WO2024170791A1

WO2024170791A1 - Means and methods for determining cellular avidity of primary t cells and target cells

Info

Publication number: WO2024170791A1
Application number: PCT/EP2024/054106
Authority: WO
Inventors: Patrycja Anna WAWRZYNIECKA; Trillian Ashley GREGG; Mirjam Rebekka DALENBERG; Lotte Susanne GROEN
Original assignee: Lumicks CA Holding BV
Current assignee: Lumicks CA Holding BV
Priority date: 2023-02-17
Filing date: 2024-02-19
Publication date: 2024-08-22
Anticipated expiration: 2025-08-17
Also published as: EP4665843A1

Abstract

The current invention relates to cell - cell interactions and in particular to cellular avidity. Provided are improved means and methods to study cell – cell interactions mediated and characterizing cellular avidity. Highly advantageously, intricate methods and protocols are provided to provide for T cells allowing for efficiently and accurately assess interactions between T cells and target cells.

Description

Title Means and methods for determining cellular avidity of primary T cells and target cells

Introduction

Despite tremendous clinical success of T cell therapy, including CAR-T cell therapy, for haematological malignancies, many obstacles still remain for the therapy in treatment of a wider range of cancers. Predicting success of T-cell therapy using in vitro data alone is still a challenge. Affinity of a binding molecule used for CAR generation towards the target antigen is frequently considered during the in vitro testing, but this does not define CAR-T cell activity. With the increasing complexity of constructing novel methodologies to further improve the clinical outcome of immunotherapies, methods to e.g. quickly identify and characterize candidates is becoming even more essential. To date, it has become evident that merely measuring the affinity between a CAR and its target will not accurately predict in vitro and in vivo outcomes. On the contrary, cellular avidity, the overall cellular binding strength between an effector cell and a target cell, provides a more complete and physiologically relevant parameter that reflects the bona fide interaction between T cells and target cells.

One of the main obstacles in the process of measuring avidity as being a critical parameter is the lack of fast, specific, and accurate tools to assess cellular avidity. The z-Movi® Cell Avidity Analyzer, a platform for measuring cell-cell binding strength facilitates a direct analysis of CAR-T cells either against surface immobilised antigens or a monolayer of target cells. Using this system, cell-cell interactions are perturbed using resonant sound waves generated by a piezoelectric element and a cell tracking system is employed to measure the required disruption force. This way, cellular avidity measurements can be done which provide for a cellular avidity score which allows to compare e.g. different candidate receptors.

The current inventors, in working with cellular avidity were now looking for means and methods to further improve cellular avidity measurements. In particular, the current inventors worked on providing highly optimized conditions for performing cellular avidity measurements.

Summary of the invention

The current inventors in working with primary T cells, expressing e.g. chimeric antigen receptors (CAR), and target cells, and performing cellular avidity measurements therewith, sought to optimize the means and methods that allow to provide for reproducible results. Such reproducible results are highly desirable when comparing different experimental conditions, e.g. comparing different CARs, different culture conditions, and effects on different target cells. The current inventors now provide for highly advantageous means and methods with which such reproducible results can be obtained. Within defined parameters as provided by the means and methods in accordance with the invention, cellular avidity windows are optimal (i.e. difference in cellular avidity measurement between conditions, such as negative and positive conditions, is relatively large) and statistically significant differences can be detected between transduced cells having a CAR and untransduced cells, already with experiments conducted with a few biological replicates (e.g. testing and comparing only 2 or 3 different donors). This way, highly efficiently, i.e. in relatively short time and with relatively little resources (least amounts of biological samples), highly accurate results can be provided allowing for proper assessment e.g. in scientific studies.

