NL2030378B1

NL2030378B1 - Selecting forces for means and methods involving cellular avidity

Info

Publication number: NL2030378B1
Application number: NL2030378A
Authority: NL
Inventors: Ashley Gregg Trillian; Fasci Domenico
Original assignee: Lumicks Ca Holding B V
Priority date: 2021-12-31
Filing date: 2021-12-31
Publication date: 2023-07-05

Abstract

The current invention relates to cell - cell interaction and in particular to cellular avidity. Provided are improved means and methods to study cell - cell interaction and characterizing cellular avidity. Varying forces were applied to cells of interest , that have interacted with target cells, was shown to have an effect on background binding. Hence, varying applied forces allows for the selection of optimal applied forces which is advantageous in means and methods relying on cellular avidity.

Description

Title: Selecting forces for means and methods involving cellular avidity

Introduction

In the art, binding studies have been conducted with target cells being attached to a plate, e,g. a glass plate, wherein subsequent cells of interest are added to the plate to interact (e.g. study the binding thereof) with the attached target cells. In particular, such studies can be performed to determine or assess cellular avidity, e.g. of effector cells such as CAR-T cells and cancer cells. Cellular avidity is than subsequently determined by exerting a force on the cells of interest and subsequently analysis of cells of interest that detach and/or remain attached to the target cells. However, a problem that can be observed with target cells attached to a surface, is that in addition to specific binding of the cells of interest to the target cells, background binding, not necessarily attributable to specific binding, may play a role in the binding of cells to the target cells and/or surface. Such background binding, to which also can be referred to as aspecific binding, can be observed for example when control cells of the cells of interest are tested under the same conditions of binding studies of the cells of interest and show a substantial binding to the target cells attached to the surface. For example, when control cell binding is relatively high as compared with specific binding of cells of interest, when different cells of interest are to be compared it becomes much more difficult to differentiate to detect variation in cellular avidity.

Hence, there is a need in the art to provide for means and methods that can reduce binding, of control cells of cells of interest to target cells attached to a surface, while at the same time allowing for effective binding of cells of interest to the target cells under the same conditions. This is highly useful when it is for example desirable to select for and identify candidate effector cells that can bind to target cells and/or modulate the cellular avidity of effector cells to target cells. This is in particular highly useful in methods in which cellular avidity is used as a means, e.g. by applying a force to cells of interest that interact with target cells. In such cellular avidity methods, it is highly desirable and important to have the ability to differentiate well between specific binding of cells of interest and background binding of control cells to target cells.

Hence, there is need in the art to provide for means and methods that may enlarge the window between positive and negative binding such that differentiation in cellular avidity between various cells of interest can be much improved, and/or cells of interest can be more efficiently identified.

Summary of the invention

As said, when target cells are attached to a surface, and subsequently allowed to interact with control cells or cells of interest, e.g. with a receptor specific to the target cells, substantial background binding can confound specific cellular avidity, in particular in methods wherein cellular avidity is an important parameter. For example, the current inventors observed that the detachment of cells of interest and control cells, when applying a force thereon, sometimes results in a large portion of control cells to remain attached to the target cells (see e.g. Figure 1). Hence, when studying the binding of cells of interest to target cells by exerting a force on the cells of interest, such substantial background binding may confound the assessment and/or use of specific cellular avidity of (candidate) cells of interest with target cells (e.g. when selecting or sorting cells). This is because a large portion of the cells of interest that bind to target cells may be cells that bind aspecifically or at least cannot be differentiated therefrom, which is highly undesirable. Hence, the current inventors sought to improve, i.e. reduce, background binding in means and methods relying on cellular avidity.

Therefore, an object of the current invention is to provide for improved means and methods reducing observed background binding to target cells attached to a surface. As shown in the examples, it was surprisingly found that when different forces were applied to cells of interest and control cells thereof, after interacting with target cells for different defined periods, the percentage of control cells that remain attached may vary considerably, as opposed to the cells of interest which were shown to vary much less. Hence, this observation allows the selection of an applied force to improve, i.e. increase, the difference between the percentage of cells of interest and control cells. By varying the applied force and/or the force loading rate, the so called window between negative and positive binding (i.e. background binding of control cells vs. binding of cells of interest, to target cells), can be improved which is highly advantageous in means and methods that rely on differentiating between cellular avidity of various cells of interest, and on ranking thereof. Hence, the current invention provides for means and methods in which different forces are applied, upon which a suitable force can be selected that allows for a much improved positive/negative window for subsequent cellular avidity methods.

Figures

Figure 1. Aspecific binding in cellular avidity experiments: Untransduced (UNT, grey lines) or FMC863-transduced Jurkat (CAR, black lines) were layered on Z-Movi chips coated with Poly-L-lysine (PLL), seeded with Raji cells and cellular avidity was measured. The left side displays the avidity curves where the y axis indicates the percentage of bound cells, the x axis indicates the force (pN). The right side displays the percentage of effector cells bound to the target cells at the end of the force ramp (1000 pN) as bar graphs. The data represents mean + SD, n=2, and is representative of two independent experiments. In the graph on the left mean is indicated as a solid line and the standard deviation of the experiments is indicated as dotted lines around the mean. Shown in the figure is that the aspecific/background binding of the untransduced control cells on the Raji cells (bottom) is relatively high. This leads to a small positive/negative window on the Raji cells (c.a. 50-85%) making it more difficult to detect differences in specific binding properties, i.e. cellular avidity, in the Raji setting.

Figure 2. Increase on target end force reduces the aspecific binding on NALM6 in cellular avidity experiments: Cellular avidity of untransduced (UNT) or FMC863- transduced Jurkat (CAR) cells in NALM6-seeded Z-Movi chips. The measurements were performed applying a force ramp of 2.5 minutes from 1 to 1000 pN or 1 to 3000pN.

A higher end force resulted in a drop of the aspecific binding (lower bottom lines, of which the lower line is from 1-3000 pN)) while maintaining the binding of the positive (CAR) sample (upper two lines). The bottom panels display the percentage of cells bound at 1000 pN (left) or at 3000 pN (right), and highlight an increased and improved positive/negative window for samples measured with higher end force. The data represents mean + SD, n=2, and is representative of two independent experiments.

Figure 3. Increase on target end force improves the positive/negative window in cellular avidity experiments: Cellular avidity of Untransduced (UNT) or FMC83- transduced Jurkat (CAR) cells in Raji-seeded Z-Movi chips. The measurements were performed applying a force ramp of 2.5 minutes from 1 to 1000 pN or 1 to 3000pN .

