Method to define a dose-response

Abstract

The present invention relates to an in vitro method for defining a D / R dose- response curve, measured on target cells in a microfluidic system. The method is particularly advantageous when the therapeutic agent is an effector cell, or the therapeutic effect is mediated by an effector cell, as is the case with immunotherapies.

Description

[0001] METHOD TO DEFINE A DOSE-RESPONSE

[0002] TECHNICAL FIELD

[0003] The present invention relates to an in vitro method for obtaining a dose-response curve to define the efficacy or potency of immunotherapies, including cell therapies.

[0004] BACKGROUND

[0005] The development of a new medicine follows a long and complex path. Starting from the first experimental observations in which promising therapeutic applications are proposed, it is proceeded with an extensive pre-clinical phase in which the safety and efficacy of the medicine must be ascertained through in vitro and laboratory animal studies (in vivo studies). If the experimental data obtained are sufficient, the clinical trial phases are proceeded with. At the same time, the production process of the medicine is perfected, first on a small scale and then on an industrial scale.

[0006] The critical transition from preclinical to clinical studies in humans must ensure the maximum possible safety for the subjects undergoing the trial. The experience of the last twenty years has led to the standardization of validated animal models in order to guarantee the legitimacy of the results as much as possible. While for chemical molecules "similarity" can often guide such studies, the greater degree of complexity of the "biological" drugs (antibodies, enzymes, hormones, growth factors, cells) has taken more than a decade to move from the first studies, however promising, to the registration of the actual medicinal product. By way of example, in immunotherapies the heterogeneity of the immune system makes the transition from preclinical to clinical particularly complex.

[0007] Medicinal products for cell therapy, gene therapy and tissue engineering, classified as Medicinal Products for Advanced Therapies (ATMP), are included in the legislative framework of the drug and not in that of transfusion or transplant medicine. This requires that the trial must comply with the GLP (Good Laboratory Practice), GMP (Good Manufacturing Practice) and GCP (Good Clinical Practice) quality standards. The complexity of these new medicines, which, due to their biological characteristics and their heterogeneity (different histological types, variety of genes, variety of clinical applications, etc.), are unlikely to be subject to some of the preclinical and clinical trial standards applicable to traditional drugs, has created a new scenario and a new challenge.

[0008] As far as cell therapy is concerned, the guideline "Human cell-based medicinal products" (CHMP / 410869 / 06) was published in 2008 where the method for identifying which studies are necessary, providing a list of factors that can increase or reduce the risk associated with the cell therapy medicinal product, was introduced. In addition, it defines terms such as pharmacodynamics or pharmacokinetics applied to products containing live cells, and describes the possible necessary safety studies.

[0009] It is therefore evident that the heterogeneity of ATMPs adds an element of complexity due to the need to fine-tune the production process, normally developed ad hoc, an activity that requires monitoring the product during production in a simple, fast and low material expenditure.

[0010] The need is much felt to have in vitro methods that can provide information on the potency of the product under development in a consistent and stable way, so that they can be used in the early stages of research and development and also during the development of the production process, where it is necessary to test the samples obtained quickly and robustly, with the least possible expenditure of material.

[0011] DESCRIPTION

[0012] The present invention relates to a method implemented in a microfluidic system to obtain in vitro a dose-response curve functional to define the efficacy or potency of immunotherapies, including cellular therapies.

[0013] The method of the invention can be used to predict whether administration of a therapeutic agent to a patient will trigger a response to the therapeutic agent or to monitor a patient's response to an ongoing therapy or to determine the potency of the therapeutic agent, using a reference cell line as a target. In a further application, said method may be used to test the efficacy of an agent on a target of potential pharmacological interest.

[0014] DESCRIPTION OF THE FIGURES

[0015] Figure 1: Flowchart of an embodiment of the method according to the present invention.

[0016] Figure 2: (A, B) Flowchart of an embodiment of the method according to the present invention.

[0017] Figure 3: D / R curves obtained with the method according to the present invention on NK cells: (A) curve obtained by interpolating the points obtained for each of the E:T ratios analysed. (B) curves obtained with stimulated (solid line) and unstimulated (dashed line) cells. Figure 4: (A) Ability of NK cells to kill K562 cells, evaluated inter -experiment (normalized data on Oh, n=16); (B) by focusing on the points collected with E:T=2 (8E and 4T), an excellent correlation is derived between the method according to the present invention and the imaging of the known art.

