Means and methods for determining cell affinity

Abstract

The present invention relates to cell-cell interactions, and in particular to cell affinity. Provided are improved means and methods for studying cell-cell interactions and characterizing cell affinity. More particularly, the methods involve studying cell-cell interactions and applying forces in order to disrupt cell-cell bonds in a controlled manner. In particular, the present inventors provide means and methods with which highly reliable and consistent cell affinity experiments can be performed in a high throughput manner.

Description

Background Art

[0001] Although T cell therapy (including CAR-T cell therapy) has achieved great clinical success for hematological malignancies, the therapy still has many obstacles in treating a wider range of cancers. It is still a challenge to predict the success of T cell therapy using in vitro data alone. The affinity of the binding molecules for CAR generation to the target antigen is often considered during in vitro testing, but this cannot define CAR-T cell activity. With the increase of the complexity of building a new methodology to further improve the clinical outcomes of immunotherapy, the screening method for quickly identifying the best lead candidates becomes more important. So far, it has become obvious that simply measuring the affinity between CAR and its target will not accurately predict the outcome in vitro and in vivo. On the contrary, cell avidity (overall cell binding strength) can provide a more complete and physiologically relevant measurement reflecting the true interaction between T cells and target cells.

[0002] Cellular avidity measurements can better predict in vitro cellular responses and drive better, more informed decisions at the early stages of drug selection and potentially improve clinical outcomes. One of the major obstacles in the process of measuring avidity, which is considered a critical parameter, is the lack of rapid, specific, and accurate tools to assess cellular avidity. The cell affinity analyzer (a platform for measuring cell-cell binding strength) facilitates the direct analysis of CAR-T cells against monolayers of surface-fixed antigens or target cells. Using this system, cell-cell interactions are perturbed using resonant acoustic waves generated by piezoelectric elements, and a cell tracking system is used to measure the required rupture force. In this way, cell affinity measurements can be performed, which provide cell affinity scores that allow comparison of, for example, different candidate receptors.

[0003] The present inventors in their work on cell affinity are seeking means and methods to further improve cell affinity measurements, particularly in a high throughput format. Summary of the invention

[0004] The inventors now provide new and improved methods for assessing cell affinity. In particular, the inventors provide means and methods with which highly reliable and consistent cell affinity experiments can be performed in a high-throughput manner, utilizing forces applied away from attached cells. These means and methods include affinity sample plates with one or more holes, whereby measurements in a highly controllable manner can be performed, the affinity sample plates including multiplexing, such as combining different samples and / or passing through multiple holes. By providing a cell affinity measurement system utilizing the affinity sample plates, the method according to the present invention can be performed highly and effectively. This is in stark contrast to means and methods known in the art that do not allow for high-throughput measurements in a highly controllable manner. BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Figure 1 . Schematic diagram showing the well design of an affinity sample plate. The upper panel shows a single well, with an inlet and an outlet. Depicted in black is a well, for example a well made of a suitable polymer. Depicted on the well surface is a grey antifouling coating, a coating for cell attachment is dark grey, to which target cells attach (dark grey circles). The well is oriented in an upright position. In the well, the fluid is contained, in this case a suspension of effector cells depicted as light grey circles. The middle panel shows a plurality of wells on an affinity sample plate in an upright position, the lower panel shows the sample plate in an inverted position. Between the upper and lower panels, pictograms are shown indicating a change in position from an upright position to an inverted position (or vice versa).

[0006] Figure 2 . A schematic diagram describing the steps of the method according to the present invention. Figure 1 The well design and its schematic representation shown are representative for use in this flow chart. In (1), an empty well is shown in an upright position, with a ceiling surface coated for target cell attachment and an antifouling coating. Target cells are added to the well (2), the affinity sample plate is placed in an inverted position (3) and the cells are allowed to settle (4) and attach. The affinity sample plate is placed in an upright position (5), and target cells attached to the ceiling surface coated for attachment are retained, while target cells that are not attached and / or settled in the antifouling coated area in (4) are not retained and settle downward in (5), (5) may include a centrifugation step. Target cells that are not attached can be removed (6) and images of target cells attached to the ceiling surface in an upright position (6) or in an inverted position (7) can be captured, as depicted on the right, with a microscope located below the well. Next, effector cells are introduced into the well (8), and the effector cells are allowed to settle downward to the bottom of the well (9). The affinity sample plate is placed in an inverted position (10) and the effector cells now settle downward on the target cells (11). Images of effector cells interacting with the target cells can be captured (11). The affinity sample plate is placed in an upright position after a specified time (12). Effector cells that are not bound to the target cells settle downward to the bottom of the wells (13), where they can be removed or collected from the wells (14). Images of effector cells bound to target cells can be captured in an upright position (14) or in an inverted position (15). The affinity sample plate is placed in an upright position (16) and centrifuged, where the applied force can cause the effector cells to become unbound (17), where the unbound cells can be collected or removed from the wells (18). After the centrifugation step, images of effector cells that remain bound to target cells can be captured in an upright position (18) or inverted position (19). Steps (17)-(19) can be repeated with increasing centrifugal force.

[0007] Figure 3 . Cross-section of a well design suitable for an affinity sample plate. A) Depicted is a pipette (1) introducing fluid at the inlet channel (2). Depicted in black are cells attached to the top plate surface. This design includes a reservoir (3) that allows more stable cell culture conditions, reduces osmotic shifts with evaporation, or reduces the risk of drying out the channel. The outlet channel (4) has a short diffusion distance from the top plate surface to the reservoir that will allow effective gas exchange, which is useful for adapting to appropriate cell culture conditions. The well design allows adaptation to different liquid levels (5) and (7), which can change (6), which is useful, for example, for introducing fluid into the well and / or removing fluid therefrom in the method of the present invention. B) On the left, the well is shown in an upright position, and on the right, the well is shown in an inverted position. The surface tension and capillary forces of the fluid at the inlet and outlet channels (depicted in black) allow the fluid to be retained in the well.

[0008] Figure 4 . A well 300 (contained on an affinity sample plate) is schematically shown in cross section, with a flow channel 310 extending between a channel inlet 320 and a channel outlet 320 on the open side, and defining a top plate 312 and a bottom plate 314 in relation to the open side OS. In the upright orientation of the sample holder, the open side OS is upward. Thus, the channel can be loaded from above via the inlet 320. In A), the channel is loaded with fluid and target cells 330. In B), the affinity sample plate is turned over and a layer of target cells is formed. In C), the well is turned back to the upright position, cleaned, and inserted into a centrifuge holder, and in D), centrifugal force is then applied to prevent the ingress of air bubbles. DETAILED DESCRIPTION

[0009] Therefore, the present invention provides means and methods for evaluating the cell affinity of effector cells and target cells. In one embodiment, a method for evaluating the cell affinity of effector cells and target cells is provided, the method comprising the following steps:

[0010] a) providing target cells at a specified concentration;

[0011] b) optionally, staining the target cells;

[0012] c) providing an affinity sample plate having one or more wells, the wells having a top plate surface coated for target cell attachment, wherein optionally at least the bottom surface is anti-fouling coated, wherein the affinity sample plate is optionally provided with an identifier, and optionally provided with one or more sensors;

[0013] d) with the affinity sample plate in an upright position, introducing the homogenized target cells into one or more wells of the affinity sample plate for obtaining a monolayer of the target cells;

[0014] e) placing the affinity sample plate in an inverted position, thereby allowing the target cells to settle to the top plate surface and attach thereto to form a monolayer;

[0015] f) optionally, culturing the target cells in a controlled incubator with the affinity sample plate in an inverted position or an upright position;

[0016] g) optionally, blocking the top plate surface having attached target cells with a blocking agent;

[0017] h) placing the affinity sample plate in an upright position, optionally in a centrifuge and centrifuging to spin down target cells that are not attached to the top plate surface;

[0018] i) optionally, washing the cells with the affinity sample plate in an upright position to remove target cells that are not attached to the top plate surface;

[0019] j) Optionally, acquiring an image of a monolayer of target cells attached to the surface of the top plate.

[0020] k) optionally, inspecting the quality of the monolayer of target cells based on the image of the monolayer;

[0021] 1) providing effector cells at a specified concentration;

[0022] m) optionally, and preferably, staining effector cells;

[0023] n) introducing the homogenized effector cells into one or more wells of an affinity sample plate in an upright position, and optionally allowing the effector cells to settle to the bottom of the affinity sample plate;

[0024] o) placing the affinity sample plate in an inverted position to allow the effector cells to settle on the monolayer of target cells attached to the top plate of the affinity sample plate, and incubating the effector cells with the target cells for a specified time;

[0025] p) optionally, with the affinity sample plate in an inverted position, capturing an image of the monolayer of effector cells and target cells;

[0026] q) placing the affinity sample plate in an upright position;

[0027] r) optionally, allowing effector cells that have not bound to target cells to settle to the bottom of the affinity sample plate, and optionally, collecting or washing away the effector cells that have settled to the bottom;

[0028] s) optionally, with the affinity sample plate in an upright position, capturing images of effector cells bound to the monolayer of target cells;

[0029] t) placing the affinity sample plate in an upright position in a centrifuge and centrifuging at a specified centrifugal force, such as 1000 x g, for a specified time;

[0030] u) with the affinity sample plate in an upright position, capturing an image of effector cells bound to the monolayer of target cells;

[0031] v) optionally, before or after capturing the image, collecting or washing away effector cells that have become unbound to the target cells;

[0032] w) optionally, repeating steps t) to v) one or more times, increasing the centrifugal force with each successive number;

[0033] x) Optionally, after capturing the image, effector cells that remain bound to the target cells are collected.

[0034] As described herein, effector cells include effector cells of the immune system that can act via receptors. For example, a T cell carrying a T cell receptor can bind to an antigen on a cancer cell, after which it can, for example, exert a cytotoxic effect and kill the target cell. Effector cells can be derived from nature, such as from a host such as a human, and can also include genetically modified cells, wherein, for example, a particularly useful receptor is provided to the effector cell.

[0035] Target cells according to the present invention are cells to which effector cells play a role, such as being combined with and triggering an immune response therein. Target cells include cancer cells presenting antigens. Antigens can be presented by MHC, i.e., human HLA, and MHC is a specialized receptor (e.g., for MHC I, a length of 8-11 amino acids) presenting peptides, such as peptides derived from digested proteins expressed by cells. Antigens can also be proteins or other biomolecules displayed on the surface of cells, such as epidermal growth factor receptors or checkpoint proteins, which are overexpressed in the case of cancer cells, so that distinguishing features are provided when compared with non-lesioned cells. Target cells can also include cells expressing autoantigens, such as cells known to participate in autoimmune diseases or cells infected with pathogens such as viruses.

[0036] It should be understood that the cell affinity as used herein as a whole includes the overall intensity of the interaction occurring in the cell-cell contact of effector cells and target cells, and relates to a variety of molecules at the surface of the interacting cells.Such interactions may include a variety of receptor-ligand pairs, wherein, for example, specific receptor-ligand interactions occur at the membrane surface of the cell.For example, when the T cell receptor of the effector cell triggers the formation of an immune synapse by identifying an antigen presented by an MHC molecule on an antigen presenting cell, synapse formation involves such numerous interactions, because other membrane-bound molecules also participate in the interaction (such as integrins, etc.).Therefore, cell affinity is not limited to, for example, the interaction of the α chain and β chain of TCR (or CAR, etc.) with the antigen presented by MHC, but involves working together to form, for example, numerous interactions of strong binding between cells.Cell affinity may also relate to active signal transduction and processes inside the cell, such as, for example, during the formation of an immune synapse.It should be understood that the cell affinity of effector cells can be defined relative to their target cells.

[0037] In the means and methods according to the invention, cell avidity assessment is made by determining, e.g., quantifying, the effector cells moving away from a target cell attached to a surface when a force is applied to the effector cell away from the target cell. In the present invention, in particular, the force to be applied is a centrifugal force. Since the formation of an immune synapse (and cell avidity in general) is a dynamic process, the time period between establishing contact between an effector cell and a target cell and the subsequent application of a force is an important parameter that needs to be well controlled in order to achieve reproducible results.

[0038] In view of the above, it should be understood that in the method according to the present invention, the cell affinity of effector cells and target cells is evaluated. In the method, the target cells are provided at a specified concentration, for example, a cell suspension having substantially single cells in a suspension. The target cells are optionally stained. It should be understood that staining the target cells with fluorescent markers can be highly advantageous in the method of the present invention because it allows the target cells to be conveniently detected using fluorescent microscopy. It should be understood that any suitable fluorescent staining can meet the requirements, such as, for example, described in the Examples section. It should be understood that the target cells can also, for example, constitutively express fluorescent markers, such as GFP, etc. instead. In any case, it can be considered to provide target cells with fluorescent markers in the method according to the present invention.