As observed in Figure 2, a higher end force resulted in a substantial improvement of the positive/negative assay window. The lower bottom lines represent untransduced cells, of which the lower line represents a force from 1-3000 pN which is substantially reduced as compared with the line above which represents 1-1000 pN, while the binding of the positive (CAR) samples were similar (upper two lines). The bottom panels display the percentage of cells bound at 1000 pN (left) or at 3000 pN (right).

The data represents mean + SD, n=10r2, and is representative of two independent experiments.

Figure 4. The NALM6 and Raji target cell monolayer viability and integrity is preserved after multiple force ramps to 3000 pN: Z-Movi chips seeded with A)

NALMS®G or B) Raji cell monolayers were subjected to five force ramps of 2.5 minutes each to 1000 pN or to 3000 pN. The monolayer integrity was evaluated taking a snapshot before and after the force ramps, and viability was assessed qualitatively with Trypan Blue staining (right side snapshots). No differences in target cell monolayer confluency, cell morphology or staining could be detected indicating the target cells tolerated the procedure well. Data is representative of two independent experiments

Figure 5. Schematic showing target cells and cells of interest and cellular avidity. Target cells (2) are provided on a surface (1), which as depicted in Figure 5 isin this case a flat surface. The target cell expresses ligands and receptors, likewise the cell to be targeting the target expresses ligands and receptors as well (5). A specific ligand-receptor interaction (2 and 3) can be the driving force for the forming of a cell-cell bond with multiple ligand-receptor interactions combined resulting in strong cell-cell binding. To rupture this cell-cell bond, a force (6) is exerted on the cell (5) away from the target cell (2), which can be in the z-axis direction e.g. when the flat surface is defined as being in the x-y plane. Alternatively, this can also be in the x or y-axis direction. When the cell-cell bond is ruptured, the cell moves away from the cell surface and / or the target cell and this event can be detected and/or detached cells can be collected and quantified and/or further analyzed.

Detailed description

WO 2018/083193 discloses a method, system and sample holder for manipulating and/or investigating cellular bodies; it makes use of acoustic forces generated by ultrasound standing waves in a microfluidic flow-cell. The acoustic force allows application of force to thousands of cells in parallel, for example, to thousands of T-cells in contact with a monolayer of cancer target cells. By detecting cell-cell rupture events (e.g. (CAR-)T cells releasing from a tumor cell monolayer) as a function of the acoustic force and/or by application of fluid flow in relation to an applied acoustic 5 force, cell-cell binding interactions may be efficiently analyzed and/or cells may be sorted according to cell-cell binding interactions. For example, an avidity curve can be generated by plotting the percentage of bound CAR T-cells as a function of the applied force (see e.g. Figure 1). Such a system, which is commercially available from

LUMICKS and known as z-Movi, has been used in the example section as described herein. However, the means and methods in accordance with the invention as disclosed herein are not restricted to the use of acoustic force, but are of use with other types of forces that can be applied on cells, such as centrifugal/acceleration force or shear flow, and which can provide for similar graphs and plots as shown in the examples herein, all relying on the same inherent properties of cellular avidity. As long as a force can be applied and controlled on cells of interest that bind/interact with target cells attached to a surface such a force is contemplated in accordance with the invention.

As said, a problem in means and methods involving cellular avidity and applying forces on cells of interest that interact with target cells that has been observed by the current inventors is that it can be difficult to differentiate between specific interactions and background binding. In accordance with the current invention, means and methods are provided that solve that problem, therewith providing for improvement in the capability to differentiate between specific interactions and background binding.

It is understood that “cellular avidity” as used throughout herein comprises the overall strength of interactions occurring in a cell-to-cell contact, involving a diversity of molecules at the surfaces of the cells that interact (See e.g. Figure 5). Such interactions may include a diversity of receptor-ligand pairs, among which e.g. a specific receptor-ligand interaction, occurring at the membrane surface of a cell. For example, when a T-cell receptor triggers the formation of an immune synapse by recognizing an antigen presented by an MHC molecule at an antigen presenting cell, the synapse formation involves such multitude of interactions, as also other membrane bound molecules are involved in the interactions (such as integrins and the like).

Hence, “cellular avidity” may not be restricted to the interaction of e.g. the alpha and beta chain of the TCR and the antigen presented by MHC, but rather involves a multitude of interactions working jointly forming a strong bond between e.g. cells. It may also involve active signalling and processes internal to the cells such as e.g. during immune synapse formation. It is understood that the cellular avidity of a cell of a certain type is defined relative to the target cells and conditions tested.

Hence, in one embodiment, a method is provided of selecting a force to be applied, for differentiating between binding of cells of interest and control cells to target cells, comprising the steps of: a) providing target cells attached to a surface; b) providing cells of interest and control cells of the cells of interest; c) contacting the control cells with the target cells to allow the control cells to interact with the target cells; d) applying a first force to the control cells, wherein the force is in a direction away from the target cells; e) determine the percentage of control cells contacted in step ¢) that remain attached to the target cells; f) perform steps c}, d) and e) with the cells of interest instead of the control cells; g) repeat steps ¢), d), e) and f) by applying a second force ; h) optionally, perform steps c, d) e) and f) with one or more further forces; i) select the force to be applied from the first, second and optional further forces, by comparing, with the same force applied, the percentages of cells of interest and control cells as determined in step e) that remained attached.

In one embodiment, the method in accordance with the invention is for selecting a force to be applied in measuring binding of cells of interest and/or control cells to target cells. In another embodiment, the method in accordance with the invention is for selecting a force for sorting control cells and/or cells of interest. In another embodiment, the method in accordance with the invention is for selecting a force for separation of cells of interest. In yet another embodiment, the method in accordance with the invention is for selecting a force for enrichment of cells of interest.

In step a) of the method, target cells are provided, and in step b) cells of interest and control cells of the cells of interest are provided. The target cells in accordance with the invention are defined to be the cells immobilized, i.e. attached to a surface.