[0018] Figure 5: (A) representative plot of three cell / well distribution curves, obtained with three different loading concentrations; (B) Linear regression that allows to interpolate the desired peak and obtain the resulting concentration to be loaded into the microfluidic device.

[0019] Figure 6: Comparison between distribution of experimental and theoretical effector cells. (A) experimental NK distribution between a triplicate D / R curve (n=3) in wells with 4T / well. (B) correlation between theoretical and experimental distribution in wells with E:T equal to 0.25, 0.5, 1, 2 and 4.

[0020] Figure 7: Schematic representation of the microfluidic device used in an embodiment of the method according to the present invention.

[0021] Figure 8: The plot shows the optimization of the concentration of cells to be loaded, based on theoretical distribution curves of target and effector cells in the receptacles. Dashed line: expected distribution in the receptacles of E cells, when loaded in two channels at the two different concentrations A, B, which concentrations lead to an expected distribution represented by the solid black lines A and B. Solid grey line: expected Poisson distribution based on the concentration of loaded target cells.

[0022] Figure 9: D / R curves on NK cells: (A) curve obtained by interpolating the points obtained for each of the E:T ratios analysed, without selecting the receptaclesa. (B) curve obtained according to the method of the present invention.

[0023] DEFINITIONS

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention relates.

[0025] The term "about" or "approximately" as used in the present document denotes a variability within 10%, more preferably within 5%, of a given value or range.

[0026] By "microfluidic" system is meant a device that employs small volume fluid samples, for example from 10 nl to 30 pl. In such microfluidic devices, the fluid samples are typically contained and transported in microchannels with sizes in the range of, for example, 10 |im to 500 m. By way of example, a microfluidic device used in the method according to the present invention is the device described in WO2017216739A1. By "receptacle" is meant a volume in the micrometre scale defined by a culture medium that comprises or does not comprise cells. By way of example, said receptacle has sizes between 10 and 100 pm, for example in droplet-based systems, or between 100 pm and 1000 |im, for example in microwell systems capable of accommodating complex structures such as spheroids. For example, the definition of "receptacle" according to the present invention includes the volume of culture medium present inside a well or a microwell, or of a hanging drop, or a droplet inside a microfluidic system capable of generating droplets of a first fluid inside a second fluid immiscible with the first or of microfluidic structures capable of trapping cells transported by a fluid, also through the use of forces capable of moving cells, such as dielectrophoresis, electro-acoustic forces, forces capable of modifying the wettability of surfaces, light waves (e.g. optical tweezers) and optofluidic forces.

[0027] By "microwell" as used in the present document is meant a vessel of micrometre size (less than 1000 |im), including the height, cross-sectional area, e.g. diameter when the microwell is tubular, and the volume.

[0028] "Hanging drop" refers to the hanging-drop culture method as described by Rasouli M. et al. (2024) Principles of Hanging Drop Method (Spheroid Formation) in Cell Culture. In: Methods in Molecular Biology. Springer, New York, NY. https: / / doi.org / 10.1007 / 7651_2024_527.

[0029] By the term "with high content" is meant a method of phenotypic analysis conducted in cells which involves the analysis of whole cells or cellular components with simultaneous reading of different parameters, typically performed by acquiring phasecontrast and / or fluorescence microscope images and analysing them.

[0030] By "treatment" is meant the therapeutic treatment of cells in vitro or of a subject where the goal is to ameliorate or slow down (reduce) the condition or disorder of the target disease, or one or more symptoms associated therewith.

[0031] "Response" or "responsive" refers to a cell or subject exhibiting at least one altered characteristic after treatment. Similarly, "responsive to" or "responds" and similar terms refer to indications that the target disease condition, or one or more symptoms associated therewith, is prevented, ameliorated, or diminished in the in vitro cell or in the subject. By way of example, the reduction of the number of tumour cells or of a tumour mass rather than the haematological response, defined according to criteria known to the experts in the field, are considered responses.

[0032] "Therapeutic agents" or "agent" according to the invention are a type of treatment consisting of molecules including, without limitation, polypeptides, peptides, antibodies, monoclonal antibodies, glycoproteins, nucleic acids, drugs of synthetic or natural origin, polyenes, macrocytes, glycosides, terpenes, terpenoids, aliphatic and aromatic compounds and their derivatives, cells. In a preferred embodiment, the therapeutic agents are ATMP, even more preferably they are cells.