[0039] In addition, the affinity sample plate is provided with one or more holes, and the hole has the top plate surface coated for target cell attachment, wherein at least the bottom surface antifouling coating, wherein the affinity sample plate is optionally provided with an identifier, and is optionally provided with one or more sensors. In addition, the affinity sample plate is provided with one or more holes, and the hole has the top plate surface coated for target cell attachment, wherein optionally at least the bottom surface antifouling coating, wherein the affinity sample plate is optionally provided with an identifier, and is optionally provided with one or more sensors. In one embodiment, the affinity sample plate as used herein includes the bottom surface of antifouling coating. In one embodiment, the affinity sample plate as used herein includes the bottom surface made of the material with antifouling properties. In one embodiment, the affinity sample plate as used herein includes the bottom surface made of the material without antifouling properties and / or the bottom surface of not antifouling coating. It should be understood that identifier and / or sensor can be detachably connected to the affinity sample plate. As used herein, affinity sample plates are plates suitable for the means and methods according to the present invention, i.e., these are sample plates compatible with microscopy, include a surface to which target cells can attach, and allow switching between upright and inverted positions without drying of attached target cells, and which also allow exchange of fluids from the wells, such as exchange of culture medium, cell suspension, or washing fluids, etc. Since affinity sample plates are highly useful for cell affinity measurements, instead of the term affinity sample plate, cell affinity sample plates may also be referred to. Non-limiting examples of suitable affinity sample plate designs include, for example, Figure 1 The depicted plate has one or more wells, e.g. in a standard 96-well format, etc., with inlets and outlets compatible with a multi-channel pipetting device, etc. The affinity sample plate is provided with one or more wells, which may have, e.g. Figure 1 , 3 and 4, for example, with an inlet and an outlet. In a preferred embodiment, the height of the hole or flow channel is from 200 to 400 μm. This height is optimal because the volume to area ratio is sufficient to allow the cells to form a monolayer and not to exhaust nutrients too quickly. A height of 200 to 400 μm typically results in a cell sedimentation time of from 2.5 to 4.5 minutes. Increasing the height means increasing the sedimentation time of the cells, which is undesirable because the longer the sedimentation time of the cells, the higher the heterogeneity of the cell sedimentation time becomes. For example, the effector cells will be distributed at multiple heights throughout the hole or channel when rushing in. Cells starting from a smaller height will take less time to sink to the target cell monolayer and therefore take less time to settle than cells starting from a larger height. The larger the channel height, the greater this difference becomes.

[0040] Since the holes on the plate can have inlets and outlets, and it is necessary to allow the plate to be inverted without drying the target cells attached to the top plate surface, it should be understood that the inlet and outlet are designed so that the fluid is retained in the hole during the inversion step, for example, preferably due to surface tension or optionally by closing the inlet and outlet with a cap or plug. For closing the inlet and outlet with a cap or plug, the inlet and / or outlet can also be closed by other means such as sealing films, films, foils or pads. In one embodiment, the sealing film, film, foil or pad is, for example, penetrable by a syringe, needle or pipette, but does not lose its sealing ability. In one embodiment, the fluid present in the inlet and / or outlet is removed.

[0041] It should be understood that the affinity sample plate according to the present invention can have a top plate surface coated for target cell attachment, and a bottom surface that is at least antifouling coated and / or at least has antifouling properties. It should be understood that the affinity sample plate according to the present invention can have a top plate surface coated for target cell attachment, and a bottom surface that is not antifouling coated or does not have antifouling properties. It should be understood that bottom and top plate are relative terms that depend on the starting position of the affinity sample plate.

[0042] The antifouling properties of a surface (such as the surface of the bottom) can be achieved by selecting a polymer with inherent antifouling properties or by applying a physically adsorbed antifouling layer, for example, a hydrophobic surface can be coated by applying an aqueous solution containing a PEG block copolymer such as Pluronic-F127, which physically adsorbs and presents a PEG brush surface with low biofouling properties. Chemical functionalization can also be achieved by first attaching a chemically reactive linker to the surface and then solution coating the device with antifouling molecules that react with the activated linker molecules. Patterned coatings can be achieved by using a photoactivated linker and activating the linker only in selected areas of the device using a photomask and UV illumination. In any case, a suitable antifouling agent or antifouling property of at least the bottom surface can be appropriately selected and applied to an affinity sample plate as described herein.

[0043] Suitable coating agents for the attachment of target cells are known in the art. Such coatings preferably comprise polypeptides. The polypeptides may be naturally occurring polypeptides and / or synthetic polypeptides. Such coatings may comprise polypeptides selected from the group consisting of: fibronectin, poly-L-lysine, poly-D-lysine, poly-L-ornithine, laminin, collagen, fibronectin, fibrinogen, vitronectin, osteopontin, thrombospondin, VEGF, VCAM-1, ICAM. In another further embodiment, the coating comprises one or more of the group consisting of: fibronectin, poly-L-lysine, poly-D-lysine, poly-L-ornithine, laminin, collagen, fibronectin, fibrinogen, vitronectin, osteopontin, thrombospondin, VEGF, VCAM-1, and ICAM. These polypeptides may be attached to the surface, and they in turn subsequently attach to the target cells, thereby attaching the target cells to the surface. In any case, a suitable coating can be selected so that target cells attached to the surface allow the target cells to remain attached to the surface when a force is applied to the effector cells bound thereto. In other words, the target cells remain substantially attached to the surface when a centrifugal force is applied to the effector cells, allowing these cells to detach from the target cells.

[0044] The purpose of this differential coating is that in an upright position, the target cells (or effector cells) do not adhere to the bottom of the affinity sample plate due to antifouling, while when in an inverted position, the target cells can adhere to the top plate surface coated for this purpose, and the effector cells can interact and bind to the target cells attached to the top plate surface. This allows a good time control method, because the interaction time between the effector cells and the target cells can be controlled and kept the same on the entire plate. In addition, because the affinity sample plate according to the present invention is compatible with centrifugation, it should be understood that in the case of using an inlet and an outlet, these inlets and outlets are preferably located opposite the bottom surface, so that when the plate is centrifuged to apply a force away from the target cells, the fluid contained in the hole is retained and there is no risk of leakage. In addition, due to this type of positioning of the inlet and outlet, as long as the surface tension of the fluid contained in the hole is sufficient to retain the fluid during the inversion of the plate, it is not even necessary to have a plug in the inlet and outlet throughout the entire procedure, which is highly advantageous because it reduces the number of processing steps of the plate, allowing a more convenient process (partial) automation, such as automatic pipetting with a multichannel. In addition, not using a cap or plug can avoid the undesirable fluid flow induced when placing such a cap or plug on the fluid inlet and / or outlet. As indicated above, a suitable alternative to using a cap or plug on the inlet and / or outlet can be the use of other means such as a sealing film, film, foil or pad. In one embodiment, the sealing film, film, foil or pad is, for example, penetrable by a syringe, needle or pipette, but does not lose its sealing ability.

[0045] In the case where caps or plugs are not used, and inlets and outlets are used in the affinity sample plate design, it may be preferred to rotate the affinity sample plate along an axis of the plate when switching between the inverted and upright positions, the axis being selected to be parallel to the axis between the inlets and outlets, with the inlets and outlets of all wells also aligned on the plate. In this way, undesirable fluid flow can be further reduced and / or avoided during handling of the plate.

[0046] As described, the affinity sample plate is optionally provided with an identifier and optionally also provided with one or more sensors. Since the aim of the means and methods according to the invention is to efficiently and well control the process, the inclusion of an identifier as well as one or more sensors highly advantageously allows the control of the steps of the method performed according to the invention. The sequence of the steps of the method can be automated, and the inclusion of an identifier as well as one or more sensors allows the tracking of the affinity sample plate and the method steps according to the variables set and from a quality control point of view.

[0047] In any case, the affinity sample plate according to the present invention is provided. Next, with the affinity sample plate in an upright position, the target cells are introduced and homogenized into one or more holes of the affinity sample plate. It should be understood that the target cells are homogenized before being introduced into one or more holes to ensure that a similar number of target cells are provided to each hole. It should be understood that the amount of cells provided is to provide a monolayer of target cells, that is, the target cells are spread on the surface of the top plate to provide a single layer of target cells. It should be understood that it is highly preferred to obtain a monolayer of target cells with high confluency. Obtaining high confluency can be achieved by introducing many target cells, whereby the target cells, once attached to the top plate surface, directly obtain high confluency. On the contrary, it is also possible to select to introduce cells and allow the target cells to grow until a high confluency is obtained. Suitable confluency can be selected from at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or more.

[0048] Once target cells are introduced, affinity sample plates are positioned in inverted positions, thereby allowing target cells to settle to the top plate surface and attach thereto to form a monolayer. Once target cells are attached to the top plate surface, it is possible to select to cultivate target cells for a period of time, for example, in order to reach high confluence as described above or in order to allow cell recovery (for example, in the case where target cells are introduced after trypsinization). Affinity sample plates can remain in inverted positions, but it is also possible to select to cultivate target cells in upright positions. Cultivating in upright positions can be advantageous, for example, because the risk of fluid leakage can be minimized, but affinity sample plates can also be maintained in inverted positions. Therefore, the next step, optionally, relates to cultivating the target cells attached in a controlled incubator in the case of affinity sample plates in inverted positions or upright positions.

[0049] When the monolayer of target cells has been formed after the optional culture, the top plate surface with attached target cells can then be blocked with a blocking agent. In the case where the coating agent can induce undesirable nonspecific binding of effector cells to the coating agent, the use of a blocking agent can be useful. Suitable blocking agents include, for example, serum, such as FBS, BSA, which can be contained in a suitable culture medium, and can also include polymers, such as pluronic-f68, etc. Molecules with linking groups reactive with the coating agent used can also be used as blocking agents. For example, since the coating agent in the present invention can include a polypeptide with a primary amine group, an agent that forms a stable conjugated bond with the primary amine, i.e., an NHS ester reagent, can be used to effectively block the remaining available coating agent. For example, a suitable NHS ester reagent can be a compound comprising at least one succinimide group, which functional group is capable of forming an ester bond, such as an ester bond with a primary amine group on a polypeptide.

[0050] In any case, as described above, an affinity sample plate in which target cells are attached to the top plate surface is provided. Subsequently, the affinity sample plate is placed in an upright position. This itself has applied 1xg, causing the target cells not attached to the top plate surface to settle downward to the bottom of the hole. The affinity sample plate is preferably placed in a centrifuge in an upright position, thereby subsequently rotating the target cells not attached with at least 20xg downward. A higher force can be preferred, for example more than 200xg. The centrifugation step can also be carried out under a force (acceleration), which is similar to or exceeds the acceleration rate (acceleration rate) applied to the effector cells in the later centrifugation step of the method for assessing cell affinity. Therefore, as described, subsequently, the affinity sample plate is placed in an upright position, optionally in a centrifuge and centrifuged to rotate the target cells not attached to the top plate surface downward.

[0051] Subsequently, with the affinity sample plate in an upright position, the target cells attached to the top plate surface are cleaned to remove the target cells that are not attached to the top plate surface. It should be understood that cleaning the cells includes at least exchanging the culture medium with, for example, fresh culture medium. Cleaning the cells may also include exchanging any fluid compatible with the cells, such as PBS buffer, etc. Of course, after cleaning, the wells are preferably provided with a suitable culture medium to ensure the viability of the target cells. It should be understood that the cleaning step may be optional in certain embodiments, for example, when the target cells provided are all attached to the coated top plate surface, in which case a cleaning step will not be required.

[0052] Next, optionally, an image of the monolayer of target cells attached to the surface of the top plate is acquired. Acquiring images at this stage is optional, but can be highly useful, for example in quality control, especially (ia) for checking the confluence and / or quality of the monolayer of target cells attached to the surface of the top plate. Of course, the monolayer of target cells attached to the surface of the top plate can also be imaged at a later stage. Providing target cells with fluorescent markers can be highly useful for acquiring images. Of course, other dyes, such as non-fluorescent (absorbing) dyes can also be considered. Acquiring images is highly preferably performed using a microscope. As will be appreciated, the microscope can be located below the affinity sample plate, or above the affinity sample plate (see Figure 1 , Figure 2 In the case of the microscope being in the lower position). It should be understood that the plate may have protruding inlets and outlets, which may interfere with providing a good view of the target cell monolayer. Therefore, the positioning of the microscope and / or the affinity sample plate design can take into account such requirements. The images obtained include images taken at a single focal plane, for example using fluorescence microscopy. It is also contemplated that multiple focal planes may be obtained. Microscopy images may include multi-color and / or multi-fluorescence microscopy images taken at one or more focal planes. When using a multi-color fluorescence microscope, it is more convenient to focus on the effector cell and target cell monolayers, and multiple focal planes may not be required. Multiple focal planes allow focus stacking (also known as z-stacking) to provide a composite image of a monolayer of target cells.

[0053] Once the target cells attached to the top plate surface as a monolayer in the affinity sample plate are provided, then the effector cells can be provided thereto. Thus, next, the effector cells are provided at a specified concentration. The concentration can depend on many factors including the height of the well or flow channel used, for example, the concentration of the effector cells can be 0.5-1e6 / ml for a height of 400 μm and 1-2e6 / ml for a height of 200 μm. The indicated concentrations are optimal for the indicated heights because they provide a number of cells that can be distinguished as single cells with imaging software used in the art and at the same time have good data quality.