The cells of interest are defined not to be immobilized. It is understood that in accordance with the invention target cells and cells of interest relate to two different cells which are to interact specifically with each other. Which cell is immobilized may not be of importance for determining cellular avidity between the cells. As long as a force can be applied to the cells binding to the immobilized cells, and detachment and/or attachment of cells can be determined, determination of the cell numbers thereof allows one to determine cellular avidity. Hence, one of the target cells and cells of interest expresses a ligand on its surface and the other cell expresses a receptor for that ligand. As control cells, the same cells as the cells of interest or target cells may be utilized but these cells e.g. do not express such a ligand or receptor or express a variant thereof which is not functional. If the control cells are control cells of the target cells, these may be immobilized as well. If the control cells are control cells of the cells of interest, these may not be immobilized. Of particular interest and as further described below, in accordance with the invention, cells of interest (or, conversely target cells) can be T-cells or the like, which are to specifically target e.g. a cancer cell or other antigen presenting cell presenting a defined antigen on its surface. Hence, accordingly, in methods of the invention the target cells attached to the surface, i.e. the immobilized cells, may be cancer cells, and the cells of interest may be T-cells.

Conversely, in methods of the invention, one may also choose to have the T-cell immobilized, as a target cell, and have the cancer cell as a cell of interest. The latter may complicate cell sorting of T-cells, but may be advantageous in scenarios where certain cancer cells are difficult to immobilize and/or different cancers cells may be studied.

The target cells are provided attached on a surface. Providing the target cells attached to a surface is well known in the art. For example, a glass or plastic surface may be utilized to attach cells thereto. A surface material may be preferred which allows for detection of attachment to and/or detachment of the target cells, i.e. of cells of interest and/or control cells thereof. Such a surface material for instance also may allow for microscopy methods (e.g. by being a transparent material). In order to attach cells to the surface, the surface may be pre-treated with a coating such as a polypeptide. Suitable polypeptides include e.g. poly-L-lysine or the like. Suitable polypeptides for attachment of target cells that may be contemplated and are known in the art include for example fibronectin, poly-L-lysine, poly-D-lysine, poly-L-ornithine, laminin, collagen, fibronectin, fibrinogen, vitronectin, or osteopontin. In any case, a suitable polypeptide or other suitable coating if needed may be selected such that the target cells that are attached to the surface allow for the target cells to remain attached to the surface when applying a force on the control cells or cells of interest. In other words, when a force is applied on the control cells or cells of interest which allows for these cells to detach from the target cells, the target cells are to substantially remain attached to the surface (see e.g. as shown in Figure 4).

In an alternative embodiment, instead of providing control cells of the cells of interest, control cells of the target cells may be provided attached to a surface. In such a scenario, cells of interest are contacted with the target cells and subsequently, cells of interest are contacted with the control cells of the target cells, and in each case the cells of interest that have detached and/or remained attached determined and cellular avidity scores provided for the cells of interest with the control cells of the target cells and with the target cells. These cellular avidity scores can be likewise compared, and from different forces applied, optimal force conditions may be selected.

In another embodiment, instead of providing target cells attached on a surface, a functionalized wall can be provided which provides the control cells and / or the cells of interest with a suitable surface to attach to in the means and methods in accordance with the invention as described herein throughout. A functionalized wall in accordance with the invention presents ligands/receptors in a similar fashion as they are presented on a cell surface, e.g. in a lipid bilayer, or the like. A functionalized wall thus preferably is functionally equivalent to target cells attached to a surface, and mimics target cells attached to a surface.

In step c) the control cells are contacted with the target cells to allow the control cells to interact with the target cells. Hence, in this step, the control cells are introduced on the target cells, e.g. by layering the control cells thereon. It is understood that this step of contacting is well controlled. For example, as shown in the example section, the step of contacting may be a defined period of 5 minutes. Of course, the step of contacting may be shorter or longer, for example in the range of about 2 minutes to about 15 minutes.

Subsequently in step d) a first force is applied to the control cells, wherein the force is in a direction away from the target cells. It is understood that in this step, the force applied may be perpendicular (in the direction of z-axis) to the surface (x,y) to which the target cells are attached, for example when a centrifugal force or acoustic force is applied. The force may also be lateral (x-axis or y-axis), for example when a shear force is applied. See e.g. Figure 5). In any case, the force is applied and is controlled such that a defined force is exerted on the cells that interact with the target cells. It is understood that the force that is exerted on the cells attached to the target cells is to be substantially equal, such can be achieved e.g. when using a flat surface as depicted in figure 5. Other suitable surface shapes may be used (e.g. a tube with exerted concentrical force or laminar flow force in the direction of the length of the tube), as long as the force exerted can be substantially equal at a defined surface area, such a surface shape may be contemplated. The force required to move a cell away from the target cell preferably can be detected, e.g. via microscopy or other means, to which may be referred to as a cell detachment event. This way, cell detachment events can be monitored and counted.

In step e) the control cells that have detached and/or remain attached in step d) are determined and provide a cellular avidity score for the control cells. By knowing the number of cells that have interacted with the target cells, and knowing the number of cells that remain attached to the target cells, the percentage of control cells that remain attached to the target cells can be determined. The percentage of control cells from the contacting step c) that remain attached to the target cells can be well determined. This can be done by relatively simple means known to the person skilled in the art, e.g. by simply providing a defined number of cells to interact with the target cells and, after incubation and applying the force, subsequently collecting detached cells and determining the amount of cells collected, and calculating the percentage that remains bound therefrom. Of course, it may be advantageous and convenient to use microscopy, with which attached cells can be identified and quantified and detachment can be likewise monitored and quantified. As shown in the examples, the z-Movi device applying an acoustic force is well equipped to do so. Likewise, similar devices may be provided with microscopy or other means to quantify cells, and attachment and/or detachment events, and also utilizing e.g. shear-force or centrifugal forces instead of acoustic force).

With regard to the cellular avidity score, it is understood that this is to express the strength of binding of cells of interest (or control cells thereof) to the target cells.

