is between 1 and 50, preferably between 1 and 20; d. maintaining the cells in culture for a period of time t; e. determining the number of dead target cells in each microwell selected to contain a single effector cell; and f. Calculate the potency score, which consists of the weighted average of the number of target cells killed by each effector cell.

2. The method according to claim 1, wherein: n is between 5 and 15, or between 7 and 10, or between 10 and 15.

3. The method according to claim 1 or 2, wherein: The potency score was calculated by subtracting the average number of target cells killed in the second set of microwells from the average number of target cells killed by each effector cell.

4. The method according to claim 1, wherein: The time t is 0 hours to 120 hours, or about 72 hours or about 24 hours.

5. The method according to any one of claims 1 to 4, wherein: The method is performed in a microfluidic system, wherein the method comprises: - providing an inverted open microwell system (1), said inverted open microwell system (1) comprising an array of open microwells (2), at least one microchannel (3), at least one input port (8) for reagents and / or for one or more biological samples and at least one output port (10) for reagents and / or for one or more biological samples, said input port and said output port being in microfluidic communication with one or more of said microchannels (3), wherein said microchannel (3) has a cross-sectional area of ​​micrometer size and provides fluid to said microwells (2); - Providing an automated management system for the inverted open microwell system, the automated management system comprising: an incubator with controlled temperature, humidity and CO2, a fluid distribution system, phase contrast and fluorescence image acquisition; - placing the inverted open microwell system (1) in the automated management system; - loading reagents through one or more of the input ports (8), wherein the reagents include: filling buffer and / or washing solution and / or one or more drugs and / or one or more dyes and / or one or more labeled antibodies and / or one or more cell viability markers; - Load at least two cell populations; - staining the cells with one or more dyes and / or one or more labeled antibodies and / or one or more cell viability markers; and - Regularly re-perfusing the culture with fresh medium, optionally comprising one or more drugs and / or one or more dyes, and / or one or more labeled antibodies and / or one or more cell viability markers.

6. The method according to any one of claims 1 to 5, wherein: Each microchannel was inoculated with 30 μl of 1.2×10 5 Resuspend the effector cells in culture medium at a concentration of 3 × 10 cells / mL and 30 μL of 5% CO 6 Resuspend the target cells in culture medium at a concentration of 10 cells / mL.

7. The method according to any one of claims 1 to 6, wherein: The effector cells are NK lymphocytes and / or T lymphocytes, and the effector cells are optionally genetically modified.

8. The method according to claim 1, wherein: The cells are cultured for at least 24 hours, such as 48 hours or 72 hours, with regular reperfusion of the culture, wherein the reperfusion is performed once within a period of between 1 hour and 30 hours, such as once every two hours, or once every 12 hours, or once every 24 hours.

9. The method according to claim 5, wherein: The microfluidic system comprises 16 microchannels (3).

10. The method according to claim 5, wherein: The microfluidic system comprises 1200 open microwells (2).

11. A method for determining long-term cell-mediated cytotoxicity, wherein: The method comprises: - providing at least two cell populations, wherein at least a first cell population comprises effector cells grown in suspension and at least a second cell population comprises target cells; - co-culturing the cells for at least 24 hours, such as 48 hours or 72 hours, regularly re-perfusing the culture, wherein the re-perfusion is performed once within a period of between 1 hour and 30 hours, such as once every two hours, or once every 12 hours, or once every 24 hours; and -Assess cell viability.

12. The method according to claim 11, wherein: The co-cultivation continued over at least 72 hours, and the reperfusion was performed once within a period between 10 hours and 26 hours.

13. The method according to claim 11, wherein: The method is performed in a microfluidic system.

14. The method according to claim 13, wherein: The method comprises: - providing an inverted open microwell system (1), said inverted open microwell system (1) comprising an array of open microwells (2), at least one microchannel (3), at least one input port (8) for reagents and / or for one or more biological samples and at least one output port (10) for reagents and / or for one or more biological samples, said input port and said output port being in microfluidic communication with one or more of said microchannels (3), wherein said microchannels (3) provide fluid to said microwells (2); - Providing an automated management system for the inverted open microwell system, the automated management system comprising: an incubator with controlled temperature, humidity and CO2, a fluid distribution system, phase contrast and fluorescence imaging; - placing the inverted open microwell system (1) in the automated management system; - loading reagents through one or more of the input ports (8), wherein the reagents include: filling buffer and / or washing solution and / or one or more drugs and / or one or more dyes and / or one or more labeled antibodies and / or one or more cell viability markers; - Load at least two cell populations; - staining the cells with one or more dyes and / or one or more labeled antibodies and / or one or more cell viability markers; and - Regularly re-perfusing the culture with fresh medium which ultimately comprises one or more drugs and / or one or more dyes, and / or one or more labeled antibodies and / or one or more cell viability markers.

15. The method according to claim 14, further comprising: - at time point T0, acquiring an image from one or more of said microwells (2); - at time point T1, acquiring an image from one or more of said microwells (2); Therein, the steps of loading and acquiring images are repeated over time.

16. The method according to any one of claims 11 to 15, wherein: The at least two cell populations are 1.2×10 5 The effector cell population was loaded at a concentration of 3.3 × 10 cells / mL and 6 The target cell population was loaded at a concentration of cells / mL.

17. The method according to any one of claims 11 to 16, wherein: The at least first cell population comprises NK lymphocytes and / or T lymphocytes, and the at least first cell population is optionally genetically modified.

Images