In one embodiment, a method is provided for providing transduced CD8+ T cells, for determining cellular avidity of said transduced CD8+ T cells with target cells, by exerting a force, wherein said method comprises the steps of: a) providing PBMCs, on day 0; b) selecting CD8+ T cells from the PBMCs, wherein the selected population is at least 90% CD8+; c) seeding the CD8+ T cells in an appropriate cell culture medium, and activating cells for 2 days by providing an activation stimulus such as a CD3/CD28 activator in the medium and supplementing IL- 2 in the medium; d) transducing the activated CD8+ T cells with a retroviral vector, on day 2, while supplementing the medium with IL-2, and allowing the transduction to proceed for 72 hours; e) on day 5, optionally, removing the activation stimulus, optionally determining transduction efficiency and/or optionally enriching for transduced CD8+ T cells for providing transduced CD8+ T cells having a transduction efficiency of at least 80 percent; f) on day 5, further seeding and culturing the transduced CD8+ T cells in the presence of IL-2, wherein the culture medium is supplemented with IL-2 every 2-3 days; g) determining cellular avidity of the cultured transduced CD8+ T cells to target cells, by providing target cells attached to a surface, allowing the cultured transduced CD8+ T cells to interact therewith and subsequently exerting a force away from the attached target cells, wherein cellular avidity is determined on day 6, up to, and including, day 21 ; wherein, optionally, after step f) and, 24 hours, or less, prior to step g), determining transduction efficiency and/or optionally enriching for transduced CD8+ T cells for providing transduced CD8+ T cells having a transduction efficiency of at least 80 percent. Hence, in this embodiment, the method starts with PBMCs and continues through to measuring cellular avidity while the cells are kept in culture.

As shown in the examples, in a further embodiment, cellular avidity as determined in step g) is performed on days 7, 8 or 9. Most optimal results were obtained on those days.

In another embodiment, a method is provided for providing transduced CD8+ T cells, for determining cellular avidity of said transduced CD8+ T cells with target cells, by exerting a force, wherein said method comprises the steps of: a) providing PBMCs, on a day 0; b) selecting CD8+ T cells from the PBMCs, wherein the selected population is at least 90% CD8+; c) seeding the CD8+ T cells in an appropriate cell culture medium, and activating cells for 2 days by providing an activation stimulus, such as a CD3/CD28 activator, in the medium and providing IL-2 in the medium; d) transducing the activated CD8+ T cells with a retroviral vector, on day 2 while supplementing the medium with IL-2, and allowing the transduction to proceed for 72 hours; e) on day 5, optionally, removing the activation stimulus, optionally determining transduction efficiency and/or optionally enriching for transduced CD8+ T cells for providing transduced CD8+ T cells having a transduction efficiency of at least 80 percent; and, f) cryopreserving the transduced CD8+ cells on day 5, or on day 5, further seeding and culturing the transduced CD8+ T cells in the presence of IL-2, wherein the culture medium is supplemented with IL-2 every 2-3 days, and cryopreserving at a day up to, and including, day 9 after providing the PBMCs; g) thawing the frozen cells, and further seeding and culturing the transduced CD8+ T cells in a medium which is supplemented with IL-2 every 2-3 days; h) determining cellular avidity of the cultured transduced CD8+ T cells to target cells, on day 2, day 3, or day 4 after step g) of thawing the frozen transduced CD8+ T cells, by providing target cells attached to a surface, allowing the cultured transduced CD8+ T cells to interact therewith and subsequently exerting a force away from the attached target cells; wherein, optionally, after step g) and, 24 hours, or less, prior to step h), determining transduction efficiency and/or optionally enriching for transduced CD8+ T cells for providing transduced CD8+ T cells having a transduction efficiency of at least 80 percent. In this method, after the PBMCs are provided and subjected to stimulation and transduction, prepared T cells are cryopreserved, such that these can be stored for some time, e.g. at -196°C, and provided at a later timepoint for cellular avidity measurement. Advantageously, this allows for more flexibility in experimental design and also allows e.g. for inclusion of suitable reference T cells in experimental design. After the cells are appropriately frozen, cellular avidity can be measured after allowing the cells to briefly recover in culture.

As shown in the examples, in a further embodiment, cellular avidity as determined in step h) is performed on day 3. Most optimal results were obtained on that day.