[0054] Optionally, and preferably, the effector cells are stained, for example with a fluorescent marker. As described, the cells are fluorescently labeled, for example as described in the Examples section or by stable transduction, for example with GFP, etc., allowing the effector cells to be conveniently detected using fluorescence microscopy. The effector cells are introduced into one or more wells of the affinity sample plate in an upright position after homogenization, and the effector cells are allowed to settle to the bottom of the affinity sample plate. In this step, the affinity sample plate is in an upright position so that the effector cells have not yet substantially interacted with the target cells attached to the top plate surface. By allowing the effector cells to settle to the bottom of the affinity sample plate, all effector cells have the same starting position and distance to the top plate surface. In one embodiment, the step of allowing the effector cells to settle to the bottom of the affinity sample plate is optional. Next, the affinity sample plate is placed in an inverted position, and the effector cells are allowed to settle to the monolayer of the target cells attached to the top plate of the affinity sample plate, and the effector cells are allowed to incubate on the target cells for a specified time. Incubation can be performed in a controlled incubator. By carrying out the method in this way, it is highly advantageous that the duration of placing the affinity sample plate in an inverted position, and the subsequent centrifugation, to the end of the incubation is the same for all wells of the affinity sample plate and can be well controlled.

[0055] It will be appreciated that in the methods and the like according to the invention, the steps of the method are preferably highly well controlled, from placing the affinity sample plate, introducing and optionally settling the effector cells to the bottom of the affinity sample plate, in an inverted position until centrifugation, e.g., to allow comparison of different cell affinity assessments performed with different affinity sample plates. It will be appreciated that the duration of the step of allowing the effector cells to settle on the target cell monolayer and the duration of the time after placing the affinity sample plate in an upright position (during which, e.g., images can be captured and the plate needs to be placed in a centrifuge) are preferably well controlled and can be largely determined by the time required for the procedural steps. The time of incubation of the effector cells settled on the target cells can be selected from the range of 1 second to 4 hours, from 1 second to 3 hours, from 1 second to 1 hour, from 1 second to 40 minutes, from 1 second to 30 minutes, from 1 second to 25 minutes, from 1 second to 20 minutes, from 1 second to 15 minutes, from 1 second to 10 minutes. In this way, and highly preferred, the total time from placement of the affinity sample plate, introduction and optional sedimentation of effector cells to the bottom of the affinity sample plate, in the inverted position until centrifugation is well controlled and reproducible between cell affinity assessments.

[0056] Optionally, and highly preferably, an image of the monolayer of effector cells and target cells is captured with the affinity sample plate in an inverted position. Capturing images at the end of the incubation time is preferred because this allows the definition of target cells attached to the top plate surface and effector cells in contact with the target cells, for example, in a defined field of view. This includes effector cells bound to target cells, and also effector cells not bound to target cells. It is also possible to consider capturing multiple images, or even videos, during a defined time or a portion thereof. In any case, it may be preferred to capture images at the end of the incubation, for example to capture bright field images, and also to capture fluorescent colors. For example, multicolor images may be captured, for example, multicolor images of target cells and effector cells that are differently labeled. It is possible to consider determining images from multiple focal planes and performing z-stacks to provide more detailed or contrast-enhanced images. As for determining the cell affinity score, the number of effector cells in a defined field of view, for example, of the top plate surface with target cells attached, is important, and when the amount of effector cells provided in each well is known, it is also possible to consider calculating the number of effector cells that should theoretically be present in a defined field of view. Therefore, capturing images is optional, but still highly preferred, as it allows for more accurate control of measurements of, for example, cell affinity.

[0057] Next, the affinity sample plate is placed in an upright position. In the upright position, a gravity force of about 1xg is applied to the effector cells, which means that the effector cells that are not bound to the target cells or are not substantially bound to the target cells can settle down to the bottom of the affinity sample plate. Therefore, the effector cells that are not bound to the target cells are optionally allowed to settle to the bottom of the affinity sample plate, and optionally, the effector cells that settle to the bottom are collected or washed away. Since it may be interesting to collect a portion of the effector cells, it may be considered to collect cells that are not bound to the target cells. Alternatively, these effector cells can be simply removed by washing them away. Now, subsequently, as described above, optionally, an image of the effector cells bound to the monolayer of target cells is captured with the affinity sample plate in an upright position. This image is actually the same as the above-mentioned picture taken in the inverted position, except that the unbound effector cells are not present in the captured image. Therefore, instead of the above-mentioned image, this image can be captured just as well.

[0058] Next, the affinity sample plate is placed in a centrifuge in an upright position and centrifuged for a prescribed time at a prescribed centrifugal force, such as a centrifugal force of 1000xg. In this step, a force is applied to the effector cells bound to the target cells to keep them away from the top plate surface to which the target cells are attached. When the strength of the binding between the effector cells and the target cells is low compared to the centrifugal force, the effector cells can become unbound and move away from the top plate surface to the bottom of the hole. After this step, subsequently, with the affinity sample plate in an upright position, an image of the effector cells bound to the monolayer of the target cells is captured. The affinity sample plate is maintained in an upright position to avoid repeated interactions with target cells attached to the top plate surface. Optionally, the effector cells that become unbound in this step can be collected or washed away before or after capturing the image.

[0059] The above-mentioned steps of centrifugation, capturing images, and optionally washing or collecting the effector cells that become unbound can be repeated one or more times, increasing the centrifugal force with each successive number. In an optional embodiment, centrifugation can be repeated with the same centrifugal force, increasing the time that the effector cells are subjected to the centrifugal force during each continuous centrifugation. In this way, for example, a cell affinity graph or curve can be determined, for example, the number of cells that become unbound is cumulatively plotted, or different parts of the cells can be collected. Finally, the effector cells that remain bound to the target cells after capturing the image can be collected. It should be understood that in the case where the steps of centrifugation, capturing images, and optional washing or collecting are repeated one or more times, the collection step is of course performed after the last imaging step. It may be interesting to collect these cells because these cells are able to form the strongest binding between the effector cells and the target cells and / or are able to form strong bonds during the selected incubation time.

[0060] With respect to capturing images, it should be understood that images can be captured from below the affinity sample plate or from above the affinity sample plate, wherein the affinity sample plate can be in an upright or inverted position (see, in particular, Figure 1 ).exist Figure 2 In, schematically depicted as having a microscope located below the affinity sample plate. For example, with respect to target cells attached to the surface of the top plate, the affinity sample plate can be made to collect images with a microscope in an upright position or in an inverted position. In the case where the microscope is located below the affinity sample plate, the working distance between the microscope in the upright position and the effector cells / target cells attached to the surface of the top plate is larger than that in the inverted position. In the case of collecting images in the upright and inverted positions of the affinity sample plate, it is highly preferred that the microscope is equipped with multiple objective lenses to adapt to the acquisition of images in these different positions. When collecting images in an inverted configuration (e.g., when the objective lens is imaged through the top plate rather than through the bottom), it may be possible to use an objective lens with a shorter working distance and therefore a higher numerical aperture and / or a larger magnification. This can be beneficial for obtaining more detailed images of cells containing more information (e.g., when determining markers as described below). It is preferred to have a microscope in the following position, such as in an affinity measurement unit as provided herein, because it allows the capture of images from below while allowing access to the affinity sample plate from above, such as with a (multi-channel and / or robot) fluid handling system. Therefore, in a preferred embodiment, images can be captured from below the affinity sample plate using a microscope. In another embodiment, images are captured from above the affinity sample plate using a microscope. Highly preferably, the microscope comprises multiple fluorescence channels, such as two, three, four or more fluorescence channels. This is highly preferred because it allows multiplexed fluorescence microscopy to be performed, making it possible to increase the number of samples for each affinity sample plate.

[0061] It should be understood that the affinity sample plate is preferably made of or comprises a material allowing microscopy as envisioned herein. Suitable materials are known in the art, such as polymers such as polystyrene, which can adapt to microscopy. It is highly preferred that the affinity sample plate comprises cyclic olefin copolymer (COC) or cyclic olefin polymer (COP) or is composed of cyclic olefin copolymer (COC) or cyclic olefin polymer (COP), the latter using a single type of monomer, which is highly useful for microscopy applications and is highly suitable for accommodating cells, such as for cell culture. Therefore, in further embodiments, the affinity sample plate is made of or comprises polymers such as COC or COP. Preferably, the affinity sample plate adopts standardized multi-well format, such as 48 holes, 96 holes or 384 hole formats. Such formats allow easy integration into high-throughput formats and equipment, such as, utilizing multi-channel fluid handling systems, etc.

[0062] In another embodiment, in the method according to the invention, the ratio of the number of target cells to the number of effector cells is in the range of 1:1 to 100:1, preferably about 10:1. It will be appreciated that in the wells in which the target cells are to be attached to the surface of the top plate, the number of target cells is preferably equal to or greater than the number of effector cells. In this way, at least if the monolayer of target cells has a high confluence, the effector cells will always come into contact with the target cells during the incubation. Therefore, in a first embodiment, a ratio of at least 1:1 is selected. It may be advantageous to select an upper limit of about 100:1, so that a field of view that is not too large can be selected and a sufficient number of effector cells can be evaluated, for example, when counting the number of effector cells. Preferably, a ratio of about 10:1 is selected for the number of target cells to the number of effector cells, because such a ratio allows an average surface area representing about 10 target cells to be available for each effector cell.

[0063] It should be understood that, since the affinity sample plate can include a large number of holes, each hole can be provided with different effector cell types or different effector cell clones.For example, with respect to different effector cell types, these can be effector cells obtained from different donors, for example, when the CAR of the regulation is provided to the effector cells separated from the PBMC obtained from different donors.In addition, different effector cell types or cell groups can be provided from the same donor, for example, by providing T cells and NK cells from the same donor, wherein the T cells of the donor can be further differentiated into T helper cells (CD4+ / CD3+) and cytotoxic T cells (CD8+ / CD3+).In addition, effector cells can also be provided with different receptors, such as different CAR receptors.The effector cells carrying the same CAR receptor can be defined as representing or representing effector cell clones, although it should be understood and accepted that the effector cells introduced therein by the same CAR receptor may not necessarily be the same in terms of phenotype or even genotype.Therefore, in another embodiment, a variety of effector cell types or a variety of effector cell clones are provided, wherein each effector cell type or clone is provided to different holes.

[0064] Since the present invention allows fluorescent multiplexing, it is not necessary to provide each effector cell clone or each effector cell type to different holes. Highly advantageously, in a further embodiment, a variety of effector cell types and / or a variety of effector cell clones are provided, each effector cell type or each effector cell clone is provided with different staining, and wherein a variety of effector cell types or clones are combined and provided to a single hole. For example, in the case of wishing to perform three replicates and using 4 different fluorescent stains, 4 different effector cell clones and / or types in each hole can be combined. In an affinity sample plate with a 96-well format, this means that (96 / 3)x4=124 different effector cell clones and / or types can be analyzed on one plate. Because a variety of different cell clones and / or types are used on a single hole and the entire affinity sample plate, cell affinity assessment is also better verified on the entire plate, because each hole is controlled by having multiple samples, and because each unique sample is provided on the entire plate.

[0065] Furthermore, in case images are taken at different time points and / or after repeated centrifugation steps, cells with different labels also allow improved tracking of individual cells in the images. It should be understood that in cases where tracking of cells is important, this also facilitates identical or highly similar effector cells, e.g. having the same phenotype and carrying the same receptors.

[0066] Further complexity can be added to the analysis when cells are allowed to have, for example, dual staining, thereby allowing even more different effector cells per cell. Dual staining with red, blue, yellow and green (r, b, y, g) allows the generation of ten unique combinations (g, b, y, r, g / b, g / y, g / r, b / y, b / r, y / r). Thus, having already four different fluorescence channels in microscopy, a wide variety of experimental conditions can be analyzed highly efficiently, involving, among other things, different effector genotypes and phenotypes, donor types and / or different effector cell clones.

[0067] In addition, control effector cell clones may be included in each well or in selected wells, and controls are also provided on all plates. Control effector cell clones include positive effector cell controls, i.e., having high cell affinity with target cells, or negative effector cell controls, i.e., having low or undetectable cell affinity with target cells. It is even possible to consider combining positive effector cell controls and negative effector cell controls in wells, for example, in most wells of an affinity sample plate, or even in all wells. Affinity sample plate runs may include such controls, and when the values ​​obtained in the cell affinity assessment meet the specified reference range, such affinity sample plate runs may be considered to be validated. Therefore, in one embodiment, in addition to effector cells that may be a variety of effector cell types and / or a variety of effector cell clones, control effector cells are also provided. In a further embodiment, the control effector cells are provided to separate wells. In yet another further embodiment, the control effector cells are included in one or more wells, or in each well, wherein the control effector cells are stained differently compared to the effector cells. In yet a further embodiment, the ratio of the number of control effector cells to the number of effector cells in the well is in the range of 1:1 to 1:10.

[0068] It should be understood that the target cells may also be stained, for example with fluorescent markers or the like. Such staining is highly preferably different from the staining of the effector cells, so that in the image the effector cells are not bound to the target cells, but rather are unintentionally bound directly to the top plate surface, for example due to the absence of target cells in certain areas within the monolayer of target cells on the top plate surface. Therefore, when evaluating the cell affinity between effector cells and target cells, areas within the image field of view in which target cells are not detected may not be taken into account. It is also conceivable to provide different target cells, for example, to provide target cells originating from different donors, each presenting the same antigen. Such different target cells may include control target cells, for example, target cells that do not express the selected antigen and / or highly efficiently express / present the selected antigen. More preferably, the different target cells are provided in separate wells and are not mixed. However, it is conceivable to mix different target cells, for example, by providing a group of different target cells in the well. For example, control target cells to which the effector cells do not bind, as well as target cells, may be included. In the method according to the invention, this may allow the selection of effector cells that do bind to the target cells but at the same time do not bind to the control target cells. Thus, in a further embodiment, the target cells are also stained, the staining being different from that of the effector cells. In yet another embodiment, different target cells are provided, optionally including control target cells, which are provided to separate wells of an affinity sample plate. In yet another embodiment, different target cells are provided, optionally including control target cells, which are provided and combined in the wells of an affinity sample plate.