It is understood that where we refer to specific forces applied to cells this may refer to average forces, e.g. such forces may not be fully homogeneous, for example over the contact surface as may be the case with acoustic forces and shear-flow forces (See e.g. Nguyen, A., Brandt, M., Muenker, T. M., & Betz, T. (2021). Lab on a Chip, 21(10), 1929-1947) for a description of force inhomogeneities in acoustic force application)

For shear-flow forces the forces may also not be fully homogeneous, for example since the flow speed near the side walls of a flow channel (e.g. with a rectangular cross section) may be lower than in the center of the flow cell (due to the no-slip boundary condition). By choosing a cross section with a high aspect ratio (low and wide) these flow effects may be minimized such that only a few percent of the cells experience a substantially smaller force than the cells in the center of the flow cell. Other methods to mitigate such effects and to specifically select cells that have experienced similar forces may include using flow cell geometries with multiple fluid inlets and/or outlets such that the properties of laminar flow can be used to ensure cells of interest only land in regions of homogeneous force and / or are only selected from regions of homogeneous force. In one example, by using three channel inlets side by side one can use the side channels as sheath flow channels to focus cells of interest inserted into the center channel where the acoustic and /or shear force may be substantially homogeneous. The sheath flow fluid may be the same buffer fluid as is used for the sample cells but then free of sample cells. By increasing the flow speed through the sheath flow channels the cells are more focused and confined to the center of the channel while by reducing the sheath flow speed the cells are allowed to spread out more. Similarly, on the collection side flows in three side-by-side collection channels may be controlled to possibly discard cells flowing close to the channel boundaries and only collecting cells from the center of the channel. By controlling the relative flow speeds of such side channels and the center channel asymmetrically the location of the effective interaction region of the sorting device can be further controlled and cells that have underwent defined forces can be selected and/or detected.

Further means to enable collection of cells from a specific interaction region (and therefore collection of cells that experienced a defined force) include means and methods wherein cells of interest may be provided with a photoactivatable label which may be subsequently activated by illumination with light of a suitable wavelength only in a well-defined interaction region of the device (e.g. near the center of a flow channel or in a center region under an (acoustic) force transducer) to photoactivate and/or switch the dye. Subsequently the cells can be sorted for example using fluorescence activated cell sorting (FACS) and only those cells which are activated are further used according to the methods described herein thereby obtaining the cells on which defined forces have been exerted. This may for example be highly useful for collecting cells that remained attached to the target cells. For example, the target cells and cells bound thereto may be trypsinized thereby obtaining both the target cells and cells that remained bound thereto in a suspension. Alternatively, attached cells can also be simply collected with physical means (e.g. scraping) from the are of interest, i.e. the surface area with a well defined nominal force.

For centrifuge forces it is easier to ensure that the force applied is homogeneous across the whole interaction region since such a force does not depend strongly on a location on a surface with respect to a force transducer and / or the wall of a flow channel or sample holder.

Accordingly, in connection to the subject matter disclosed herein, means and methods exist which allows one to exert forces on cells attached to a surface and collect the cells, detached and/or attached cells, on which defined forces have been exerted and determine i.a. the amount thereof, i.e. number of cells..

It is understood that with regard to the force exerted, the exact forces experienced by cells may also depend on cell size and or other cell properties such as density and compressibility (The force may be a nominal force and not the true force experienced by the control cells or the cells of interest. E.g. it may be hard to precisely predict the average cell size, density, compressibility, etc. of the cells and the force may have been calculated based on theory alone or may have been calibrated using test particles with specific (preferably known) properties [see e.g. Kamsma, D.,

Creyghton, R., Sitters, G., Wuite, G. J. L., & Peterman, E. J. G. (2016). Tuning the

Music: Acoustic Force Spectroscopy (AFS) 2.0. Methods, 105, 26-33). The force may be such a calculated or calibrated force expressed with units of N (e.g. pN) but it may also be expressed without calibration as the input power (Vpp) applied to a piezo element [see Sitters, G., Kamsma, D., Thalhammer, G., Ritsch-Marte, M., Peterman,

E.J. G., & Wuite, G. J. L. (2014). Acoustic force spectroscopy. Nature Methods, 12(1), 47-50), as angular velocity squared (w?) in the case of centrifugal force application or as flow speed v and or as shear stress (Pa) in applications using shear forces. As long as the forces exerted by the devices, e.g. shear force, acoustic force, or centrifugal forces, but not limited thereto, can be varied and controlled and reproduced in such devices such devices are suitable for the means and methods in accordance with the invention.

As the percentage of cells that remains bound at a certain applied force is indicative of cellular avidity, i.e. the larger the percentage of cells that is bound the higher the cellular avidity, it is useful to refer to such a percentage as a cellular avidity score. Of course, one may use a different measure which relates to cellular avidity.

One may also refer to the percentage of detached cells instead, wherein conversely a low number is indicative of a relative higher cellular avidity. Instead of percentage, one may also provide the ratio of cells that remain attached divided by the total number of cells that interacted, or provide the ratio for detached cells. One may also, in case of a cellular avidity plot as shown herein determine the area under (or above) the curve.

One may also, in case a fixed number of cells is to be provided to a target surface, simply provide the number of cells that have detached and/or remained attached. In any case, as long as a unit is provided that is representative of the number of cells that have detached or cells that have remained attached, relative to the total number of cells that have interacted, such a unit may be contemplated. Such a unit allows for ranking cellular avidities, when comparing e.g. different cells of interest. Providing such a unit may be referred to as providing a cellular avidity score.

Hence, instead of determining the percentage of cells that remain attached to the target cells, relative to the cells of the contacting step, in the means and methods any unit may be provided that is representative of the number of cells that have detached or cells that have remained attached, relative to the total number of cells that have interacted, as such a unit can be regarded as a cellular avidity score. A preferred unit of cellular avidity, to which also may be referred to as a cellular avidity score, may be the percentage of cells that remains attached, relative to the cells that have interacted, the latter being set at 100%. While in the current examples a defined end force of 1000 pN is chosen for comparing the cellular avidities, other forces, force ramps or combinations of forces may also be contemplated as forces at which the fractions of bound vs unbound cells are compared.

Step f) comprises the same steps as described above for control cells of cells of interest, but instead utilizes the cells of interest. Hence, in accordance with the method as described above, step f), in carrying out steps c), d) and e), provides for the percentage of cells of interest contacted in accordance with step c} that remain attached to the target cells. This way, for both the control cells and cells of interest, a measure or unit representative of cellular avidity is provided. It is the difference, or ratio, between these two that is important in the current invention.