[0033] The term "Tissue-engineered products" means cells and / or tissues of human or animal origin, expanded "in vitro" or that in any case have undergone "extensive manipulation" for therapeutic purposes or for the purpose of transplantation on patients.

[0034] By "Immunotherapy" is meant the treatment of a disease by activating or suppressing the immune system. Immunomodulators include a wide range of recombinant, synthetic and natural preparations, such as interleukins, cytokines, chemokines. A further class of immunotherapies comprises monoclonal antibodies that act by favouring the activity of immune cells against a target, for example the checkpoint inhibitors, the so-called engagers, bi-specific antibodies, antibodies capable of activating ADCC (Antibody-Dependent Cell-Mediated Cytotoxicity). Then there are cell-based immunotherapies. Immune effector cells are e.g. lymphocytes, macrophages, dendritic cells, natural killer cells (NK cells), cytotoxic T lymphocytes (CTLs).

[0035] "Cell therapy" is, in particular, understood as the administration to humans for therapeutic, diagnostic and prophylactic purposes of autologous or heterologous live human cells that have undergone any type of ex vivo manipulation, including their propagation in vitro. Another type of cell therapy is given by the in-vivo cell therapies, capable of inserting genetic material by acting directly in the human body, for example mRNA technologies or using nanoparticles or adeno-associated viruses.

[0036] Autologous is defined as using cells and / or tissues from the same patient, allogeneic as using donor-derived cells and / or tissues, and xenogenic as using cells and / or tissues of animal origin.

[0037] In the "Tissue-engineered products" the cells can be the only constituents or can be associated with biomaterials of different origin and characteristics.

[0038] "Extensive manipulation" of cells and tissues means all those manipulations that can lead to cell activation and / or stimulation of cell proliferation. The cells that, although not specifically activated or stimulated to proliferate, are associated with biomaterials, whether synthetic biomaterials or biomaterials of extractive origin, are also considered "extensively manipulated". All cells that have undergone a manipulation of their genetic heritage are considered "extensively manipulated".

[0039] "Minimal manipulation" means all those manipulations that do not lead to cell activation and / or stimulation of cell proliferation. In particular, the following are considered "minimal manipulation": cutting, shape modification, cry opreservation, sterilization by gamma radiation, centrifugation and treatment of a tissue with antibiotics. Similarly, "minimal manipulations" are considered the extraction or separation of cells from a tissue, when this does not alter the main characteristics of the cells and the remaining tissue from which the cells were extracted or separated.

[0040] A "dye" or "marker" indicates a molecule, compound, or substance that can provide an optically detectable signal, such as a colorimetric, luminescent, bioluminescent, chemiluminescent, phosphorescent, or fluorescent signal. In a preferred embodiment of the invention, the dye is a fluorescent dye. Non-limiting examples of dyes include CF dyes (Biotium, Inc.), Alexa Fluor dyes (Invitrogen), DyLight dyes (Thermo Fisher), Cy dyes (GE Healthscience), IRDyes (Li-Cor Biosciences, Inc.), and HiLyte dyes (Anaspec, Inc.). In some embodiments, propidium iodide, calcein, annexin V, caspase 3- 7 are markers used to determine the viability / mortality of the cells or their state of apoptosis. In some embodiments, the excitation and / or emission wavelengths of the dye are between 350 nm and 900 nm, or between 400 nm and 700 nm, or between 450-650 nm. In one embodiment, a marker is an antibody used to characterize the immunophenotype, a marker of viability, apoptosis, an antibody that exhibits protein phosphorylation and pathway activation.

[0041] The term "imaging time-lapse" in the present document means the acquisition of multiple images of the same field performed at later times.

[0042] A "dose-response curve" is a semi-logarithmic plot. The x-axis shows, in logarithmic form, the concentration of the therapeutic agent under study, where in case the therapeutic agent consists of an immunotherapeutic agent, the concentration is usually defined by the ratio between effector cells and target cells. The dose-response curve allows to obtain, for said therapeutic agent, parameters such as maximum efficacy, potency, EC50 (concentration at which 50% of the effect occurs), EC99, Hill coefficient. By way of example, a Hill coefficient =1 indicates that from 5% to 95% of the response there are two orders of magnitude, i.e. the dose must be increased 100-fold.

[0043] DETAILED DESCRIPTION

[0044] The method described herein is an in vitro method for defining the D / R doseresponse curve, measured on target cells. The method is particularly advantageous when the therapeutic agent is an effector cell, or the therapeutic effect is mediated by an effector cell, as is the case with immunotherapies.