In yet another embodiment, a method is provided for determining cellular avidity between effector cells and target cells, by providing target cells attached to a surface, allowing the effector cells to interact therewith, and by subsequently exerting a force away from the attached target cells, wherein 24 hours or less prior to determining cellular avidity, effector cells or target cells are subjected to a selection step, wherein said selection step comprises a positive selection using MACS. Highly surprisingly, the current inventors found that while optimizing means and methods for cellular avidity measurement, subjecting T cells to selection procedures which involved magnetic beads, typically referred to in the art as microbeads, which bind to T cell subpopulation to thereby allow for enrichment of such cell subpopulations, such selection procedures were compatible with cellular avidity measurements that rely on exerting a force on the T-cell subpopulations. This was highly surprising as the magnetic microbeads could thus remain attached/present and not interfere with cellular avidity measurements. Highly advantageously, this allows to apply for enrichment steps just prior to measurement of cellular avidity in any of the methods for cellular avidity measurements in accordance with the invention as described herein.

Figures

Figure 1 . Shown are cellular avidity measurement results obtained comparing different cytokines used for T cell culture in assays as described in example 1 . The percentage of bound cells remaining at the end of different cellular avidity runs is shown for untransduced cells (UNT) and transduced cells (CAR) (left and right bars respectively, and datapoints shown as circles and squares respectively) on either Raji cells (1A) or Nalm6 cells (1 B) with either IL2 or IL7/IL15. Highly comparable results, with regard to statistical significance and averages of bound cells remaining, were obtained when comparing IL2 vs IL7/IL15.

Figure 2. Shown are cellular avidity measurement results comparing different culturing times postthaw and culturing in the presence of IL-2, immediately added post-thaw (A) or added 1 day postthaw (B) as described in example 2. Cellular avidity is shown as percentage of bound cells remaining at the end of a run for untransduced cells (UNT) and transduced cells (CAR) on Nalm6 cells at different time points post thaw (0, 1 , 2, 3 and 4 days, from left to right). Results show that when comparing cellular avidity measurements of untransduced cells with transduced cells, best results are obtained 72 hours post-thaw (3 days), with best statistically significant differences observed in both experimental set-ups (A ***; B *). With regard to the other time points, statistically significant differences (*) were also observed at 24, 48, and 96 hours post-thaw (i.e. Day 1 , 2 and 4), when IL- 2 was added immediately post-thaw.

Figure 3. Shown are cellular avidity measurement results obtained from untransduced (UNT, circles and left) and transduced (CAR, triangles and right) CD3+, incubated with or without CD4 and CD8 magnetic microbeads, with Nalm6 cells. Cellular avidity is shown as percentage of bound cells remaining at the end of a run, As can be observed by comparing the datapoints, highly similar results were obtained under Regular conditions (without microbeads) and in the Beads (microbeads) condition. This was confirmed by statistical analysis, which showed no statistically significant difference when comparing cellular avidity measurements of either UNT or CAR in the presence or absence of beads (ns). The difference between UNT and CAR in either condition reached the same statistical significance (**). Figure 4. Cellular avidity measurement at different days post-transduction. Shown are cellular avidity measurement results at days 3, 4, 5, 6 and 7 (i.e. days 5, 6, 7, 8 and 9 after isolation/stimulation), plotted as percentage of bound cells remaining at the end a run for untransduced cells (UNT, squares, lower line) and transduced cells (CAR, triangles, upper line) on Nalm6 cells.

Figure 5. Cellular avidity measurement during culturing cells for up to 4 weeks post-transduction of T cells. Shown are cellular avidity measurement results at days 6, 9, 13, 14, 16, 20, 21 , 23, 27, 28, 30 after isolation/stimulation, plotted as percentage of bound T cells remaining at the end a run for untransduced cells (UNT, lower line) and transduced cells (CAR, upper line) on Nalm6 cells..