[0069] In yet another embodiment, the image is analyzed in the assessment of cellular affinity between effector cells and target cells. It should be understood that such analysis can be performed at the location where the affinity sample plate is subjected to the method of the invention, for example by capturing the image and processing and evaluating the image with analysis software. It should be understood that such analysis can also be performed at a location remote from the location where the affinity sample plate is subjected to the method of the invention.

[0070] Image analysis includes analyzing one or more captured images, regarding the number of effector cells in the captured images, for example, in a specified field of view. It should be understood that when multiple images of a well are captured, the same specified field of view is used in the analysis. Based on the captured images, a cell affinity score can be determined. For example, when the ratio of the number of effector cells remaining after centrifugation to the number of effector cells that initially interacted with the target cell (for example, in the field of view) is calculated, a cell affinity score is provided. Of course, it may not always be necessary to calculate the ratio relative to the number of effector cells initially provided (determined by calculation or by analysis of an image such as a specified field of view). It is also possible to calculate a score relative to, for example, a reference value or a determined number of effector control cells, which also provides relevant information related to the cell affinity between effector cells and control cells. Therefore, in a further method according to the present invention, analyzing the captured images includes determining the number of effector cells in the captured images. In yet another embodiment, if a plurality of effector cell types and / or effector cell clones are introduced into the wells as described above, the number of effector cells determined is used to calculate the cell affinity score, for example, for each well and / or for each effector cell type and / or effector cell clone contained in each well. In one embodiment, the cell affinity score is the ratio of the number of effector cells that remain bound to the number of effector cells initially provided. In another embodiment, the cell affinity score is the ratio of the number of effector cells that remain bound after centrifugation to the number of effector cells initially provided. In yet another embodiment, the cell affinity score is the ratio of the number of effector cells that remain bound after centrifugation to the number of effector control cells that remain bound. In any case, different cell affinity scores can be calculated and considered that take into account the number of effector cells that remain bound after the centrifugation step. As long as the cell affinity score can provide a useful relative parameter that allows ranking or correlation of different cell affinity scores determined for different effector cell target cell combinations, such cell affinity scores can be considered.

[0071] It should be understood that synapse is a specialized structure formed when the plasma membrane of two cells is close to transmit signals.Synapse can be formed between cells expressing receptors and target cells, such as when cells with receptors are effector cells.Immune system cells form synapses, which are essential for cell activation and function.Lymphocytes such as T cells, B cells and natural killer (NK) cells form synapses that can be referred to as immune synapses.Such synapses are usually formed between effector cells and target cells, such as cells presenting antigens.Non-limiting examples are, for example, T cells or CAR-T cells and cancer cells.The formation of synapses such as effector cells and target cells such as APC (antigen presenting cells) is a landmark event and signal of the existence of specific interactions (i.e., specific interactions between, for example, TCR or CAR and the antigens thus identified) between effector cells and target cells involved in the formation of such immune synapses.

[0072] In the case of T cells, synapses can be formed between lymphocytes and antigen presenting cells (APCs) during recognition of peptide antigens-major histocompatibility complex (pMHC) ligands by T cell antigen receptors (TCR). Both TCR and pMHC are membrane-bound, so TCR will be triggered by its ligand only at the interface between T cells and APC. Synapses can be observed at the T cell-APC interface as concentric rings by confocal microscopy, commonly referred to as "bull's eye" (Huppa, JB, & Davis, MM (2003). T cell-antigen recognition and the immunological synapse. Nature Reviews Immunology, 3 (12), 973-983). These rings are named as central, peripheral, and distal supramolecular activation clusters (i.e., cSMAC, pSMAC and dSMAC, respectively). It has been reported that the TCR is present in the cSMAC, while other lymphocyte-specific proteins such as lymphocyte function-associated antigen-1 (LFA-1) are integrated into the pSMAC ring surrounding the TCR. However, the formation of this ring structure ("bull's eye") is not universal, and other formations have been described, such as "multifocal immune synapses" between T cells and dendritic cells.

[0073] Immune synapse can be considered as any structure formed at the interface produced by functional and specific effector cell-target cell interaction, such as, for example, T cell-APC contact. Markers associated with effector cells and synapse formation include one or more of the following: CD43, CD44, CD45, LFA-1, talin, F-actin, ZAP70, CD2, CD4, CD8, CD3, CD28, PD-1, ICOS, and TCR. Markers of target cells include one or more of the following: ICAM-1 (associated with LFA-1), CD48 / 58 (interacting with CD2), CD80 / CD86 (interacting with CTLA-4 and CD28), PDL1 / PDL2 (associated with PD-1), and MHC presenting antigens (specifically interacting with TCR). These markers and their concentrations can be detected, for example, at the interface between effector cells and target cells or in cells near the synaptic interface with fluorescent labels.

[0074] For example, the markers of effector cells associated with dSMAC are CD43, CD44, CD45. Effector cell markers LFA-1, talin, F-actin, CD2, CD4 and CD8 are associated with pSMAC. Effector cell markers CD3, CD28, PD-1, ICOS and TCR are associated with cSMAC. The markers associated with synapses on target cells are ICAM-1 (associated with LFA-1), CD48 / 58 (interacting with CD2), CD80 / CD86 (interacting with CTLA-4 and CD28) and PDL1 / PDL2 (associated with PD-1), and of course MHC (specifically interacting with TCR) presenting antigens. These markers have been shown to be associated with synapses in TCR-MHC interactions. Likewise, cell engagers that engage effector cells with target cells (such as bispecific antibodies, e.g., binding CD3 on effector cells to one arm and an antigen on the target cell to the other arm) can trigger the formation of synapse-like structures as observed with classical TCR-MHC interactions.

[0075] About CAR such as CAR T cells, these are chimeric antigen receptors simulating TCR, etc. CAR is engineered. The first generation of CAR is provided with an antigen recognition portion, which is often fused to a transmembrane region and an intracellular region (such as a transmembrane region and an intracellular region of a CD3 ζ chain) The region of the antibody source (such as scFv). Subsequent generations combine the intracellular signaling domains of various costimulatory protein receptors (for example, CD28, 41BB, ICOS) and are integrated into the cytoplasmic tail of CAR to further enhance signal transduction. Subsequent generations also integrate the induced release of transgenic immunomodulators such as IL-12 in their design, to shape the tumor environment by strengthening, for example, T cell activation, attraction and activation of innate immunity. Because CAR generally has an integrated antibody variable region, these can target, for example, receptors themselves (such as Her2, PD-1, etc.) presented at the surface of the cell, or can also target antigens presented by MHC derived from intracellular proteins such as those processed by the ubiquitin-proteasome system. Such peptides presented by MHC include proteins processed and presented by MHC internally, which may be derived from receptors, secreted proteins, intracellular proteins, or internalized proteins. The synapse formed between CAR and target cells may not provide a classic bull's-eye structure with a well-characterized SMAC domain, but may result in a less ordered pattern. Multiple CAR microclusters are formed, and signaling molecules are dispersed in the center of the synaptic interface.

[0076] Synapse formation is a spatiotemporal process that begins, for example, with the binding of TCR to MHC or the binding of antigen to CAR or cell engagement, followed by phosphorylation of the cytoplasmic tail of CD3 leading to a triggered state. This set of cascade processes leads to an activated T cell state, which also depends on the effector cell type and its phenotypic state. Key processes include: calcium signaling, changes in the internal cell structure and / or cytoskeleton of effector cells, involving changes in F-actin, talin, and microtubules, centrosomes, lytic granules, nuclear position, and mitochondrial position. Transactivation of adhesion molecules, cytokines, and marker expression. IFNγ, granzymes, and perforins can be released by effector cells, thereby inducing especially target cell killing. Moreover, in addition, apoptotic markers can be found in target cells, including ICAM-1 clustering, phosphatidyl translocation, mitochondrial depolarization, caspase-3 activation, and DNA fragmentation. Therefore, specific effector cells and target cells interact and various stages of immune activation can be detected.

[0077] In any case, synapse formation or synapses can be determined by staining, i.e., using fluorescent markers, ligands, antibodies or probes, etc., which can be used on living cells or on fixed cells, targeting mechanisms involved in, for example, T cell activation and / or synapse formation.

[0078] In any case, in the means and methods according to the invention, one or more markers associated with synapse formation may also be determined. Thus, in a further embodiment, markers associated with synapse formation are determined in target cells or effector cells. In another embodiment, in the methods according to the invention, one or more markers associated with synapse formation are determined and one or more markers are determined in effector cells and / or in target cells.

[0079] In one embodiment, a method according to the invention is provided, wherein the marker associated with synapse formation is selected from the group consisting of: calcium signaling signature; spatial clustering of synapse localization molecules such as LFA-1, CD28, CD3, Agrin, etc.; changes in the internal structure of the cell and / or the cytoskeleton such as F-actin, talin, microtubules, centrosomes, lytic granules, nuclear position, mitochondrial relocation; changes in the motility of effector cells; changes in external cell morphology and / or cell shape; and apoptosis of target cells. Synapse formation includes the initiation of synapses up to and including the establishment of synapses, wherein as described above but not necessarily limited thereto, markers associated with synapse formation are relevant.

[0080] In a further embodiment, the marker for synapse formation is calcium signaling, which can be detected with a fluorescent calcium indicator such as Fura2 AM (available from Invitrogen, Cat. No. F1221), which is suitable for detection in effector cells and in living cells. Other suitable dyes for detecting calcium signaling can be selected from the group consisting of: Fura Red AM, Indo-1, pentapotassium, Fluo-3, fluo-4, Calcium Green-1, Rhod-2 and X-Rhod-1, Oregon Green 488BAPTA. Therefore, in one embodiment, the marker for synapse formation is calcium signaling, which is detected with an indicator selected from the group consisting of: Fura2 AM, Fura Red AM, Indo-1, pentapotassium, Fluo-3, fluo-4, Calcium Green-1, Rhod-2 and X-Rhod-1, and Oregon Green 488BAPTA. With these markers, calcium signals can be detected, which is a hallmark of synapse formation.

[0081] In addition, membrane potential dyes can also be used as calcium signaling indicators, such as the Invitrogen FluoVolt TMMembrane Potential Kit (Cat. No.: F10488). Depolarization of synaptic cells is performed using a slow-response potential-sensitive probe such as Invitrogen DiSBAC2(3) (Bis-(1,3-Diethylthiobarbituric Acid) Trimethine Oxonol), Cat. No.: B413. Thus, in another embodiment, markers for synapse formation are detected using membrane potential dyes.

[0082] In another embodiment, a marker for synapse formation is cytoskeletal rearrangements, which can be detected in effector cells using cell staining with live or fixed cells, for example, F-actin can be detected using Phalloidin conjugates or CellMask TM (Invitrogen, Catalog No. A57243). Other staining agents that can be used include SiR-Actin, CellLight TM Talin-GFP, BacMam 2.0, or Tubulin Tracker Deep Red. Thus, in one embodiment, the marker for synapse formation is cytoskeletal rearrangement detected with a stain selected from the group consisting of: Phalloidin conjugate, CellMask TM 、SiR-Actin、Cell Light TM Talin-GFP, BacMam 2.0, or Tubulin Tracker Deep Red. Using these markers, cytoskeletal rearrangements can be detected.

[0083] In another embodiment, markers for synapse formation involve monitoring effector cell motility. Detection of effector cell motility can be performed by video / time-lapse monitoring of effector cells that remain in contact with target cells after force has been applied. Detection of synapse formation includes detecting effector cell immobility, i.e., after synapse formation, the effector cell will stop moving and remain in contact with the target cell with which it formed a synapse. Cell motility can be detected by membrane staining and imaging of effector cells, or using bright field, dark field, or phase contrast microscopy.

[0084] It should be understood that some methods for detecting markers for synapse formation using, for example, microscopy and monitoring of motility or short-lived signals can be combined with providing cells with, for example, photoactivatable markers to activate such markers in cells following detection of the markers, so that cells in which markers associated with synapse formation are detected can be easily tracked in subsequent steps such as sorting, or FACS analysis, etc.

[0085] As described above, according to the present invention, markers associated with synapse formation can be determined, for example, via the use of labels, etc. Therefore, in further embodiments, means and methods are also provided for detecting immune synapses, and fluorescent labels are also highly useful for detecting immune synapses. When analyzing captured images, the cell affinity score can be calculated based on the number of effector cells that form synapses with target cells. Therefore, in another embodiment, immune synapses are detected. Such detection preferably occurs using captured images. In a further embodiment, analyzing the captured images includes determining the number of effector cells that form synapses. In a further embodiment, the determined number of effector cells that form synapses is used to determine the cell affinity score.