[0045] In one embodiment, summarized in the block diagram of Figure 1, the method according to the present invention comprises the following steps: Step [1]: imaging time-lapse , with the acquisition of at least one image at time 1 (tl) and at least one image at time 2 (t2) of a plurality of receptacles comprising target cells and effector cells, where said images show in a punctual manner the cells contained in each receptacle of said plurality of receptacles, obtaining a multiplicity of images at tl and a multiplicity of images at t2;

[0046] Step [2]: a step of acquisition of a numerical value representative of the number of target cells and the number of effector cells, where present, included in said multiplicity of images;

[0047] Step [3]: a step of acquisition of a numerical value representative of the number of live target cells and the number of dead target cells included in said multiplicity of images at t2;

[0048] Step [4]: a step of selection of the receptaclesa, which are the subset of receptacles comprising a target cells at tl;

[0049] Step [6]: acquisition of information that defines the dose-response curve, where said dose-response curve is obtained by considering:

[0050] - ratio of live target cells in said multiplicity of images at t2 in said subset of receptaclesa to total target cells in said multiplicity of images at tl or t2 in said subset of receptaclesa; number of effector cells in each of said images of said multiplicity of images tl.

[0051] In one embodiment, in said Step [6] said ratio is calculated as a ratio of live target cells in said multiplicity of images at t2 in said subset of receptaclesa to total target cells in said multiplicity of images at tl in said subset of receptaclesa.

[0052] In one embodiment, in said Step [6] said ratio is calculated as a ratio of live target cells in said multiplicity of images at t2 in said subset of receptaclesa to total target cells in said multiplicity of images at t2 in said subset of receptaclesa. Said embodiment is particularly useful to compensate for the possible variation of the total target cells over time due, for example, to the proliferation thereof.

[0053] In one embodiment, said tl is the time immediately following seeding of the coculture.

[0054] In one embodiment, said method also comprises a Step [5] where, within the subset of receptaclesa, N subsets of receptaclesN are identified, wherein each of said subsets of receptaclesN contains an equal number of effector cells, the number of E cells being between m E cells and n E cells. In one embodiment, n - m = N.

[0055] The ratio of E cells to T cells found in the selected receptacles is between m / a and n / a. By way of example, this ratio is between:

[0056] 1:10 and 1:1; or

[0057] 1:1 and 10:1 or

[0058] - 1:10 and 5:1.

[0059] In a preferred embodiment, m = 1 and n = a2and said E / T ratio is between 1 / a and a. In this embodiment, the D / R curve is symmetrical with respect to the value 1 on the x axis. By way of example, this ratio is between 1:4 and 4:1 and N = 16, i.e. there are 16 subsets of receptacles, each subset representing a different E / T ratio.

[0060] Having N subsets of receptaclesN, in said Step [6] the information defining said dose-response curve are N points, each of which is obtained by analysing a multiplicity of images for each of said N subsets. By way of example, where N = 16, said doseresponse curve is defined by 16 points, each calculated from the analysis of the images of the corresponding subset of receptaclesN. In one embodiment, this analysis is the mean of the T cell viability at t2 measured in each of said receptacles belonging to the subset of receptaclesN.

[0061] In one embodiment, said images are acquired from at least 100 receptacles. In one embodiment, said images are acquired from at least 100 receptaclesaeach comprising a T cells. In one embodiment, said images are acquired from at least 100 receptaclesaeach comprising a number of E cells between m and n.

[0062] In one embodiment, multiple brightfield images at different focus levels and in multiple fluorescence channels are acquired by a fluorescence microscope for a same receptacle at the same time.

[0063] In one embodiment, Figure 2A, said Step [2] comprises identifying in said images the areas corresponding to each receptacle, each comprising one or more cell types, and assigning to each receptacle an ID reference.

[0064] In one embodiment, Figure 2A, said Step [2] comprises identifying the cells contained in each receptacle at each time, where each cell corresponds to a region of interest (ROI), i.e. an area of the image containing a series of pixels. Preferably, said identification is carried out automatically, through a machine learning process.

[0065] In one embodiment, Figure 2A, each cell identified in said Step [2] is correlated to the ID of the receptacle to which said cell belongs. In one embodiment, Figure 2A, said Step [2] comprises classifying the cell type. Such classification is performed using a combination of morphological properties of each cell (e.g., ROI size, elongation) and / or intensity of the pixels contained in the ROI for one or more fluorescence channels.