Figure 6. IL2 timing. IL2 was supplemented after thawing (DO) and was supplemented after thawing and after 48 hours (D0+D2), subsequently, 72 hours after thawing, cellular avidity measurements were performed. Cellular avidity was plotted as percentage of bound T cells remaining at the end of a run for untransduced cells (UNT, circles/left) and transduced cells (CAR1 , squares/middle, and CAR2, triangles/right) on Nalm6 cells. Results indicate that there were no statistically significant (ns) differences between IL-2 supplementation on DO and D0+D2.

Figure 7. Cell density and cellular avidity. Cellular avidity is plotted as percentage of bound T cells remaining at the end of a run for untransduced cells (UNT, circles/left) and transduced cells (CAR, squares/right) on Nalm6 cells. The same amount of cells in the same volume were seeded in different well-formats having different surface areas. From left to right, 6-well, 12-well, 24-well, 48-well results are respectively plotted. Results indicate that best statistical significances was obtained with the 24- well and 48-well format (****).

Figure 8. Culture medium volume and cellular avidity. Cellular avidity is plotted as percentage of bound T cells remaining at the end of a run for untransduced cells (UNT, circles/left) and transduced cells (CAR, squares/right) on Nalm6 cells. The same amount of cells were seeded in different volumes as indicated at the same surface area, i.e. in 24-well plate format. Results indicate that different volumes (0.5 ml, 1 ml or 2 ml, from left to right) did not result in differentiating resultsNote that there were no statistically significant (ns), differences and almost identical bar heights. Statistical significance between UNT vs. CAR for individual donor in each volume was **** (not shown in the graph), which is in alignment with results previously obtained and shown in Fig. 7.

Figure 9. Recommended primary T cell transduction and culture protocol timeline. Fresh blood product is obtained on Day 0, and CD8+ T cells are isolated, stimulated and supplemented with IL2. On day 2, retroviral transduction is performed, and on day 5 transduced T cells are harvested, transduction efficiency is checked, and the cells are sorted for transduction marker if needed. The optimal recommended days for cellular avidity measurement are days 7, 8 and 9. Cellular avidity measurements can be performed any time from day 6 to day 21 . In case of cryopreservation of the transduced T cells, the cryopreservation is performed on day 5, up to day 9. The optimal recommended time for cellular avidity measurement post T cell thaw is day 3, although the avidity measurement can also be successfully performed on days 2 and 4.

Detailed Description

Accordingly, in one embodiment a method is provided for providing transduced CD8+ T cells, for determining cellular avidity of said transduced CD8+ T cells with target cells, by exerting a force, wherein said method comprises the steps of: a) providing PBMCs, on day 0; b) selecting CD8+ T cells from the PBMCs, wherein the selected population is at least 90% CD8+; c) seeding the CD8+ T cells in an appropriate cell culture medium, and activating cells for 2 days by providing an activation stimulus such as a CD3/CD28 activator in the medium and supplementing IL- 2 in the medium; d) transducing the activated CD8+ T cells with a retroviral vector, on day 2, while supplementing the medium with IL-2, and allowing the transduction to proceed for 72 hours; e) on day 5, optionally, removing the activation stimulus, optionally determining transduction efficiency and/or optionally enriching for transduced CD8+ T cells for providing transduced CD8+ T cells having a transduction efficiency of at least 80 percent; f) on day 5, further seeding and culturing the transduced CD8+ T cells in the presence of IL-2, wherein the culture medium is supplemented with IL-2 every 2-3 days; g) determining cellular avidity of the cultured transduced CD8+ T cells to target cells, by providing target cells attached to a surface, allowing the cultured transduced CD8+ T cells to interact therewith and subsequently exerting a force away from the attached target cells, wherein cellular avidity is determined on day 6, up to, and including, day 21 ; wherein, optionally, after step f) and, 24 hours, or less, prior to step g), determining transduction efficiency and/or optionally enriching for transduced CD8+ T cells for providing transduced CD8+ T cells having a transduction efficiency of at least 80 percent.