It is understood that in accordance with the invention, the repeating of the steps c), d) and e) may involve utilizing the same target cells attached to a surface and/or may involve utilizing multiple provided target cells attached to a surface. For example, as shown in the examples, a chip (such as described i.a. in Fernandez de Larrea, C., et al. (2020). Blood Cancer Discovery, 1(2), 146-154, WO2018083193, and such as available from LUMICKS for the z-Movi device (see i.a. z-Movi-Brochure_2021.pdf, available from <https://lumicks.com/products/z-Movi-cell-interaction- studies/#brochure>) with target cells attached to its surface may be repeatedly used in the z-Movi device and cells that have remained bound to the target cells and remain bound to the target layer in a subsequent measurement may e.g. be masked) in analysis (e.g. by the software or by manually identifying these cells and excluding these in a subsequent measurement). Hence, it is understood that repeating steps may be performed with the same target cells attached to a surface or may be performed with further provided target cells attached to a surface. When multiple surfaces with attached target cells are provided it is understood that of course these are prepared in the same way such that different measurements with each of the multiple surfaces can be compared and are substantially reproducible. Hence, in one embodiment, the method may be repeated with the same target cells attached to a surface, e.g. as a monolayer. In step g), steps c), d), e) and f) are repeated by applying a second force. It is understood that the second force is off course different than the first force that is applied. Optionally, in step h) one or more further forces are applied by repeating said steps c), d), e) and f) with one or more further forces.

It is understood that of course multiple measurements may be optionally performed of the different forces applied, e.g. as shown in Figures 2 and 3, measurements were carried out in duplicate, and averages and/or standard deviations determined, or the like of the determined percentages and/or units provided in step e).

In step g), once all the measurements have been carried out by applying different forces, a force can be selected by comparing, with the same force applied, the percentages of cells of interest and control cells as determined in step e) that remained attached, or likewise the units representative thereof. The force to be selected involves comparing the percentages/units of cells that remain attached.

Preferably this difference is the largest difference between the cells of interest and control cells. For example, when the measure/unit determined of control cells and cells of interest is the percentage of cells that remain attached after the force has been applied, ideally, the percentage of control cells is preferably below 15% and of the cells of interest preferably above 85%. Hence, preferably a force is selected which allows for obtaining such percentages. It may also be preferred if different forces result in similar percentages, to select the lowest force applied to obtain such similar percentages and/or differences. Selecting the lowest force may allow for more repeated use of target cells attached to a monolayer and/or may be less stressful to cells. Of course, it may depend on the application with which cellular avidity is employed as a means to differentiate between background binding (of control cells) and specific binding (of cells of interest). For example, in a scenario, wherein of a particular cell of interest, e.g. a defined CAR-T, cellular avidities are to be subsequently selected which are to be improved and/or reduced, in order to provide for a range of different cellular avidities, it may be desirable to select for a cellular avidity representative of about 50/60% for the cells of interest and about 10% or lower of the control cells thereof. In any case, by varying the forces to be applied and comparing detachment of control cells and cells of interest, a suitable and advantageous force for subsequent means and methods relying on cellular avidity can be selected.

As already described above, instead of determining the percentage of cells that remain attached, other units representative thereof or (inversely) correlating therewith may likewise be used in the means and methods in accordance with the invention.

Hence, in one embodiment, a method is provided of selecting a force to be applied, for differentiating between binding of cells of interest and control cells to target cells, comprising the steps of: a) providing target cells attached to a surface; b) providing cells of interest and control cells of the cells of interest; c) contacting the control cells with the target cells to allow the control cells to interact with the target cells; d) applying a first force to the control cells, wherein the force is in a direction away from the target cells; e) determine control cells that have detached and/or remain attached in step d) and provide a unit representing the number of cells that have detached or cells that have remained attached, relative to the total number of cells that have interacted in step ©); f) perform steps cj, d) and e) with the cells of interest instead of the control cells; g) repeat steps c)-f) by applying a second force ;

h) optionally, perform steps c¢, d) and e) with one or more further forces; i) select the force to be applied from the first, second and optional further forces, by comparing, with the same force applied, the units as determined in step e) with the cells of interest and of the control cells.

In another embodiment, a method is provided of selecting a force to be applied, for differentiating between binding of cells of interest and control cells to target cells, comprising the steps of: a) providing target cells attached to a surface; b) providing cells of interest and control cells of the cells of interest; c) contacting the control cells with the target cells to allow the control cells to interact with the target cells; d) applying a first force to the control cells, wherein the force is in a direction away from the target cells; e) determine control cells that have detached and/or remain attached in step d) and provide a cellular avidity score for the control cells; f) perform steps c}, d) and e) with the cells of interest instead of the control cells; g) repeat steps c¢)-f) by applying a second force ; h) optionally, perform steps c, d) and e) with one or more further forces; i) select the force to be applied from the first, second and optional further forces, by comparing, with the same force applied, the cellular avidity scores as determined in step e) with the cells of interest and of the control cells.

As said, preferably, the cellular avidity score or unit determined of control cells and/or cells of interest represents the percentage of cells that remain attached after the force has been applied. In one embodiment, a method is provided of selecting a force to be applied, for differentiating between binding of cells of interest and control cells to target cells, comprising the steps of: a) providing target cells attached to a surface; b) providing cells of interest and control cells of the cells of interest; c) contacting the control cells with the target cells to allow the control cells to interact with the target cells; d) applying a first force to the control cells, wherein the force is in a direction away from the target cells;

e) determine the percentage of control cells contacted in step ¢) that remain attached to the target cells; f) perform steps ¢), d) and e) with the cells of interest instead of the control cells; g) repeat steps c)-f) by applying a second force ; h) optionally, perform steps c, d} and e) with one or more further forces; i) select the force to be applied from the first, second and optional further forces, by comparing, with the same force applied, the percentages of cells of interest and control cells as determined in step e) that remained attached.

Preferably, the percentage difference is at least 30%, or more. As said, preferably, a force may be selected with the largest difference between the percentages. Preferably, the percentage of control cells remaining attached is respectively 15% or below, and of cells of interest this preferably is in the range of 85% or higher. More preferably, in a scenario wherein e.g. a force is to be selected for sorting purposes and the like, it may be desirable to have no control cells remaining attached, or at least a low percentage, such as 5% or less, 4% or less, or 3% or less.

With any other unit or cellular avidity score determined, optimal differences can likewise be selected.

The method as described above involves providing cells of interest. It is also possible to only provide control cells, and accordingly, not include cells of interests in the steps of the method. Hence, in another embodiment, the means and methods as described herein for selecting a force to be applied may comprise not providing cells of interest in the method nor determining an avidity score thereof. For example, one may solely determine the force with which a low percentage of control cells remain attached to the target cells. This way, by selecting such a force, under these conditions it may be possible to select for cells of interest subsequently, i.e. cells of interest with desirable or predefined desirable cellular affinities relative to the control cells.