[0066] In one embodiment, Figure 2A, in said Step [3] each cell is classified as live or dead using a live / dead fluorescent marker and setting an associated threshold where the dead cells are those negative / positive to said live / dead marker.

[0067] By way of example, said cell viability marker is selected between Propidium Iodide (PI) and Calcein.

[0068] In one embodiment, Figure 2B, said Step [6] comprises preparing a plot on the Cartesian plane containing a2data points, one data point for each subset of receptacles a2, where the x-value of each point is E / T at tl and the y-value is the ratio between the number of live T-cells and the total number of T cells contained in said subset at t2.

[0069] In one embodiment, Figure 2B, said Step [6] comprises determining the equation of the sigmoid D / R curve (dose-response) that best fits the points on said plot, thereby defining the key parameters of the curve that reflect the power measurements (e.g. EC50, IC50).

[0070] In one embodiment, the method is performed in the open-well microfluidic system described in WO2017 / 216739.

[0071] Briefly, said microfluidic device is an inverted open microwell system schematized in Figure 6 comprising a series of open microwells (2), at least one microchannel (3), at least one inlet port (8) for reagents and / or for one or more biological samples and at least one outlet port (10) for them, said inlet and outlet ports being in microfluidic communication with one or more of said microchannels (3), wherein said microchannel (3) has a cross-sectional area of micrometric sizes and provides fluid to said microwells (2), wherein said inverted open microwell system is, in one embodiment, inserted into an automated management system comprising the following features: a controlled temperature, humidity and CO2 incubator, fluid distribution system, phase-contrast and fluorescence image acquisition. Said automated management system is obtained by assembling elements known in the art such as temperature, humidity and CO2 control incubator, microplate pipetting systems, fluorescence and phase-contrast microscopy lenses connected to an image acquisition camera, such as a CMOS or CCD camera, where said elements are managed in whole or in part by software known to the experts in the field through hardware connected thereto.

[0072] In a particularly preferred embodiment, each microchannel (3) is associated with an inlet port (8) and an outlet port (10).

[0073] In a preferred embodiment, the microfluidic device (1) also comprises reservoirs, wherein said reservoirs are at least one reservoir for reagents and at least one reservoir for one or more biological samples. Said reservoirs are selected from the group comprising: plates, one or more multi-well plates, such as 96-well plate, Eppendorf tubes. Said reservoirs may be 2, or 4, 8, 16, 24, 48, 96, 384.

[0074] In one embodiment, as reported by the plot of Figure 8, when said method is performed in the microfluidic device described above, two or more of said microchannels (3) are loaded with T cells, where the suspension of said T cells used for loading each of said channels is at the same concentration, such as to generate a Poisson distribution (grey line). Conversely, E cells are loaded using cell suspensions at different concentrations for each microchannel (channel A and channel B, black solid lines). The authors demonstrated how selecting these concentrations based on expected Poisson distributions maximizes the number of microwells containing the desired number of E and T cells (dashed line). Advantageously, this means that, for each experiment, the selected subset of receptacles will be as large as possible, thus limiting the number of receptacles to be discarded as not satisfying the selection criteria.

[0075] Advantages:

[0076] In vitro analysis of the potency by means of conventional immune cell killing assays requires a large number of cells and considerable manual work to construct doseresponse curves that are reproducible. Typically, there is a need to evaluate each type of E cell against 3-5 T cell lines, with different sensitivity to cell-mediated cytotoxicity. For each E-cell - T-cell pair, typically up to eight E: T ratios are tested, each in triplicate. This, with the methods known in the art, requires the availability of over a million effector cells, making it extremely expensive and consequently reducing the feasibility of complete studies.

[0077] The method proposed herein, allowing to conduct miniaturized assays that allow to measure thousands of samples, where, for each sample, only one or a few effector cells are used on a number in the order of units of target cells, generates dose-response curves with a drastic reduction in the quantities of cells required and working time. The results obtained are statistically robust, as also evidenced by the examples below, since the measurements are carried out on a large number of samples, where in each of these the parameters are defined.

[0078] By way of example, to obtain a result that, with the methods of the prior art, would require the use of at least 1 million effector cells, about 300,000 effector cells are sufficient with the method described herein, which also enables to reduce the steps of manual intervention, for example, of use of the pipettor, from over 300 to less than 20, thus allowing a saving of time and above all a reduction in errors attributable to the operator.