Hence, in the first step of the method, on day 0, PBMCs are provided. PBMCs refers to peripheral blood mononuclear cells. These cells include immune cells with a single, round nucleus that originate in bone marrow and are secreted into peripheral circulation. These cells are components of the immune system and are involved in both humoral and cell-mediated immunity. These cells can be obtained e.g. from a buffy coat or from fresh blood. Such PBMCs highly preferably are human PBMCs. From the provided PBMCs, CD8+ cells are preferably selected. Although it may be contemplated to use primary T cells, CD3+ cells or pan -T cells, instead in alternative methods in accordance with the invention, using CD8+ cells allows to provide for more consistent results. The CD8+ T cells, also referred to as cytotoxic T cells, are preferred as they are highly preferred effector cells to be provided with a chimeric antigen receptor (CAR) in the methods of the invention. The selected CD8+ cell population preferably represents a high percentage of the selected population. The CD8+ cell population may be selected from PBMCs with any suitable means available, such as FACS, MACS or alternative cell sorting protocol. It may be preferred to use MACS or alternative magnetic cell separation protocol as this may be less stressful for the cells and high yields of CD8+ T cells are obtained. In any case, the percentage of isolated CD8+ cells is preferably high, e.g. in some embodiments at least 60%, at least 70%, at least 80%, at least 90%, or more of CD8+ T cells may be provided. In case different donors are used, preferably, the percentage of CD8+ selected between donor cells is to be similar. It is highly preferred to use a method that provides for at least 90% CD8+ cells in order to provide consistent quality of CD8+ cells, such as MACS and as utilized in the examples herein or alternative magnetic cell separation protocol.

As said, in case CD3+ T cells are to be used, these can be selected from provided PBMCs instead using methods such as MACS, e.g. positive MACS with CD3+, or negative MACS, i.e. such as with the a pan-T cell selection kit such as described in the examples herein. Hence, it is understood that in embodiments wherein CD3+ T cells are contemplated, instead of selecting CD8+ T cells from the PBMCs, selected in step b) are CD3+ T cells from the PBMCs, wherein the selected population is at least 90% CD3+.

Subsequently, the CD8+ T cells are seeded in an appropriate cell culture medium, and subjected to activation for 2 days by providing an activation stimulus through a CD3/CD28 activator in the medium and supplementing IL-2 in the medium. Providing an activation stimulus to the CD8+ T cells activates the cells and triggers their expansion. Suitable activation stimuli are known in the art and any may be contemplated. A suitable activation stimulus or activator can be CD3 and CD28 targeting antibodies or beads, such as described in the examples herein, which are widely used in the art. Such an activation stimulus may be highly preferred in the embodiments as contemplated herein. In one embodiment, in the means and methods in accordance with the invention, the activation stimulus that is provided is DynaBeads™ Human T-Activator CD3/CD28. Furthermore, a highly suitable interleukin is to be added for culturing T cells, i.e. Interleukin 2, (also referred to as IL- 2 or IL2).

Following up on the stimulation, subsequently, the CD8+ cells are to be transduced with a retroviral vector. Transduction is to be performed on day 2, while supplementing the medium with IL- 2, and allowing the transduction to proceed for 72 hours. Retroviral vectors, such as based on MMLV, require division of cells for transduction as the provirus cannot cross the nuclear membrane. Allowing the transduction to proceed for 72 hours allows for retroviral vectors to be integrated into the cellular genome and to express the transgene. It is understood that as a retroviral vector, lentiviral vectors may be contemplated as well. As a further embodiment, in means and methods in accordance with the invention as described herein, it can be preferred to aid the transduction with the retroviral vector by inclusion of a fibronectin reagent, such as RetroNectin, such as described in the example section herein.