[0086] It will be appreciated that detection of synapses and / or further markers may also be performed on effector cells that remain bound to target cells. The cells may be fixed or live. In such cases, it is also conceivable that synapses are not detected in the images obtained from the affinity sample plate. Effector cells that remain bound to target cells may be collected from the affinity sample plate. Thus, in one embodiment, the method of the invention comprises a further collection step, wherein effector cells that remain bound to target cells are collected, which comprises subjecting the effector cells bound to the attached target cells to a trypsin treatment and / or a resuspension step.

[0087] When using affinity sample plates in the methods of the present invention, in order to monitor the steps of the method, the duration of one or more steps can be recorded. The duration of all steps of the method can be recorded. However, it is particularly important to monitor the time when the affinity sample plate is placed in an inverted position to allow effector cells to settle on the target cells attached to the top plate surface of the affinity sample plate, and then incubate the specified time until the affinity sample plate is placed in an upright position (e.g., as described herein, especially steps o) to q) of the above-mentioned method). In addition, it is highly preferred to control the duration of each step of the method. This can be done manually, but highly advantageously, this can be done with an automated process, such as using a robot or the like to handle the affinity sample plate, which allows good control of switching the plate between upright and inverted positions. It should be understood that not all treatments can be performed with an automated process, some steps of the method can be performed manually, and other steps of the method can be performed automatically. Therefore, in one embodiment, the duration of one or more steps is recorded, such as the duration of each step. In a further embodiment, the duration of one or more steps is preferably controlled by an automated process. In a further embodiment, the duration of each step is controlled. It will be appreciated that recording the duration of the steps of a method may be used as a means of controlling the method or validating the method.

[0088] As described herein, in the method according to the present invention, the affinity sample plate can be further provided with one or more sensors. Providing one or more sensors for the affinity sample plate further allows the steps of monitoring and / or verifying the method, for example, when (partially) performed as an automated process. In particular, one or more sensors can be accelerometers, which allow the direction of the monitoring plate and the applied force, such as during the change of the position of the affinity sample plate between an upright and an inverted position, and also allow monitoring, for example, during one or more centrifugation steps, of the force applied to the effector cells. Therefore, it is highly useful to have an accelerometer as a sensor in the plate. Further useful sensors that the affinity sample plate can be provided with are, for example, thermometers, humidity sensors, and / or CO2 sensors, which can be useful for ensuring cell culture conditions, that the affinity sample plate is stored in accordance with a specified parameter range, and further controlling the entire process. It should be understood that, it is highly preferred that the sensor readings are registered in time. Therefore, in one embodiment, the affinity sample plate is provided with one or more sensors and the one or more sensors are accelerometers, and wherein in the method, the sensor readings are registered in time. In another embodiment, the affinity sample plate is provided with one or more sensors and the one or more sensors are selected from accelerometers, thermometers, humidity sensors, CO2 sensors, and wherein in the method, sensor readings are registered in time. It should be understood that sensor readings such as captured images can be used in subsequent analysis and / or verification of cell affinity assessment. As described, it should be understood that such analysis can be performed at a location where the affinity sample plate is subjected to the method of the present invention. For example, such analysis can include capturing images, which can also include sensor readings, and processing and evaluating images and further data provided by analysis software. It should be understood that such analysis can also be performed away from the location where the affinity sample plate is subjected to the method of the present invention.

[0089] As mentioned above, the method of the present invention is highly useful for determining the cell avidity between effector cells and target cells. As understood, additional molecules may also be included in the method of the present invention, because these may also affect the cell avidity between effector cells and target cells. In particular, cell binders may be of interest. Cell binders include antibodies, etc., which can bind to target cells and cells of interest such as effector cells. Such antibodies may include single-chain antibodies comprising two binding domains such as scFv domains, and include BiTE (i.e. bispecific T cell binders) etc. Conventional antibody design includes heavy chain and light chain, wherein half of the antibody (a heavy chain and a light chain) engages with the target cell, and the other half of the antibody (another heavy chain and another light chain) engages with the effector cell, wherein preferably, the Fc domain is made inert. In any case, suitable cell binders are well known in the art, and the present invention allows research and / or determination of cell avidity, which may include formation of synapses such as induced by the cell binder between the target cell and the effector cell. Therefore, accordingly, in the means and methods according to the present invention, a cell binder can be additionally provided, and for example the cell affinity score induced by the cell binder between the cell of interest (e.g., effector cell) and the target cell is determined, wherein the cell binder has a binding region capable of binding to the cell of interest (e.g., effector cell) and a binding region capable of binding to the target cell. It should be understood that the incubation step in the method of the present invention can therefore be carried out in the presence of a cell binder to allow the cells to interact, i.e., the effector cell and the target cell, and to form a bond, such as a synapse, via the cell binder.

[0090] Thus, in one embodiment, in a method according to the invention, a cell binder capable of binding to effector cells and target cells and inducing synapse formation is provided, and the cell binder is included in the incubation step. In addition, such methods can be highly useful for screening cell binders, for example, by identifying cell binders that are particularly capable of inducing, for example, synapse formation.

[0091] Of course, the methods of the present invention are not limited to the use of cell binders, and other molecules or combinations thereof that can affect the interaction between effector cells and target cells can be used therein. Thus, in further embodiments, additional molecules (e.g., cell binders) and / or compounds are added before, during, or after the effector cells are introduced into the affinity sample plate.

[0092] Also provided according to the present invention is an affinity measurement system, which may also be referred to as a cell affinity measurement system. In one embodiment, such an affinity measurement system comprises a centrifuge unit, an affinity measurement unit, an affinity sample plate holder, and one or more affinity sample plates assembled in the affinity sample plate holder. In another embodiment, the affinity measurement system further comprises an incubator unit. It should be understood that the centrifuge unit and / or the affinity measurement unit may have climate control like an incubator. However, in the case where multiple affinity sample plates need to be processed, it may be advantageous to have a separate incubator unit, particularly in the case where, for example, an incubation step and / or a culture step that takes a lot of time is selected.

[0093] The affinity sample holder is designed so that it is compatible with the centrifuge unit, the affinity measurement unit, and optionally the incubator unit. In yet a further embodiment, the affinity measurement unit comprises a microscope and a fluid handling unit. It should be understood that the affinity measurement unit may also be provided with means allowing the orientation of the affinity sample plate to be switched between an upright and an inverted position, for example by a robot or the like. Furthermore, the microscope of the affinity measurement unit is in a further embodiment equipped with a fluorescence microscope having more than one fluorescence channel.

[0094] In yet another embodiment, an affinity measurement unit compatible with the affinity sample plate is provided, wherein the affinity measurement unit comprises a microscope and a fluid handling unit. It should be understood that the affinity measurement unit may also be provided with means for allowing the orientation of the affinity sample plate to be switched between an upright and an inverted position, such as by a robot or the like.

[0095] Furthermore, the microscope of the affinity measurement unit is in a further embodiment equipped with a fluorescence microscope having more than one fluorescence channel.

[0096] The affinity measurement unit and the affinity measurement system as described herein are particularly suitable for performing the method of the present invention.

[0097] Instead of preparing an affinity sample plate as described above with target cells attached, a sample plate comprising a monolayer of target cells may also be provided using the steps as described herein. Thus, in another embodiment, a method for assessing the cell affinity of effector cells and target cells is provided, the method comprising the following steps:

[0098] a) providing an affinity sample plate having one or more wells, the wells having a top plate surface comprising a monolayer of target cells, wherein at least the bottom surface is antifouling coated, wherein the affinity sample plate is optionally provided with an identifier, and optionally provided with one or more sensors;

[0099] wherein the target cells are optionally stained;

[0100] b) providing effector cells at a specified concentration;

[0101] c) optionally, and preferably, staining effector cells;

[0102] d) introducing the homogenized effector cells into one or more wells of an affinity sample plate in an upright position, and optionally allowing the effector cells to settle to the bottom of the affinity sample plate;

[0103] e) placing the affinity sample plate in an inverted position to allow the effector cells to settle on the monolayer of target cells attached to the top plate of the affinity sample plate, and incubating the effector cells with the target cells for a specified time;

[0104] f) optionally, with the affinity sample plate in an inverted position, capturing an image of the monolayer of effector cells and target cells;

[0105] g) placing the affinity sample plate in an upright position;

[0106] h) optionally, allowing effector cells that have not bound to target cells to settle to the bottom of the affinity sample plate, and optionally, collecting or washing away the effector cells that have settled to the bottom;

[0107] i) optionally, with the affinity sample plate in an upright position, capturing images of effector cells bound to the monolayer of target cells;

[0108] j) placing the affinity sample plate in an upright position in a centrifuge and centrifuging at a specified centrifugal force, such as 1000 x g, for a specified time;

[0109] k) with the affinity sample plate in an upright position, capturing an image of effector cells bound to the monolayer of target cells;

[0110] 1) optionally, before or after capturing the image, collecting or washing away effector cells that have become unbound to the target cells;

[0111] m) optionally, repeating steps j) to l) one or more times, increasing the centrifugal force with each successive number;

[0112] n) Optionally, collecting effector cells that remain bound to target cells after capturing the image.

[0113] In an embodiment of the above method, the antifouling coating of the bottom surface is optional. In an embodiment of the above method, the bottom surface is made of a material with antifouling properties. In an embodiment of the above method, the bottom surface does not comprise an antifouling coating and / or is not made of a material with antifouling properties. In another embodiment of the above method, in step (b), the cells provided at a specified concentration are tumor cells such as, for example, tumor B cells.

[0114] It should be understood that, instead of having a top plate surface coated with target cells, the top plate surface may also be provided with any other suitable surface, and it may be of interest to study the interaction with the surface, such as the interaction with effector cells or any other cells of interest. Therefore, in another embodiment, instead of providing target cells attached to the top plate surface, a functionalized wall is provided instead, which provides a suitable surface to study the interaction of effector cells according to the present invention as described throughout this article. The functionalized wall according to the present invention can present ligands / receptors in a similar manner to that they present on the cell surface, such as in lipid bilayers, etc. Therefore, the functionalized wall in one embodiment may preferably be functionally equivalent to the target cells attached to the surface, and may simulate the target cells attached to the surface. The functionalized wall according to the present invention may also present polypeptides, proteins, binding molecules (such as antibodies or parts or derivatives thereof), peptides, antigens, ligands, receptors, compounds, to name a few. Such functionalized walls provide suitable surfaces to study the interaction with cells or other objects, as described throughout this article according to the present invention.

[0115] Therefore, in another embodiment, a method for assessing the cellular affinity of effector cells to a functionalized wall is provided, the method comprising the steps of:

[0116] a) providing an affinity sample plate having one or more wells, the wells having a top plate surface comprising functionalized walls, wherein at least the bottom surface is anti-fouling coated, wherein the affinity sample plate is optionally provided with an identifier, and optionally provided with one or more sensors;

[0117] b) providing effector cells at a specified concentration;

[0118] c) optionally, and preferably, staining effector cells;

[0119] d) introducing the homogenized effector cells into one or more wells of an affinity sample plate in an upright position, and optionally allowing the effector cells to settle to the bottom of the affinity sample plate;

[0120] e) placing the affinity sample plate in an inverted position to allow the effector cells to settle on the functionalized walls of the affinity sample plate, and incubating the effector cells with the functionalized walls for a specified time;

[0121] f) optionally, with the affinity sample plate in an inverted position, capturing an image of the effector cells;

[0122] g) placing the affinity sample plate in an upright position;

[0123] h) optionally, allowing effector cells that are not bound to the functionalized wall to settle to the bottom of the affinity sample plate, and optionally, collecting or washing away the effector cells that have settled to the bottom;

[0124] i) optionally, with the affinity sample plate in an upright position, capturing an image of effector cells bound to the functionalized wall;

[0125] j) placing the affinity sample plate in an upright position in a centrifuge and centrifuging at a specified centrifugal force, such as 1000 x g, for a specified time;

[0126] k) with the affinity sample plate in an upright position, capturing an image of the effector cells bound to the functionalized wall;

[0127] 1) optionally, before or after capturing the image, collecting or washing away effector cells that have become unbound to the functionalized wall;

[0128] m) optionally, repeating steps j) to l) one or more times, increasing the centrifugal force with each successive number;

[0129] n) Optionally, collecting effector cells that remain bound to the functionalized wall after capturing the image.

[0130] In an embodiment of the above method, the antifouling coating of the bottom surface is optional. In an embodiment of the above method, the bottom surface is made of a material with antifouling properties. In an embodiment of the above method, the bottom surface does not comprise an antifouling coating and / or is not made of a material with antifouling properties. In another embodiment of the above method, in step (b), the cells provided at a specified concentration are tumor cells such as, for example, tumor B cells.