Hence, in another embodiment, a method is provided of selecting a force to be applied, for differentiating between binding of cells of interest and control cells to target cells, comprising the steps of: a) providing target cells attached to a surface; b) providing control cells of cells of interest; c) contacting the control cells with the target cells to allow the control cells to interact with the target cells;

d) applying a first force to the control cells, wherein the force is in a direction away from the target cells; e) determine control cells that remain attached to the target cells and/or control cells that detached from the target cells; f) repeat steps c)-e) by applying a second force ; g) optionally, perform steps c, d) and e) with one or more further forces; i) select the force to be applied from the first, second and optional further forces, by comparing the determined control cells that remain attached to the target cells and/or control cells that detached from the target cells. Of course, this may also and preferably determining a cellular avidity score.

As already described above, the step of determining the control cells that remain attached may also comprise alternative measurements, e.g. determining cells that detach from the target cells in step d) above. Whichever way, a cellular avidity score or a unit is determined representing a binding strength of the target cell-cell of interest bond, such as a cellular avidity score representing the number of cells that have detached or cells that have remained attached, relative to the total number of cells that have interacted in step c). Preferably the force to be selected is a force with which the percentage of control cells that remain attach is lower than 50%, preferably lower than 35%, more preferably lower than 15%. By selecting such a low force that provides for such low background binding of control cells of cells of interest, advantageously in subsequent assays, cells of interest can be selected with a relative higher avidity as compared with the control cells and the low background binding allows to differentiate between different relative cellular avidities of potential different candidate cells of interest.

Hence, the means and methods in accordance with the invention as described herein, do not necessarily require a priori the provision of cells of interest in order to select a suitable force. Hence, it is understood that the means and methods as describe herein can be performed without providing cells of interest and solely apply the different forces on the control cells.

Any suitable force application method may be contemplated in accordance with the invention. Increasing the force can be well controlled with acceleration based methods of applying force such as centrifugation, with shear flow and with acoustic force, which are all suitable means to be used in the methods in accordance with the invention but any other means of controllably causing a force on the cells attached to the target cells thereby forcing them away from the target cells or the functionalized wall surface, may be contemplated. In a further embodiment, the applied force in the means and methods of the invention is a force ramp, preferably a linear force ramp. It is understood that the different forces that are to be applied can be constant forces applied for a defined period. The forces applied may be in various forms as a function of time. Preferably however, the applied force is an increasing force, that is, after the incubation step, an increasing force is applied for a defined period until a defined end force is reached. For example, as shown in the example section, in about 150 seconds, a defined end force is reached of 1000 pN or 3000 pN. Increasing the force is preferably done in a linear force ramp, but other ways to increase the force over time may also be used (e.g. exponential loading where the force is doubled over a certain time period and keeps doubling until a defined end force is reached).

The different forces (of the first, second and optional further forces) that are applied thus may include different force loading rates and/or a defined (end) force. A defined end force in the case of different force loading rates can be the same or can be differently selected end forces. Hence, in case of varying force loading rates, the time period of the applied force loading rate can be varied or can be selected to be the same for each force loading rate. Preferably, the force loading rate can be a linear force loading rate, which can be expressed as e.g. pN/s. It is understood that when reference is made to the forces applied this relates to the forces that are applied relative to the cells of interest or control cells of the cells of interest (see also below).

Hence, in a further embodiment, for the different forces applied as a first, second, and optional further force, wherein the forces are applied as a linear force ramp, includes providing for each linear force ramp a force loading rate and an end force.

In any case, when applying different force ramps, with different force loading rates, with different and/or the same end forces, the results obtained therewith can be compared and subsequently a force loading rate and time applied may be selected therefrom. Hence, a force loading rate and/or time period for application of the force loading rate can be selected in the step g). This may be the same force loading rate and time applied in the method. Alternately, a shorter time may be selected for applying the force loading rate when for example cellular avidity curves shows a flattening of the curve and additional time and increased force may not be much informative. For example, as shown in Figure 2, two different force loading rates were tested, 20 pN/s for 150s, and 6,7 pN/s for 150s, with defined end forces of 3000 pN and 1000 pN respectively. As can be observed in Figure 2, the biggest difference between background binding (UNT) and cells of interest (CAR) is obtained with a 20 pN/s for 150 seconds. However, when we look closely at figure 2, from about 100 seconds, the additional 50 seconds in this scenario do not contribute much, i.e. cells of interest remain bound and no substantial amount of cells are detached further.

Hence, when comparing the applied forces a shown in Figure 2, it would be advantageous to select a force loading rate of 20 pN/s and apply this for 100s, arriving at an end force of 2000 pN. This way, it is not necessary to apply a further force up to 3000 pN. Not applying a further force and increased force can be advantageous as it may allow for more measurements to be performed. It should be noted that although it has been demonstrated that cells remain viable under these types of force applications (see e.g. Figure 4) it may still be advantageous to limit the force applied to both the surface bound target cells as well as the control cells and /or cells of interest. It may be advantageous to limit the forces applied and the duration of the forces applied as much as possible as it may reduce cell perturbation. Hence, in one embodiment, the method in accordance with the invention, in the selection step of a force, from the selected force ramp the force rate and the end force is selected. In another embodiment, advantageously, in the selection step of a force, from the selected force ramp the force rate is selected and a lower end force, which lower end force being lower than the end force of the selected force ramp and showing no substantial further cell detached from the target cell attached to the surface. In another embodiment, in the selection step of a force, from the selected force ramp the force rate is selected and a lower end force. As said, the applied force, be it a force ramp, direct force, or a linear force ramp, may be an acoustic force, a shear flow or a centrifugal force. It may be advantageous to apply shear flow and/or centrifugal flow in scenarios wherein large number of cells are to be tested As shown in the examples, the steps c¢), d) and e) may be performed with control cells and with cells of interest in separate measurements. Of course, the steps c), d) and e) may also be performed by combining the control cells and cells of interest in a combined measurement, for example when it is possible to distinguish between cells of interest and control cells, e.g. carrying different (fluorescent) labels or the like. Hence, in another embodiment, in the method in accordance with the invention, the steps c), d) and e) with the control cells and step f) with the cells of interest are performed with the control cells and cells of interest combined. In another embodiment, in a method in accordance with the invention, the contacting step c) of the control cells and of the cells of interest, and subsequent steps d) and e) are performed separately or these steps are performed with the control cells and cells of interest combined.