Subsequently, on day 5, optionally, the activation stimulus can be removed. It is understood that removal may include e.g. the use of methods and/or devices that can selectively remove the stimulus. For example, as described in the examples herein, in case of magnetic beads carrying a CD3/CD28 stimulus, this may involve using a magnet. One may also wash cells I spin down cells to remove the stimulus by dilution. In any case, in some scenarios it may be contemplated not to remove an activation stimulus. On day 5, one may also determine transduction efficiency. This is optional, as it may be contemplated that one uses well controlled transduction procedures which results in highly reproducible efficiencies or one may determine transduction efficiency (and/or enrich) at a later timepoint. Nevertheless, transduction efficiency may be determined and/or one may optionally enrich for transduced CD8+ T cells to thereby provide for transduced CD8+ T cells having a transduction efficiency of at least 80%. In case transduction efficiency is sufficiently high, it may not be required to have an enrichment step. In case transduction efficiency is low, e.g. below 80%, this may warrant an enrichment step. One can easily enrich for transduced CD8+ T cells, e.g. by using FACS sorting or MACS, e.g. in case of the viral vector used also expresses a selectable marker. A selectable marker for FACs sorting may include a fluorescent marker. A selectable marker may be an antigen expressed on the cell surface that may be used for MACS sorting using beads capable of specifically binding with the antigen, e.g. via a conjugated antibody. Hence, on day 5, one optionally, removes the activation stimulus, optionally determines transduction efficiency and/or optionally enriches for transduced CD8+ T cells for providing transduced CD8+ T cells having a transduction efficiency of at least 80 percent.

On day 5, one subsequently further seeds and cultures the transduced CD8+ T cells in the presence of IL-2, wherein the culture medium is supplemented with IL-2 every 2-3 days.

Next, cellular avidity is determined of the (cultured) transduced CD8+ T cells to target cells, by providing target cells attached to a surface, allowing the cultured transduced CD8+ T cells to interact therewith and subsequently exerting a force away from the attached target cells, wherein cellular avidity is determined on day 6, up to, and including, day 21 .

Target cells in accordance with the invention are the cells on which the T cells are to exert an effect, i.a. bind therewith. Target cells include cancer cells presenting an antigen. An antigen may be presented by MHC, i.e. HLA in humans, which are specialized receptors that present peptides e.g. derived from digested proteins expressed by the cell (e.g. usually 8-1 1 amino acids in length for MHCI). An antigen may also be a protein or other biomolecule that is presented on the surface of a cell, e.g. epidermal growth factor receptors or checkpoint proteins, which in the case of cancer cells are overexpressed therewith providing a differentiating feature. Target cells may also include cells expressing auto-antigens, e.g. known to be involved in autoimmunity diseases or cells infected with a pathogen, e.g. a virus or edited by genomic engineering techniques. In accordance with the invention, effector cell may be CD8+ T cells carrying a CAR receptor. As shown in the examples, Nalm6 and Raji cells can represent suitable target cells as these are known cancer cells express the CD19 antigen.

In the context of avidity measurement, the target cells are attached to a surface. It is understood that surfaces for attaching cells may be any surface suitable for attaching cells. Suitable surfaces for attaching cells include plastic or glass surfaces. These surfaces may be coated e.g. with a protein to attach cells to the surface, such as poly-L-lysine or the like. The attachment of the cells to the surface is such that the strength of the binding to the surface is sufficient to have the cells remaining attached when applying a suitable force during a cellular avidity measurement.

It is understood that the effector cell, i.e. T cell or CD8+ T cell, is capable of binding target cells, or is studied for its capability of binding target cells. It is understood this capacity of binding target cells can include a specific interaction that can induce synapse formation, i.e. the effector cell is to bind to a target cell and ultimately exert an effect thereon, e.g. induce target cell killing. It is understood that a synapse is a specialized structure that forms when the plasma membranes of two cells come into close proximity to transmit signals. Cells of the immune system form synapses that are essential for cell activation and function. Lymphocytes such as T cells can form synapses that can be referred to as immunological synapses. Such a synapse typically forms between immune effector cells and target cells, e.g. cells presenting an antigen. An example is e.g. a T cell or a CAR- T cell and a cancer cell, such as used in the examples as described herein. The formation of synapses between e.g. an effector cell and a target cell, for example an APC (antigen presenting cell), is a hall-mark event and signals the presence of specific interactions (i.e. the specific interaction between, for example, a TCR or CAR and an antigen recognized thereby) between the effector cell and the target cell that are involved in the formation of such immunological synapses.