[0131] In yet another embodiment, a method for assessing cell affinity for a functionalized wall is provided, the method comprising the steps of:

[0132] a) providing an affinity sample plate having one or more wells, the wells having a top plate surface comprising functionalized walls, wherein at least the bottom surface is anti-fouling coated, wherein the affinity sample plate is optionally provided with an identifier, and optionally provided with one or more sensors;

[0133] b) providing cells at a specified concentration;

[0134] c) optionally, and preferably, staining the cells;

[0135] d) introducing the homogenized cells into one or more wells of an affinity sample plate in an upright position, and optionally allowing the cells to settle to the bottom of the affinity sample plate;

[0136] e) placing the affinity sample plate in an inverted position to allow the cells to settle on the functionalized walls of the affinity sample plate, and incubating the cells with the functionalized walls for a specified time;

[0137] f) optionally, with the affinity sample plate in an inverted position, capturing an image of the cells;

[0138] g) placing the affinity sample plate in an upright position;

[0139] h) optionally, allowing cells that are not bound to the functionalized wall to settle to the bottom of the affinity sample plate, and optionally, collecting or washing away the cells that have settled to the bottom;

[0140] i) optionally, capturing an image of cells bound to the functionalized wall with the affinity sample plate in an upright position;

[0141] j) placing the affinity sample plate in an upright position in a centrifuge and centrifuging at a specified centrifugal force, such as 1000 x g, for a specified time;

[0142] k) with the affinity sample plate in an upright position, capturing an image of cells bound to the functionalized wall;

[0143] 1) optionally, before or after capturing the image, collecting or washing away cells that have become unbound to the functionalized wall;

[0144] m) optionally, repeating steps j) to l) one or more times, increasing the centrifugal force with each successive number;

[0145] n) Optionally, collecting cells that remain bound to the functionalized wall after capturing the image.

[0146] In yet another embodiment, a method for assessing cell affinity for a cell to a functionalized wall or monolayer of target cells is provided, the method comprising the steps of:

[0147] a) providing an affinity sample plate having one or more wells, the wells having a top plate surface comprising a functionalized wall or a monolayer of target cells, wherein at least the bottom surface is antifouling coated, wherein the affinity sample plate is optionally provided with an identifier, and optionally provided with one or more sensors;

[0148] b) providing cells at a specified concentration;

[0149] c) optionally, and preferably, staining the cells;

[0150] d) introducing the homogenized cells into one or more wells of an affinity sample plate in an upright position, and optionally allowing the cells to settle to the bottom of the affinity sample plate;

[0151] e) placing the affinity sample plate in an inverted position to allow the cells to settle on the functionalized wall of the affinity sample plate or the monolayer of target cells, and incubating the cells with the functionalized wall for a specified time;

[0152] f) optionally, with the affinity sample plate in an inverted position, capturing an image of the cells;

[0153] g) placing the affinity sample plate in an upright position;

[0154] h) optionally, allowing cells that are not bound to the functionalized wall or the monolayer of target cells to settle to the bottom of the affinity sample plate, and optionally, collecting or washing away said cells that have settled to the bottom;

[0155] i) optionally, with the affinity sample plate in an upright position, capturing images of cells bound to the functionalized wall or monolayer of target cells;

[0156] j) placing the affinity sample plate in an upright position in a centrifuge and centrifuging at a specified centrifugal force, such as 1000 x g, for a specified time;

[0157] k) with the affinity sample plate in an upright position, capturing images of cells bound to the functionalized wall or monolayer of target cells;

[0158] l) optionally, before or after capturing the image, collecting or washing away cells that have become unbound from the functionalized wall or the monolayer of target cells;

[0159] m) optionally, repeating steps j) to l) one or more times, increasing the centrifugal force with each successive number;

[0160] n) Optionally, collecting cells that remain bound to the functionalized wall or monolayer of target cells after capturing the image.

[0161] In an embodiment of the above method, the coating of the antifouling bottom surface is optional. In an embodiment of the above method, the bottom surface is made of a material with antifouling properties. In an embodiment of the above method, the bottom surface does not comprise an antifouling coating and / or is not made of a material with antifouling properties. In another embodiment of the above method, in step (b), the cells provided at a specified concentration are tumor cells (such as, for example, tumor B cells) or effector cells.

[0162] Of course, as described in particular elsewhere herein, these embodiments utilizing the monolayer or functionalized wall of target cells as described above may also include additional molecules such as small molecules or biologics in the described methods, because these can also affect the cell affinity between the monolayer or functionalized wall of (effector cells or tumor) cells and target cells. Such molecules can be molecules of any type suitable for affecting, for example, enhancing, reducing or blocking the interaction between the monolayer or functionalized wall of (effector or tumor) cells and target cells. For example, cell conjugates may be of interest. Of course, the methods of the present invention are not limited to the use of cell conjugates, and other molecules or combinations thereof that can affect the interaction between the monolayer or functionalized wall of effector cells or tumor cells and target cells can be used therein. Therefore, in further embodiments, additional molecules (e.g., cell conjugates) and / or compounds are added before, during or after effector cells or tumor cells are introduced into the affinity sample plate. Such methods can be highly useful for screening molecules, for example, by identifying molecules that are particularly capable of enhancing, reducing or blocking the binding of cells to the monolayer or functionalized wall of target cells.

[0163] In yet another embodiment, a method for assessing cell affinity for a cell to a functionalized wall or monolayer of target cells is provided, the method comprising the steps of:

[0164] a) providing an affinity sample plate having one or more wells, the wells having a top plate surface comprising a functionalized wall or a monolayer of target cells, wherein at least the bottom surface is antifouling coated, wherein the affinity sample plate is optionally provided with an identifier, and optionally provided with one or more sensors;

[0165] b) providing cells at a specified concentration;

[0166] c) optionally, and preferably, staining the cells;

[0167] d) introducing the homogenized cells into one or more wells of an affinity sample plate in an upright position, and optionally allowing the cells to settle to the bottom of the affinity sample plate;

[0168] e) placing the affinity sample plate in an inverted position to allow the cells to settle on the functionalized wall of the affinity sample plate or the monolayer of target cells, and incubating the cells with the functionalized wall for a specified time;

[0169] f) optionally, with the affinity sample plate in an inverted position, capturing an image of the cells;

[0170] g) placing the affinity sample plate in an upright position;

[0171] h) optionally, allowing cells that are not bound to the functionalized wall or the monolayer of target cells to settle to the bottom of the affinity sample plate, and optionally, collecting or washing away said cells that have settled to the bottom;

[0172] i) optionally, with the affinity sample plate in an upright position, capturing images of cells bound to the functionalized wall or monolayer of target cells;

[0173] j) placing the affinity sample plate in an upright position in a centrifuge and centrifuging at a specified centrifugal force, such as 1000 x g, for a specified time;

[0174] k) with the affinity sample plate in an upright position, capturing images of cells bound to the functionalized wall or monolayer of target cells;

[0175] l) optionally, before or after capturing the image, collecting or washing away cells that have become unbound from the functionalized wall or the monolayer of target cells;

[0176] m) optionally, repeating steps j) to l) one or more times, increasing the centrifugal force with each successive number;

[0177] n) Optionally, collecting cells that remain bound to the functionalized wall or monolayer of target cells after capturing the image.

[0178] In an embodiment of the above method, the coating of the antifouling bottom surface is optional. In an embodiment of the above method, the bottom surface is made of a material with antifouling properties. In an embodiment of the above method, the bottom surface does not comprise an antifouling coating and / or is not made of a material with antifouling properties. In another embodiment of the above method, in step (b), the cells provided at a specified concentration are tumor cells (such as, for example, tumor B cells) or effector cells.

[0179] In one embodiment, a new affinity sample plate is used. In another embodiment, the affinity sample plate is prepared by rehydration in the following manner. A clean and dry affinity sample plate is taken out of a dry incubator and one or more wells are rehydrated with a buffer such as, for example, PBS.

[0180] Next, the surface of the hole of the affinity sample plate is functionalized or provided with a monolayer of cells. In a preferred embodiment, at least the top plate surface of the hole is functionalized or provided with a monolayer of cells. In one embodiment, functionalization is completed by coating one or more surfaces with one or more polypeptides such as, for example, glycoproteins such as fibronectin. Instead of fibronectin, monolayer cells such as monolayer stromal cells can be used. In a specific embodiment, 1-50 μg / ml, preferably 5-25 μg / ml, more preferably about 10 μg / ml of polypeptide such as fibronectin are used. Application depends on the polypeptide used and can be completed as known in the art, for example, for fibronectin in a dry incubator at 37°C for at least 1 hour.

[0181] Prior to use, the sample plate is removed and one or more wells are washed once with fresh buffer such as, for example, PBS. This is then blocked with a suitable blocking agent (e.g., BSA) to avoid non-specific binding. The conditions for incubation depend on the reagent used for blocking and are known in the art, for example, 4% (w / w) BSA incubation for 10 minutes in a dry incubator.

[0182] After a specified time, e.g., 10 minutes, one or more wells of the affinity sample plate are washed at least once with buffer, followed by another wash with affinity assay medium, e.g., culture medium such as, e.g., complete (with serum) RPMI medium, for a specified time, e.g., 10 seconds per wash.

[0183] Prior to performing an avidity assay, ie, assessing the cellular affinity of cells to a functionalized wall or monolayer, cells, such as B cells, are harvested and stained with, for example, a dye such as, for example, CellTrace TM Far red dye (Thermo Fisher) was added essentially according to the manufacturer's instructions at an initially specified density, e.g., 20 x 10 6 cells / ml for staining.

[0184] In one embodiment, during preparation of the affinity sample plate (eg, incubation with fibronectin or a monolayer of stromal cells), cells are prepared (eg, cells are harvested and stained as described above).

[0185] In one embodiment, half of the stained population is maintained in affinity assay medium without additional molecules, while the other half of the stained population is maintained in affinity assay medium with additional molecules, such as small molecule kinase inhibitors.

[0186] Conditions (eg concentration, time, temperature, etc.) depend on the additional molecule to be applied and are known in the art. For example, a small molecule kinase inhibitor can be used at a final concentration of 100 nM at 37°C for 1 hour.

[0187] In one embodiment, this is followed by at least one wash and final resuspension in affinity assay medium containing additional molecules, such as small molecule kinase inhibitors.

[0188] In one embodiment, the stained cells with and without additional molecules are introduced into the separate holes of the affinity sample plate with and without anti-IgM stimulation. Preferably, anti-IgM is introduced into the hole simultaneously with the cell. Preferably, the final concentration of the cells in each hole, such as B cells, is at a specified concentration, such as 10 million / ml for B cells, and the final concentration for anti-IgM molecules is, for example, 100ng / ml. Next, after a specified incubation time, such as 10 minutes, avidity is measured as described herein, for example, by placing the affinity sample plate in a centrifuge and centrifuging the specified time at a specified centrifugal force, such as 1000xg. Preferably, the affinity sample plate is placed in a centrifuge in an upright position and centrifuged at a specified centrifugal force, such as 1000xg, for a specified time.

[0189] The present invention also relates to a method for evaluating the cell affinity of effector cells and target cells, the method comprising the following steps:

[0190] a) providing an affinity sample plate having one or more wells, the wells having a top plate surface coated with target cells;

[0191] b) placing the affinity sample plate in an upright position;

[0192] c) introducing effector cells into one or more wells of the affinity sample plate in an upright position;

[0193] d) placing the affinity sample plate in an inverted position to allow the effector cells to settle on the target cells attached to the top plate of one or more wells of the affinity sample plate, and incubating the effector cells with the target cells for a specified time;

[0194] e) capturing images of effector cells and target cells;

[0195] f) placing the affinity sample plate in an upright position in a centrifuge, and centrifuging the affinity sample plate;

[0196] g) capturing images of effector cells bound to target cells;

[0197] h) optionally, repeating steps (f) and (g) one or more times, increasing the centrifugal force with each successive number;

[0198] i) Determine how many effector cells are bound to target cells after centrifugation.

[0199] All embodiments and features described in this application can also be applied to the just described method of assessing cellular affinity of effector cells and target cells.

[0200] In one embodiment, at least the bottom surface of one or more holes of the affinity sample plate is antifouling coated and / or made of a material with antifouling properties. In one embodiment, the bottom surface does not have any antifouling properties. In one embodiment, the affinity sample plate is provided with an identifier and / or is provided with one or more sensors. In one embodiment, the target cell is marked. In one embodiment, the top plate surface comprises a coating agent suitable for the attachment of the target cell. In one embodiment, the top plate surface is coated with a monolayer of target cells. In one embodiment, the target cells are cultured in a controlled incubator when the affinity sample plate is in an inverted position or an upright position. In one embodiment, the top plate surface with attached target cells is blocked with a blocking agent. In one embodiment, the target cells are cleaned when the affinity sample plate is in an upright position to remove the target cells loosely attached to the top plate surface. In one embodiment, an image of the target cells attached to the top plate surface is acquired.

[0201] As described above, the affinity sample plate is placed in an upright position.

[0202] In one embodiment, the effector cells introduced into one or more wells of the affinity sample plate are provided at a specified concentration. In one embodiment, the effector cells are labeled, such as fluorescently labeled. In one embodiment, the effector cells are homogenized before introduction. In one embodiment, the effector cells are introduced into one or more wells of the affinity sample plate together with one or more additional molecules such as, for example, one or more cell conjugates. The effector cells and one or more additional molecules can be combined before introduction and introduced as a mixture. Alternatively, the effector cells and one or more additional molecules can be introduced continuously in any order. In one embodiment, the effector cells and / or one or more additional molecules are first allowed to settle to the bottom of the affinity sample plate before the affinity sample plate is placed in an inverted position to allow the effector cells and / or one or more additional molecules to settle on the target cells attached to the top plate of the one or more wells of the affinity sample plate.

[0203] After placing the affinity sample plate in an inverted position to allow the effector cells to settle on the target cells attached to the top plate of one or more wells of the affinity sample plate and incubating the effector cells with the target cells for a specified time, images of the effector cells and target cells are captured. In one embodiment, the images are captured with the affinity sample plate in an inverted position.