In it is understood that e.g. when further subsequent measurements are performed using the same target cells attached to a surface that at the end of each force application, the cells are to be removed such that these cells can no longer interact with the target cells, which may disturb further measurements. Of course, one may also use after a measurement a new surface with attached target cells which has been in the same manner. Any of the cells that remained attached to the target cells may be masked in a subsequent analysis (i.e. not taken into account in the analysis).

Hence, it is understood that repeated use of surface with attached target cells may be preferred in cellular avidity measurements or the like, and perhaps less useful for sorting, and the latter may require that all cells that detached and that remained attached are to be removed.

In another embodiment, the target cells are attached to a glass surface, preferably a glass surface in a chip (i.a. as described in WO2018083193, and such as available from LUMICKS for the z-Movi device (see ia. z-Movi-

Brochure _2021.pdf, available from <hnttps://lumicks.com/products/z-Movi-cell- interaction-studies/#brochure>). The target cells that are attached to the surface, preferably are attached as a monolayer. The monolayer preferably is at high confluency. The subsequent cells of interest (and control cells thereof) that are to interact with the target cells are preferably provided in a relatively low cell density as compared with the target cells, such that substantially all cells of interest (and control cells thereof) can interact with a target cells (there are more target cells per cell of interest). Such provides for advantageous controllable conditions when applying the force on the control cells or cells of interest.

With regard to the target cells and cells of interest and control cells thereof, it is understood that this may involve cells that are to have a specific interaction, i.e. one cell carrying a receptor and the other cell having a ligand for the receptor. The term ligand and receptor in this sense and in accordance with the invention referring to defining their inter-relationship. The term receptor may not be construed to be limiting in any way and is understood to mean a protein presented at the cell surface which can (specifically) interact with another protein (ligand) presented at a another cell. The terms ligand and receptor are used to indicate a complementarity which is important for specific recognition between cells without restrictions on the complementary molecules that can be contemplated. For example, one cell may have a T cell receptor or a CAR-T, and the other cell may present an antigen for the T-cell receptor, e.g. presenting an antigen via MHC. Such interactions are of interest to study and to modify. Hence, such cells of interest, and control cells thereof not carrying e.g. the

CAR-T, and target cells are in particular of use in the means and methods of the invention, as exemplified in the example section herein. In a preferred embodiment immune effector cells are provided as cells of interest, and target cells expressing an antigen which the cell of interest is to specifically target. For example, effector cells may be selected from T cells, NK cells, dendritic cells, macrophages, or monocytes.

Such cells may be genetically modified, e.g. provided with e.g. a CAR. As said, target cells may be cancer cells, or other cells expressing an antigen, e.g. viral antigens or the like.

Once a force is selected in the last step of the method, this can be subsequently used in advantageous methods that rely on cellular avidity and for which it is important to be able differentiate between different cellular avidities. Cellular avidity may be defined as the percentage of cells that remain attached to the target cells. Of course, any other unit, such as described above, may be equally selected as a measure of cellular avidity. As in this case, the forces applied provide for further information related to the cellular avidity.

Hence, in further embodiments in accordance with the invention, a method for determining cellular avidity is provided comprising: i) providing a selected force in accordance with the invention and as described above; ii) providing target cells attached to a surface; iii} providing cells of interest; iv) incubating the cells of interest with the target cells attached to the surface; v) determining the cellular avidity score of the cells of interest to the target cells by applying the selected force on the cells of interest and determine cells of interest detaching from the target cells and/or cells of interest remaining attached to the target cells.

Such further methods relying on the selected force in accordance with the invention may be carried out with the target cells attached to the surface used in the method of selecting the force, but may of course also be performed with newly provided target cells attached to a surface. It is understood that such newly provided target cells attached to a surface are of course prepared in the same way as used in the methods of selecting the force. Of course, in these further methods, control cells of the cells of interest can be provided as well, and steps iv) and v) can be performed therewith.

A parameter of interest in addition to cellular avidity may be the ratio between the cellular avidity of the cells of interest and the control cells. For example, the higher the ratio, it may be indicative of the cell of interest and target cell interaction being more selective, and may allow e.g. to compare different cell of interest and/or different target cell interactions.

Advantageously, of a variety of cells of interest the cellular avidity may be determined. This is for example of interest when it is desirable to provide for a candidate CAR, a variety of cellular avidities. By providing a variety of CARs, candidates can be selected and subsequently assessed with regard to functionality, e.g. by in vivo experiments. As cellular avidity is an important parameter for function, this way highly efficiently, suitable candidates can be selected.

Of course, the selected force is not only useful for determining cellular avidity, but can also be utilized in other methods for other purposes. For example, further methods can include methods for identifying a candidate agent capable of modulating the cellular avidity between a cell of interest and a target cell, wherein in the steps i) to — iv) of the method as defined above, agent(s) are provided. This way, when it is for instance of importance or interest to block or enhance a particular interaction, agent(s) capable of doing so can be identified. Furthermore, a selected force can also be used in methods for screening of different cells of interest, by performing the method as defined, optionally in the presence of agent(s) as defined above, and comparing determined cellular avidities for the different cells of interest. This way, for example, in case agents are present that are known to modulate a desired interaction between a cell of interest and a target cell, receptor/ligand interactions may be selected that are not affected by such agents, or, conversely, are aided by such agents. Of course, once such a force has been selected, and/or agent, and shown to provide for selectivity with regard to binding of cells of interest to the target cells, as opposed to control cells, such can also be used for selecting and/or sorting cells of interest. Hence, in another embodiment, a method is provided for selecting and/or sorting cells of interest, comprising the steps i) to iv) of the method as defined above, and optionally in the presence of agent(s) as defined above, comprising the further step of applying the selected force on the cells of interest and subsequently selecting and/or sorting cells of interest that have detached and/or that remain attached to the target cells.

Cells of interest which can be selected and/or sorted and thus be obtained accordingly with means and methods in accordance with the invention may subsequently in a final step be admixed with a pharmaceutically acceptable buffer or otherwise pharmaceutical acceptably formulated.

Examples

Materials and methods.

Primary cells and cell lines

Untransduced or FMC63 CAR transduced Jurkat effector cells were obtained from

Creative Biolabs while Raji and NALM6 (ATCC) cells were used as target cells. All the cell lines were cultured with RPMI+Glutamax supplemented with 10% heat inactivated fetal bovine serum and Penicillin-Streptomycin, unless otherwise indicated.