The CD8+ T cells and the target cells are in proximity to each other to allow the cells to interact. This step is such that the effector cells will have sufficient time to interact with target cells and can form a bond, including synapses. It is understood that a cell-cell interaction may not always result in a cell-cell bond, which can be a synapse or aspecific bond, the contacting step is such that cell-cell bonds can be formed and appropriate conditions therefore are selected. It is understood that because the conditions are selected such that a synapse can be formed, this necessarily implies that aspecific cell-cell bonds are allowed to be formed at the same time. This interaction step is to be performed for a defined amount of time.

Once the effector cells have interacted with the target cells for a defined time, and thus have had the opportunity, if possible, to form a synapse, in a subsequent step a force is applied. The direction of the force is away from the cells attached to the surface (monolayer cells), such that at least part of the cells bound to the cells attached to the surface (monolayer cells), and unbound cells as well, move away from the cells attached to the surface (monolayer cells). This way, target cells are obtained with effector cells bound thereto, which are attached to the surface, wherein the exerted force was not sufficient to break cell-cell bonds. Of course, cells that are attached to the surface, to which no subsequent cells are bound, remain as well.

As said, the force is applied in a direction away from the attached 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 cells are attached, for example when a centrifugal force or acoustic force is applied. The force may also be lateral (in the direction of the x-axis or y-axis relative to the surface), for example when a shear force is applied. In any case, the force is applied and is controlled such that a defined force is exerted on the cells that interacted with the attached cells. It is understood that the force that is exerted on the cells interacting with attached cells is to be substantially equal, such can be achieved e.g. when using a flat surface. 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 to which cells are attached, such a surface shape may be contemplated.

The cells that are attached are preferably attached to a glass or plastic surface, preferably a surface in a chip, which allows for detection of cells e.g. via microscopy or other means. The applied force required to move a cell away from an attached cell preferably can be detected, e.g. via microscopy or other means, to which may be referred to as a cell detachment event or cells moving away. This way, cells moving away can be monitored and counted. It may be advantageous and convenient to use microscopy, with which bound cells can be identified and quantified and cells moving away can be likewise monitored and quantified, also allowing e.g. to detect markers. For example, the z-Movi® device as available from Lumicks which applies an acoustic force may be well equipped to do so. Likewise, similar devices may be provided with microscopy or other means to quantify cells, detect markers and bound cells and/or cells moving away, and also utilizing e.g. shear force or centrifugal forces instead of acoustic force.

In any case, transduced CD8+ cells that have remained bound to the target cells, and are attached to the surface after applying the force, are detected. Furthermore, cellular avidity score is determined. It is understood that cellular avidity can be determined by providing a cellular avidity score based on the number of cells that have moved away and/or remain bound after the force has been exerted. It is understood that the cellular avidity score highly preferably is to be determined based on the transduced CD8+ T cells that were not attached to the surface. For example, as shown in the example section herein, when the target cells (such as Nalm6 or Raj) were attached to the surface, and the transduced CD8+ T cells provided, cellular avidity was presented as a cellular avidity score expressed as the percentage of CD8+ T cells that remained bound after exerting the force. Such a cellular avidity score can be determined utilizing the determined number of CD8+ T cells that have moved away and/or remain bound after the force has been exerted.

A cellular avidity score can also be calculated by determining the ratio of the number of effector cells that remained bound with the target cells to the number of effector cells that was initially provided or to the number of effector cells that moved away. As is understood herein, the ratio’s determined may be based on absolute numbers, or may be calculated based on numbers determined of fractions thereof. For example, one may determine of a defined surface with cells attached, the number of subsequent cells that interact therewith and detect each cell that moves away from the attached cells and determine the number of cells at the defined surface that remain after the force has been exerted. One may represent calculated ratio’s as a number or as a percentage. In any case, determining the number of cells that have moved away and/or remain bound after the force has been exerted allows one to provide for a cellular avidity score that is a highly useful measure which is indicative of how well the effector cell carrying the receptor can binding with a target cell. The more cells that remain bound and/or the less cells that move away, the higher the cellular avidity is, which is reflected in a calculated cellular avidity score which allows for comparing e.g. different effector cells. Hence, in a further embodiment, determining cellular avidity comprises: - determining the number of transduced CD8+ T cells or effector cells that have moved away and/or remain bound after applying the force. In yet another further embodiment, based on the determined number(s), a cellular avidity score is provided.