[0204] In one embodiment, any inlet and / or outlet in one or more wells of the affinity sample plate are sealed or capped before the affinity sample plate is placed in an upright position in a centrifuge and the affinity sample plate is centrifuged. In one embodiment, effector cells that are not bound to target cells are allowed to settle to the bottom of the affinity sample plate before the affinity sample plate is placed in an upright position in a centrifuge and the affinity sample plate is centrifuged, and optionally, the effector cells that settle to the bottom are collected or washed away.

[0205] In one embodiment, centrifugation is performed at a specified centrifugal force, such as 1000 xg, for a specified time.

[0206] After centrifugation, images of effector cells bound to target cells are captured. In one embodiment, images of effector cells bound to a monolayer of target cells are captured. In one embodiment, images are captured with the affinity sample plate in an upright position.

[0207] The centrifugation step and the image capture step can be repeated one or more times, increasing the centrifugal force with each successive number of times. In one embodiment, the centrifugal force remains constant, but the centrifugation time increases with each successive centrifugation step. In one embodiment, the effector cells that remain bound to the target cells after the centrifugation step and the image capture step are collected. In one embodiment, it is determined how many effector cells are bound to the target cells after the centrifugation step and the image capture step. In one embodiment, the determination of the number of effector cells bound to the target cells after the centrifugation step and the image capture step is completed by counting the effector cells bound to the target cells before and after centrifugation. In one embodiment, the image capture step is completed by using a fluorescent microscope. In one embodiment, the captured images are bright field images and fluorescent images. In one embodiment, the post-centrifugation images are provided after each centrifugation step. In one embodiment, the percentage of effector cells bound after each centrifugation step is quantified by comparing the number of effector cells present in the pre-centrifugation image with the post-centrifugation image. In order to obtain data, the images can be analyzed using customized internal cell detection image analysis software. The percentage of effector cells bound in the pre-centrifugation condition and the post-centrifugation condition can be a measure of cell affinity. The cell avidity induced by one or more additional molecules, such as cell engagers, can be determined by subtracting the fraction of bound effector cells in the absence of the one or more additional molecules from the fraction of bound effector cells in the presence of the one or more additional molecules.

[0208] The present invention also relates to a method for evaluating the cell affinity of a cell and a polypeptide, the method comprising the following steps:

[0209] a) providing an affinity sample plate having one or more wells, the wells having a top plate surface coated with a polypeptide;

[0210] b) placing the affinity sample plate in an upright position;

[0211] c) introducing cells into one or more wells of an affinity sample plate in an upright position;

[0212] d) placing the affinity sample plate in an inverted position to allow the cells to settle on the polypeptide attached to the top plate of one or more wells of the affinity sample plate, and incubating the cells with the polypeptide for a specified time;

[0213] e) capturing images of cells and polypeptides;

[0214] f) placing the affinity sample plate in an upright position in a centrifuge, and centrifuging the affinity sample plate;

[0215] g) capturing images of cells bound to the polypeptide;

[0216] h) optionally, repeating steps (f) and (g) one or more times, increasing the centrifugal force with each successive number;

[0217] i) Determine how many cells bind to the polypeptide after centrifugation.

[0218] All embodiments and features described in this application can also be applied to the method of assessing cell affinity of a cell and a polypeptide as just described. The polypeptide can be a protein, a binding molecule (e.g., an antibody or a portion or derivative thereof), a peptide, an antigen, a ligand, a receptor, a compound, to name a few. In one embodiment, two or more, three or more, four or more, five or more different polypeptides can be used.

[0219] Implementation

[0220] 1. A method for evaluating the cell affinity of effector cells and target cells, the method comprising the following steps:

[0221] a) providing target cells at a specified concentration;

[0222] b) optionally, staining the target cells;

[0223] c) providing an affinity sample plate having one or more wells, the wells having a top plate surface coated for target cell attachment, wherein optionally at least the bottom surface is anti-fouling coated or optionally at least has anti-fouling properties, wherein the affinity sample plate is optionally provided with an identifier, and optionally provided with one or more sensors;

[0224] d) with the affinity sample plate in an upright position, introducing the homogenized target cells into one or more wells of the affinity sample plate for obtaining a monolayer of the target cells;

[0225] e) placing the affinity sample plate in an inverted position, thereby allowing the target cells to settle to the top plate surface and attach thereto to form a monolayer;

[0226] f) optionally, culturing the target cells in a controlled incubator with the affinity sample plate in an inverted position or an upright position;

[0227] g) optionally, blocking the top plate surface having attached target cells with a blocking agent;

[0228] h) placing the affinity sample plate in an upright position, optionally in a centrifuge and centrifuging to spin down target cells that are not attached to the top plate surface;

[0229] i) optionally, washing the cells with the affinity sample plate in an upright position to remove target cells that are not attached to the top plate surface;

[0230] j) optionally, acquiring an image of a monolayer of target cells attached to the surface of the top plate;

[0231] k) optionally, inspecting the quality of the monolayer of target cells based on the image of the monolayer;

[0232] 1) providing effector cells at a specified concentration;

[0233] m) optionally, and preferably, staining effector cells;

[0234] n) introducing the homogenized effector cells into one or more wells of an affinity sample plate in an upright position, and optionally allowing the effector cells to settle to the bottom of the affinity sample plate;

[0235] o) placing the affinity sample plate in an inverted position to allow the effector cells to settle on the monolayer of target cells attached to the top plate of the affinity sample plate, and incubating the effector cells with the target cells for a specified time;

[0236] p) optionally, with the affinity sample plate in an inverted position, capturing an image of the monolayer of effector cells and target cells;

[0237] q) placing the affinity sample plate in an upright position;

[0238] r) optionally, allowing effector cells that have not bound to target cells to settle to the bottom of the affinity sample plate, and optionally, collecting or washing away the effector cells that have settled to the bottom;

[0239] s) optionally, with the affinity sample plate in an upright position, capturing images of effector cells bound to the monolayer of target cells;

[0240] t) placing the affinity sample plate in an upright position in a centrifuge and centrifuging at a specified centrifugal force, such as 1000 x g, for a specified time;

[0241] u) with the affinity sample plate in an upright position, capturing an image of effector cells bound to the monolayer of target cells;

[0242] v) optionally, before or after capturing the image, collecting or washing away effector cells that have become unbound to the target cells;

[0243] w) optionally, repeating steps t) to v) one or more times, increasing the centrifugal force with each successive number;

[0244] x) Optionally, after capturing the image, effector cells that remain bound to the target cells are collected.

[0245] 2. The method according to embodiment 1, wherein the image is captured from below the affinity sample plate using a microscope.

[0246] 3. The method according to embodiment 1, wherein the image is captured from above the affinity sample plate using a microscope.

[0247] 4. The method according to embodiment 2 or embodiment 3, wherein the microscope is provided with a plurality of objective lenses, one of which can be selected for capturing an image.

[0248] 5. The method according to any one of embodiments 1-4, wherein the affinity sample plate is made of a polymer such as polystyrene.

[0249] 6. The method according to any one of embodiments 1-5, wherein the affinity sample plate is in a 48-well, 96-well, or 384-well format.

[0250] 7. The method according to any one of embodiments 1-6, wherein the ratio of target cells to effector cells is in the range of 1:1 to 100:1, preferably about 10:1.

[0251] 8. The method according to any one of embodiments 1 to 7, wherein the step of collecting the effector cells that remain bound to the target cells comprises subjecting the effector cells bound to the attached target cells to a trypsin treatment and / or a resuspension step.

[0252] 9. The method according to any one of embodiments 1-8, wherein a plurality of effector cell types or clones are provided, wherein each effector cell type or clone is provided to a different well.

[0253] 10. The method according to any one of embodiments 1-8, wherein a plurality of effector cell types or clones are provided, wherein each effector cell type or clone is provided with a different staining, and wherein the plurality of effector cell types or clones are combined and provided to a single well.

[0254] 11. The method according to any one of embodiments 1 to 10, wherein the target cells are also stained, which staining is different from the staining of the effector cells.

[0255] 12. The method according to any one of embodiments 1-11, wherein different target cells, optionally including control target cells, are provided, wherein the different target cells are provided to separate wells.

[0256] 13. The method according to any one of embodiments 1 to 12, further providing control effector cells.

[0257] 14. The method according to embodiment 13, wherein control effector cells are provided to separate wells.

[0258] 15. The method according to embodiment 13, wherein control effector cells are included in each well, wherein the control effector cells are differently stained.

[0259] 16. The method according to embodiment 15, wherein the ratio of control effector cells to effector cells in the wells is in the range of 1:1 to 1:10.

[0260] 17. The method according to embodiments 1-16, wherein the image is analyzed.

[0261] 18. The method according to any one of embodiments 17, wherein analyzing the captured image comprises determining the number of effector cells in the captured image.

[0262] 19. The method according to embodiment 17 or 18, wherein the determined number is used to calculate the cell affinity score for each well.

[0263] 20. The method according to embodiment 19, wherein the cellular avidity score is the ratio of the number of effector cells that remain bound to the number of effector cells initially provided.

[0264] 21. The method according to any one of embodiments 1-20, wherein an immune synapse is detected.

[0265] 22. The method according to embodiment 21, wherein analyzing the captured images comprises determining the number of effector cells that formed synapses.

[0266] 23. The method according to embodiment 22, wherein the cell affinity score is determined based on the number of effector cells determined to form synapses.

[0267] 24. The method according to any one of embodiments 1 to 23, wherein the duration of each step is recorded.

[0268] 25. The method according to any one of embodiments 1-24, wherein the duration of each step is controlled.

[0269] 26. The method according to any one of embodiments 1-25, wherein the affinity sample plate is provided with one or more sensors, and the one or more sensors are selected from accelerometers, thermometers, humidity sensors, CO2 sensors, and wherein in the method sensor readings are registered in time.

[0270] 27. The method according to any one of embodiments 1-26, wherein additional molecules (eg cell engagers) and / or compounds are added before, during or after introduction into the effector cells.

[0271] 28. A cell affinity measurement system comprising a centrifuge unit, an affinity measurement unit, an affinity sample plate holder, and one or more affinity sample plates mounted in the affinity sample plate holder.

[0272] 29. The cell affinity measurement system according to embodiment 28, wherein the affinity measurement system further comprises an incubator unit.

[0273] 30. The cell affinity measurement system according to embodiment 28 or 29, wherein the affinity sample plate holder is compatible with a centrifuge unit, an affinity measurement unit, and optionally an incubator unit.

[0274] 31. The cell affinity measurement system according to embodiments 28 to 30, wherein the affinity measurement unit comprises a microscope and a fluid handling unit.

[0275] 32. The cell affinity measurement system according to embodiment 31, wherein the microscope is a fluorescence microscope having more than one fluorescence channel.

[0276] Example

[0277] Example 1

[0278] The monolayer of target Nalm6 (CD19+) cells (obtained from ATCC, product number CRL-1567) was contacted with IL-2 stimulated primary human effector T cells purified from buffy coats, which were transduced with CAR-FMC63 anti-CD19 (Kramer, AM; (2017) Delineating the impact of binding-domain affinity and kinetic properties on Chimeric Antigen Receptor T cell function. Doctoralthesis, UCL (University College London)). After removing free and unbound cells by washing and centrifugation, the cells that remained bound were evaluated by microscopic examination.

[0279] Monolayer inoculation

[0280] Nalm6 (CD19+) target cells were used and seeded as a monolayer on the top plate of a 400 μm high channel slide (μ-Slides from Ibidi, catalog number: 80176). First, the cells were counted and plated at approximately 30×10 6 The concentration of cells / mL is resuspended in serum-free medium to obtain a confluence close to 100%. Next, 100 μL of resuspended cells are pipetted to the entrance of the channel slide and introduced into the channel by tilting the slide for a few seconds until it reaches the outlet of the channel slide. The channel slide is then placed horizontally so that 100 μL fill up the entire volume of the channel. The channel slide is immediately flipped over and incubated inverted for 30 minutes in a controlled incubator of humidity, temperature, and CO2. After incubation, the culture medium is exchanged by first applying 60 μL of serum-containing culture medium to the entrance and then taking out 60 μL from the outlet. The process is repeated four times. The slide is inverted and placed back in a controlled incubator and incubated for another 30 minutes. Next, the culture medium is exchanged with the PBS containing 0.5-1 μm CellTrace Violet (ThermoFisher#C34557) and incubated in an incubator for 15 minutes. Finally, the staining solution was exchanged by first applying 60 μL of serum-containing medium into the inlet and then withdrawing 60 μL from the outlet. This process was repeated four times.

[0281] Effector cell preparation

[0282] In preparation for the subsequent steps, primary T cells transduced with anti-CD19 CAR-FMC63 were cultured. Cells were counted and viability was tested according to standard protocols. Next, 3x106 Cells / mL containing 1μm CellTrace TM Green CMFDA (Thermo Fisher #C2925) in PBS was used for staining and incubated in the incubator for 15 minutes. Finally, primary T cells were pelleted and resuspended in complete medium.