[0079] EXAMPLES

[0080] The following examples are merely intended to show some embodiments of the present invention, they are not intended in any way to be limiting thereof. The scope of the invention is defined by the claims.

[0081] Example 1: dose-response curve of stimulated or unstimulated NK effector cells

[0082] The example was conducted in a device of the type described in WO2017 / 216739.

[0083] Target cells K562, human myeloid leukaemia cell line, were loaded, through the microfluidic channels, into 720 microwells. The loading took place starting from cells in suspension at a concentration such as to maximize the number of microwells so that they contained 4 target cells (see Example 3).

[0084] Two different populations of NK effector cells were then added to the same microwells, said two populations being NK cells stimulated with IL-2100U / mL, 72 hours or unstimulated (both isolated from PBMCs from healthy donors).

[0085] Images are then acquired at tl from each of the loaded microwells.

[0086] The analysis of the images obtained from each of the loaded microwells classifies each of them based on the number of T cells it contains, subset of receptaclesa, and the number of E cells it contains, subset of receptaclesN, grouping the wells based on the E:T ratios. The T cells were stained with 7pM CellTracker™ Blue CMAC, death was detected by Propidium Iodide.

[0087] 16 different E:T ratios were generated using one pair of channels per D / R curve. This makes it possible to compare up to 8 combinations of E and T cell pairs in the 16 channels available on each plate.

[0088] The dose-response curves obtained with stimulated and unstimulated NK cells are reported in Figure 3. Each curve was generated using 16 points corresponding to 16 E:T ratios, the values of which were determined from a total of 241 microwells containing E:T ratios in the range of interest (Figure 3A). From the curves, it was possible to obtain the IC50 values for each pair of target-effectors, showing a decrease in the viability of the target cells over time and an adaptation of the 24-hour D / R curves for stimulated (R2=0.94) and unstimulated NK (R2=0.82) where the maximum mortality is reached with the highest E:T ratio tested. The comparison between IC50 values (Figure 3B) confirms, as expected, that the NK cells have a higher potency, as evidenced by the lower value of the IC50 parameter for the stimulated cells (IC50=l.l) compared to the unstimulated cells (IC50=2.24).

[0089] Example 2: dose-response curve obtained with the method according to the present invention or with the method according to the known art

[0090] A same NK effector cell population was tested with 8 replicates on two different days in co-culture with K562 target cells. The viability of K562 decreases over time and in relation to the E:T ratio, as shown in Figure 4A. At the same time, the same effector cell population was tested with a traditional image-based technology for mass analysis of target cell killing by immune cells, by seeding in a 96-well plate a constant number of target cells per well equal to 10,000 and a variable number of effector cells to generate cocultures at variable ratios between 1:4 and 4:1. Comparison of the experimental data obtained with the method described here and with traditional image-based technology reported a correlation higher than 90%, as reported in Figure 4B, where we focused on the data points from the E:T ratio = 2, i.e. obtained in those wells comprising 8 effector cells and 4 target cells at the initial time.

[0091] Example 3: microwell loading method

[0092] Using the microfluidic device used in Example 1, the need arose to maximize the efficiency of loading the cells into the microwells, with the aim of obtaining as many microwells as possible comprising a predefined E:T ratio, so as to limit the number of wells to be discarded and maximize the number of wells to be used for subsequent analyses.

[0093] In this example, it was set a = 4, that is, it was chosen to work in the subset of receptacles4 and studied the loading concentration of the effectors to identify a mathematical model to correlate the distribution of the experimental effectors and the maximized peak E / well. PBMC effector cells were loaded at 8 different concentrations. Each loading concentration was correlated to the maximum peak of cells / well. From here, a linear equation was calculated from which to extrapolate the loading concentration of the effector cells so as to maximize the number of wells comprising the desired E:T ratio (Figure 5).

[0094] With the microfluidic device, the trend of the NK effector cell load on 3 different donors was evaluated by applying the above linear equation, setting two effector concentrations to cover the entire E:T range (1:4 - 4:1). The loading statistics correlate well with the theoretical Poisson distribution, as shown in Figure 6.