Avidity measurement

Z-Movi chips (obtained and as available from LUMICKS (<<https://lumicks.com/products/z-movi-cell-interaction-studies/>>), with channel dimensions of 7x2x0.1mm and made of glass) were coated the day before performing the experiment with Poly-L-Lysine (#P4707-50ML, Sigma) diluted 1:5 in PBS for 10 minutes. The following morning the target cells were seeded in serum free culture medium, incubated for 1h at 37°C, and the medium was replaced with RPMI complete medium. The target cells were incubated for 1.5 h before starting the cellular avidity experiment. The effector cells were stained with CellTrace™ Far Red dye (Thermo

Fisher Scientific) at 1 uM for 15 minutes in PBS at 37°C, then resuspended at 10 million/mL in complete medium and used for the avidity experiments. The effector cells (i.e. cells of interest) were introduced in the target cell-seeded flow cell, incubated for 5 minutes, and then different force ramps were applied throughout 1 or 2.5 minutes using Z-Movi operated of which the Ocean V1.4 software which was customized to allow control of force duration (time) and control of force increase (i.e. pN/s). The whole procedure in general can be performed in about 2-3 hours.

Varying force

The acoustic force on the effector cells was applied as a linear ramp from 0 to 1000 or from 0 to 3000 pN over 1 or 2.5 minutes.

Results

We hypothesized that different strength of binding defines the interaction between the transduced effector cell-target cell and untransduced effector cell-target cell (control cell-target cell) and that changing the way we apply the force could improve the positive/negative window while preserving the transduced-target cell binding and reducing the aspecific binding of the untransduced sample.

We tested this hypothesis by monitoring the percentage of effector cells bound to the target cells after application of different acoustic force ranges. A linear force ramp applied over the course of 2.5 minutes to 1000 pN on Jurkat effector cells layered on

NALM®G target cells resulted in a ~47% difference in percentage of bound cells at the end of the ramp between the positive and negative untransduced control. Increasing the target end force to 3000 pN while keeping constant the force ramp time to 2.5 minutes enhanced the difference in percentage of bound cells at the end of the ramp to ~68%, thus considerably improving the positive/negative window of the assay (figure 2).

We next repeated the experiment using as target Raji cells, a cell line displaying high levels of aspecific binding of the untransduced sample. While a force ramp of 2.5 minutes to 1000 pN resulted in a difference of ~26%, a ramp with an end force to 3000 pN gave a difference between the positive and negative sample of ~52% (figure 3).

We also subjected the target cell monolayer to multiple force ramps at different end forces, and qualitatively evaluated the viability and the confluency of the target cell monolayer after staining with a Trypan Blue solution at the end of the measurements and could not observe differences across three different cell lines between chips acquired with the lower or higher end force ramps (figure 4). This observation ruled out that the decrease in background binding observed and shown in Figures 2 and 3 was due to a decrease of the target cell monolayer viability or integrity.

Claims

CONCLUSIONS

A method of selecting a force to apply to discriminate between binding of cells of interest and control cells to target cells, comprising the steps of: a) Providing target cells attached to a surface; b) Providing cells of interest and control cells of the cells of interest; c) Contacting the control cells with the target cells, for allowing the control cells to interact with the target cells; d) Applying a first force to the control cells, the force being in a direction away from the target cells; e) Determining control cells that have detached and/or remained bound in step d) and providing a cellular avidity score for the control cells; f Perform steps c), d) and e) with the cells of interest instead of the control cells; g) Repeat steps c)-f) with the application of a second force; h) Optionally, perform steps c, d, and e) with one or more forces; i} Selecting the force to be applied from the first, second, and optional further forces, by comparing, with the same applied force, the cellular avidity scores as determined in step e) of the cells of interest and of the control cells.

The method of claim 1, wherein the applied force is a force ramp, preferably a linear force ramp.

The method of claim 2, wherein when the applied force is a force ramp, applying the different forces as a first, second, and optional further force includes providing for each force ramp a force load rate and an end force.

The method according to any one of claims 1-3, wherein the applied force is an acoustic force, a shear force, or a centrifugal force.

The method according to any one of claims 1-4, wherein the step c) of contacting the control cells and the cells of interest, and subsequent steps d) and e) are performed separately, or wherein these steps are performed with the control cells and cells of interest combined.

The method according to any of claims 5, wherein in the force selection step, of the selected force ramp, the force speed is selected and the final force, or the force speed is selected and a lower force final force.

The method according to any one of claims 1-6, wherein the target cells are attached to a glass surface, preferably a glass surface in a chip.

The method of any one of claims 1-7 wherein the target cells are attached to the surface as a monolayer.

A method of determining cellular avidity comprising: i) providing a selected force according to any one of claims 1-8; ii} providing target cells attached to a surface; iii} providing cells of interest; iv) incubating the cells of interest with the target cells attached to the surface; v) determining the cellular avidity score of the cells of interest with the target cells by applying the selected force to the cells of interest and determining detachment of the cells of interest from the target cells and/or cells of interest that remain bound to the target cells.

The method of claim 9, wherein the method is performed with the target cells attached to the surface used in the method of any one of claims 1-8, and/or with newly provided target cells attached to a surface.

The method of claim 9 or claim 10, wherein the control cells of the cells of interest are provided, and steps iv) and v) are performed therewith, or wherein control cells of the target cells are provided and further provided attached to a surface and steps iv) and v) are thus performed.

The method of claim 11, wherein the ratio between the avidity of the cells of interest and the control cells is determined.

The method of any one of claims 9-12, wherein the cellular avidity of a variety of cells of interest is determined.

A method of identifying a candidate agent capable of modulating cellular avidity between a cell of interest and a target cell, comprising the steps of the method as defined in any one of claims 9-13, wherein an agent or agents are provided, and wherein the agent or agents are present in the incubation step.

A method of screening different cells of interest, by performing the method as defined in any one of claims 9-13, optionally in the presence of an agent or agents as defined in claim 14, and comparing certain cellular avidities of the different cells of interest.

A method of selecting and/or sorting cells of interest, comprising the steps i) to iv) of the method as defined in claim 9 or 10, and optionally in the presence of an agent or agents as defined in claim 14, comprising the further step of applying the selected force to the cells of interest and then selecting and/or sorting the cells of interest that have detached from and/or remained bound to the target cells.