[0283] Effector cell binding

[0284] The channel entrance and exit are filled with plugs before centrifugation. The channel slide containing the inoculated Nalm6 target cells is placed upright in the custom adapter of the centrifuge bucket, and rotated for two minutes at 1000xg. Centrifugation removes the partial cells that are not well attached to the glass. Next, the slide is removed from the centrifuge. First, the effector cell suspension of 100 μL is pipetted into the entrance, after which 100 μL is taken out from the outlet to obtain the uniform distribution of the effector cells over the entire length of the channel. The slide containing target cells and effector cells is kept upside down (to allow the effector cells and the Nalm6 cells on the top plate to interact) and incubated for five minutes. After incubation, three positions of the channel are imaged using a fluorescent microscope (one in the center, and one near the entrance / exit). Each position is imaged in bright field, green and purple channels. The slide is put back into the centrifuge using a custom adapter and rotated for two minutes at 1000xg. Finally, the channel slide is removed from the centrifuge and imaged in the same position and using the same channel. It was observed that approximately 50% of the cells remained bound to the target cells after centrifugation. In general, low background binding of Nalm6 cells was observed when the centrifuge procedure was applied, which was usually in the range of 5-15%.

[0285] Example 2

[0286] Assessment of cellular avidity of effector and target cells in the presence of cell-engaging substances

[0287] The cellular avidity of effector and target cells is measured in the presence of a cell binder using the methods for assessing cellular avidity as described herein.

[0288] Nalm6 (CD19 +) cells (obtained from ATCC, product number CRL-1567) were used as target cells. Nalm6 (CD19 +) cells were grown, harvested and resuspended in serum-free RPMI1664 culture medium. Thereafter, 6 channel slides (Ibidi, product number 80606-90) were used in each channel with 0.9 x 10 6 Cells / ml were plated, slides were inverted and incubated upside down in a humidified incubator at 37°C for 30 minutes.

[0289] After incubation, the slides were turned over and the medium in each channel was exchanged twice with 150 μL RPMI1664 medium supplemented with 10% fetal calf serum (FCS). The slides were incubated upside down again at 37° C. in a humidified incubator for 30 minutes.

[0290] Thereafter, to test monolayer integrity, the slides were flipped to an upright position and they were centrifuged at 10 relative centrifugal force (rcf) for 2 minutes, washed and visually inspected for monolayer integrity using a benchtop microscope. The slides were stored upside down in a humidified incubator at 37°C until use.

[0291] Healthy donor T cells were used as effector cells. T cells were cultured at 37°C with 5 μm CellTrace TM Far red staining was performed in phosphate buffered saline (PBS) for 15 minutes and washed with 10 ml of PBS. T cells were then resuspended in RPMI1664 medium supplemented with 10% FCS.

[0292] Next, an antibody called Blinatumomab (Invivogen) and a bispecific T cell conjugate were added to T cells at the following concentrations: 1 ng / ml, 10 ng / ml, 100 ng / ml or 1000 ng / ml (where ng / ml means ng Ab / ml cell suspension). As a control, no antibody was added. The obtained mixture was added directly to the channel. After all channels were filled, the slide containing target cells and effector cells was flipped upside down to allow T cells and cell conjugates to interact with the Nalm6 cells on the top plate, and incubation was started at 37°C for 10 minutes.

[0293] After incubation, the number of T cells on the monolayer was imaged with the slide inverted and then flipped again to an upright position.

[0294] Thereafter, the slides were placed in an upright position in a centrifuge and a force was applied in a direction away from the target cell monolayer by centrifuging the slides at 10 rcf for 2 minutes.

[0295] After centrifugation, the slides were removed from the centrifuge and images of T cells that remained attached to the target cell monolayer were taken. Monolayer-bound T cells were counted using in-house made software. The percentage of T cells that remained attached was determined, and the T cells that were attached before force was applied were used to set the baseline.

[0296] The results are shown in Table 1. They show that the method according to the invention and as described herein can be successfully used to measure the cell avidity of effector cells and target cells in the presence of a cell binder. The results further show that increasing the concentration of the cell binder increases the number of effector cells that remain attached to the target cell monolayer after application of a centrifugal force directed away from the target cell monolayer.

[0297] Example 3

[0298] Assessment of cellular avidity of effector and target cells in the presence of cell-engaging substances

[0299] The cell avidity of effector and target cells was measured in the presence of cell binders using the method for assessing cell avidity as described herein. The experiments were essentially similar to those described in Example 2.

[0300] Nalm6 (CD19 +) cells (obtained from ATCC, product number CRL-1567) were used as target cells. Nalm6 (CD19 +) cells were grown, harvested and resuspended in serum-free RPMI1664 culture medium. Thereafter, a 6-channel flow cell (with a channel volume of 30 μL and a 0.6 cm 2 surface area) in each channel with 30x10 6 Cells / ml were seeded, the flow cell was inverted and incubated upside down in a humidified incubator at 37° C. for 60 min. To promote monolayer cell adhesion, the channels were pre-coated with 0.02 mg / ml of poly-L-lysine.

[0301] The flow cells were stored inverted in a humidified incubator at 37°C until use, force testing was performed on the target cell monolayer immediately prior to introduction of effector cells and cell-engagement agents, and each channel was washed to remove any detached target cells.

[0302] Jurkat E6.1 cells were used as effector cells. Cells were incubated at 37°C with 5 μM of the fluorescent dye CellTrace in phosphate buffered saline (PBS). TM The cells were stained with far red for 15 minutes and washed with 10 ml of PBS for imaging. After staining, the cells were centrifuged at 0.5 x 10 6 The cells were resuspended in RPMI1664 medium supplemented with 10% FCS at a concentration of 1:1 / ml and placed at 37°C.

[0303] Bispecific antibody belintoquinolone As a cell engager, a control bispecific T-cell engager containing the same CD3-targeting scFv as belintoumab but with a target arm directed against the bacterial-derived β-galactosidase protein (absent in Nalm6 (CD19+) cells) was used as a negative control cell engager. As another control, no antibody was added.

[0304] Immediately before use, effector cells were mixed with the appropriate cell conjugate dilution (2x10 -5 nM, 2x10 -4 nM, 2x10 -3 nM, 2x10 -2 nM, 2x10 -1 nM, 2x10 0 nM, 2x10 1 nM, 2x10 2 nM or 2x10 3 nM) were mixed and introduced into the target cell-containing channel of a 6-channel flow cell and incubated for 2, 5, 10, 20, or 40 minutes after the flow cell was inverted upside down to allow the effector cells and cell engagers to interact with the target cells on the top plate.

[0305] After incubation, the number of effector cells on the target cell monolayer was imaged using fluorescence microscopy with the flow cell inverted. In this step, pre-centrifugation bright field and fluorescence images were acquired for each channel. After applying the pre-centrifugation image collection, the flow cell was flipped again to be in an upright position.

[0306] Thereafter, the flow cell was placed in an upright position in a centrifuge and a force was applied in a direction away from the target cell monolayer by centrifuging the flow cell at 1000 xg for 2 minutes.

[0307] Following centrifugation, the flow cell was removed from the centrifuge and bright field and fluorescence images were acquired of the effector cells that remained attached to the target cell monolayer.

[0308] The percentage of bound effector cells after the centrifugation step (ie, force application step) was quantified by comparing the number of effector cells present in the pre- versus post-force application fluorescence images for all channels. Fluorescence images were analyzed using custom in-house cell detection image analysis software.

[0309] Avidity values ​​were calculated as the mean fraction of channels that remained bound to the monolayer after force application. The cell binder-induced cell avidity signal was determined by subtracting the fraction of bound effector cells for the no cell binder control channel from the fraction of bound effector cells for each cell binder-containing channel.

[0310] The results of the experiment (data not shown) show a dose-dependent response of belintoin monoclonal antibody in cell affinity, confirming the applicability of the method described herein for exploring cell-binding agent-mediated cell affinity. The highest cell affinity signal for all incubation times tested was observed at a belintoin monoclonal antibody concentration of 20 nM. At higher concentrations, the affinity signal remained stable or decreased.

[0311] The results further showed that increasing incubation time resulted in an increase in the avidity signal, demonstrating the time-dependent behavior of cell-adapter-specific cell engagement.

[0312] Based on the above, it can be concluded that the method as described herein can determine cell binder-specific affinity with high accuracy, sensitivity and reproducibility over a wide range of cell binder concentrations.

[0313] Furthermore, it can be concluded that the method as described herein allows for a high degree of control over force application in the presence of cell engagers, as well as simultaneous high-throughput analysis of cellular affinity between effector and target cells, and is therefore highly suitable for cell engager candidate screening in a high-throughput format with high sensitivity and precision.

[0314] Table 1. Cell avidity measurements of effector and target cells in the presence of cell engagers

[0315]

Claims

1. A method for evaluating the cell affinity of effector cells and target cells, the method comprising the following steps: a) providing target cells at a specified concentration; b) optionally, staining the target cells; c) providing an affinity sample plate having one or more wells, the wells having a top plate surface coated for attachment of the target cells, wherein optionally at least the bottom surface is anti-fouling coated, wherein the affinity sample plate is optionally provided with an identifier, and optionally provided with one or more sensors; d) introducing the homogenized target cells into one or more wells of the affinity sample plate to obtain a monolayer of target cells with the affinity sample plate in an upright position; e) placing the affinity sample plate in an inverted position, thereby allowing the target cells to settle to the top plate surface and attach thereto to form a monolayer; f) optionally, culturing the target cells in a controlled incubator with the affinity sample plate in an inverted position or an upright position; g) optionally, blocking the top plate surface having attached target cells with a blocking agent; h) placing the affinity sample plate in an upright position, optionally in a centrifuge and centrifuging to spin down target cells that are not attached to the top plate surface; i) optionally, washing the cells with the affinity sample plate in an upright position to remove target cells that are not attached to the top plate surface; j) optionally, acquiring an image of a monolayer of target cells attached to the surface of the top plate; k) optionally, inspecting the quality of the monolayer of target cells based on the image of the monolayer; 1) providing effector cells at a specified concentration; m) optionally, and preferably, staining said effector cells; n) introducing homogenized effector cells into one or more wells of the affinity sample plate in an upright position, and optionally allowing the effector cells to settle to the bottom of the affinity sample plate; o) placing the affinity sample plate in an inverted position to allow the effector cells to settle on the monolayer of target cells attached to the top plate of the affinity sample plate, and incubating the effector cells with the target cells for a specified time; p) optionally, with the affinity sample plate in an inverted position, capturing an image of the monolayer of effector cells and target cells; q) placing the affinity sample plate in an upright position; r) optionally, allowing effector cells that have not bound to target cells to settle to the bottom of the affinity sample plate, and optionally, collecting or washing away the effector cells that have settled to the bottom; s) optionally, with the affinity sample plate in an upright position, capturing an image of effector cells bound to a monolayer of target cells; t) placing the affinity sample plate in an upright position in a centrifuge and centrifuging at a specified centrifugal force, such as 1000 x g, for a specified time; u) capturing an image of effector cells bound to a monolayer of target cells with the affinity sample plate in an upright position; v) optionally, before or after capturing the image, collecting or washing away effector cells that have become unbound to the target cells; w) optionally, repeating steps t) to v) one or more times, increasing the centrifugal force with each successive number; x) Optionally, after capturing the image, effector cells that remain bound to the target cells are collected.

2. The method of claim 1, wherein the image is captured from below the affinity sample plate using a microscope or wherein the image is captured from above the affinity sample plate using a microscope.

3. The method according to claim 2, wherein the microscope is provided with a plurality of objective lenses, one of which can be selected for capturing an image.

4. The method according to any one of claims 1-3, wherein the affinity sample plate is in a 48-well, 96-well, or 384-well format.

5. The method according to any one of claims 1 to 4, wherein the step of collecting the effector cells that remain bound to the target cells comprises subjecting the effector cells bound to the attached target cells to a trypsin treatment and / or a resuspension step.

6. The method according to any one of claims 1 to 5, wherein a plurality of effector cell types or clones are provided, wherein each effector cell type or clone is provided to a different well.

7. The method according to claims 1-5, wherein a plurality of effector cell types or clones are provided, wherein each effector cell type or clone is provided with a different staining, and wherein the plurality of effector cell types or clones are combined and provided to a single well.

8. The method according to any one of claims 1 to 7, wherein different target cells, optionally including control target cells, are provided, wherein the different target cells are provided to separate wells.

9. The method according to claims 1-8, wherein the image is analyzed.

10. The method of claim 9, wherein analyzing the captured image comprises determining the number of effector cells in the captured image.

11. The method of claim 10, wherein the determined number is used to calculate a cell avidity score.

12. The method of any one of claims 1-11, wherein an immune synapse is detected.

13. The method according to any one of claims 1-12, wherein the affinity sample plate is provided with one or more sensors, and the one or more sensors are selected from accelerometers, thermometers, humidity sensors, CO2 sensors, and wherein in the method sensor readings are registered in time.

14. A cell affinity measurement system comprising a centrifuge unit, an affinity measurement unit, an affinity sample plate holder, and one or more affinity sample plates mounted in the affinity sample plate holder, and wherein optionally, the affinity measurement system further comprises an incubator unit.

15. The cell affinity measurement system according to claim 14, wherein the affinity sample plate holder is compatible with the centrifuge unit, the affinity measurement unit, and optionally the incubator unit, and / or wherein the affinity measurement unit comprises a microscope and a fluid handling unit.

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