[0095] Example 4: operation of the method according to the present invention versus the method of the known art

[0096] With the aim of highlighting that the method proposed here not only allows obtaining an accurate and reproducible dose-response curve but also allows a considerable saving of time and reagents, Table 1 shows a comparison of the steps required by the method according to the known art compared to those of the method according to the present invention, where the two methods were used to generate a 16- point D / R curve. It should also be highlighted that with the method according to the present invention it is passed from 1 million cells to only 300 thousand, without losing reproducibility. Table 1

[0097] Example 5: dose-response curve and factor R2

[0098] An effector NK cell population was tested with 8 replicates on two different days in co-culture with K562 target cells. The experiment was conducted according to the method of the present invention, and the results are reported in Figure 9B. For comparative purposes, the experiment was conducted in parallel following the same method but omitting Step [4], i.e. without selecting the receptaclesa, which are the subset of receptacles comprising a target cells at fl. The results of the comparative experiment are reported in Figure 9A. The experiments clearly show that the factor R2is much lower if the selection is not made, i.e. 0.80 in the plot of Figure 9A versus 0.94 in the plot of Figure 9B. The factor R2indicates how close the points are to the interpolation curve, R2is 1 in an ideal situation where each point is perfectly placed on the curve.

[0099] The comparative experiment conducted therefore shows that the selection step improves the quality of the result significantly.

Claims

CLAIMS1. An in vitro method for defining a D / R dose-response curve measured on target T cells, wherein the therapeutic agent is at least one effector E cell, said method comprising:Step [1]: imaging time-lapse, with the acquisition of at least one image at time 1 (tl) and at least one image at time 2 (t2) of a plurality of receptacles comprising T cells and E cells, where said images show in a punctual manner the cells contained in each receptacle of said plurality of receptacles, obtaining a multiplicity of images at tl and a multiplicity of images at t2;Step [2]: a step of acquisition of a numerical value representative of the number of T cells and the number of E cells included in said multiplicity of images;Step [3]: a step of acquisition of a numerical value representative of the number of live T cells and the number of dead T cells included in said multiplicity of images at t2;Step [4]: a step of selection of the receptaclesa, which are the subset of receptacles comprising a target cells at tl;Step [6]: acquisition of information that defines the dose-response curve, where said dose-response curve is obtained by considering:- ratio of live T cells in said multiplicity of images at t2 in said subset of receptaclesa to T cells in said multiplicity of images at tl or t2 in said subset of receptaclesa; number of E cells in each of said images of said multiplicity of images tl.

2. The method according to claim 1, wherein in said Step [6] said ratio is calculated as a ratio of live T cells in said multiplicity of images at t2 in said subset of receptaclesa to total T cells in said multiplicity of images at tl in said subset of receptaclesa.

3. The method according to claim 1, wherein in said Step [6] said ratio is calculated as a ratio of live T cells in said multiplicity of images at t2 in said subset of receptaclesa to total T cells in said multiplicity of images at t2 in said subset of receptaclesa.

4. The method according to one of the claims from 1 to 3, said method also comprising a Step [5] wherein, within said subset of receptaclesa, N subsets of receptaclesN are identified, wherein each of said subsets of receptaclesNcontains an equal number of E cells, the number of E cells being between m E cells and n E cells.

5. The method according to claim 4, wherein m = 1 and n = a2.

6. The method according to one of the claims from 1 to 5, wherein said Step [2] comprises identifying in said images the areas corresponding to each receptacle, each comprising one or more cell types, and assigning to each receptacle an ID reference.

7. The method according to one of the claims from 1 to 6, wherein said Step [2] comprises identifying the cells contained in each receptacle at each time, where each cell corresponds to a region of interest (RO I), i.e. an area of the image containing a series of pixels.

8. The method according to one of the claims from 1 to 7, wherein each cell identified in said Step [2] is correlated to the ID of the receptacle to which said cell belongs.

9. The method according to one of the claims from 1 to 7, wherein said Step [2] comprises classifying the cell type, performed using a combination of morphological properties of each cell (e.g., ROI size, elongation) and / or intensity of the pixels contained in the ROI for one or more fluorescence channels.

10. The method according to one of the claims from 1 to 9, wherein said Step [6] comprises preparing a plot on the Cartesian plane containing a2data points, one data point for each subset of receptacles a2, where the x-value of each point is E / T at tl and the y-value is the ratio between the number of live T cells and the total number of T cells contained in said subset at t2.

11. The method according to one of the claims from 1 to 10, wherein said Step [6] comprises determining the equation of the sigmoid curve D / R (doseresponse), defining potency parameters thereon.

12. The method according to one of the claims from 1 to 11, which is implemented in an open-well microfluidic device.

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