Furthermore, as the cellular avidity measurement between the transduced CD8+ T cells and target cells can be determined up to day 21 in culture, it may be contemplated to prior to determining cellular avidity, determine the transduction efficiency of the CD8+ T cells and/or enrich fortransduced CD8+ T cells. This way, one can control for transduction efficiency. Of course, one may always rely on established procedures known to provide for sufficient transduction efficiency within the time frame of the experiment. Thus, in case one wishes to determine transduction efficiency or enrich for transduced CD+ T cells, one optionally, after step f) and, 24 hours, or less, prior to step g), determining transduction efficiency and/or optionally enriching for transduced CD8+ T cells for providing transduced CD8+ T cells having a transduction efficiency of at least 80 percent.

In one embodiment a method is provided for providing transduced CD8+ T cells, for determining cellular avidity of said transduced CD8+ T cells with target cells, by exerting a force, wherein said method comprises the steps of: a) providing PBMCs, on day 0; b) selecting CD8+ T cells from the PBMCs, wherein the selected population is at least 90% CD8+; c) seeding the CD8+ T cells in an appropriate cell culture medium, and activating cells for 2 days by providing an activation stimulus such as a CD3/CD28 activator in the medium and supplementing IL- 2 in the medium; d) transducing the activated CD8+ T cells with a retroviral vector, on day 2, while supplementing the medium with IL-2, and allowing the transduction to proceed for 72 hours; e) on day 5, optionally, removing the activation stimulus, f) on day 5, further seeding and culturing the transduced CD8+ T cells in the presence of IL-2, wherein the culture medium is supplemented with IL-2 every 2-3 days; g) determining cellular avidity of the cultured transduced CD8+ T cells to target cells, by providing target cells attached to a surface, allowing the cultured transduced CD8+ T cells to interact therewith and subsequently exerting a force away from the attached target cells, wherein cellular avidity is determined on day 6, up to, and including, day 21 ; wherein, after step f) and, 24 hours, or less, prior to step g), determining transduction efficiency and/or enriching for transduced CD8+ T cells for providing transduced CD8+ T cells having a transduction efficiency of at least 80 percent.

In a further embodiment a method is provided for providing transduced CD8+ T cells, for determining cellular avidity of said transduced CD8+ T cells with target cells, by exerting a force, wherein said method comprises the steps of: a) providing PBMCs, on day 0; b) selecting CD8+ T cells from the PBMCs, wherein the selected population is at least 90% CD8+; c) seeding the CD8+ T cells in an appropriate cell culture medium, and activating cells for 2 days by providing an activation stimulus such as a CD3/CD28 activator in the medium and supplementing IL- 2 in the medium; d) transducing the activated CD8+ T cells with a retroviral vector, on day 2, while supplementing the medium with IL-2, and allowing the transduction to proceed for 72 hours; e) on day 5, optionally, removing the activation stimulus, and provide for transduced CD8+ T cells having a transduction efficiency of at least 80 percent; f) on day 5, further seeding and culturing the transduced CD8+ T cells in the presence of IL-2, wherein the culture medium is supplemented with IL-2 every 2-3 days; g) determining cellular avidity of the cultured transduced CD8+ T cells to target cells, by providing target cells attached to a surface, allowing the cultured transduced CD8+ T cells to interact therewith and subsequently exerting a force away from the attached target cells, wherein cellular avidity is determined on day 6, up to, and including, day 21 , wherein the cultured transduced CD8+ T cells have a transduction efficiency of at least 80 percent.

In yet another embodiment a method is provided for providing transduced CD8+ T cells, for determining cellular avidity of said transduced CD8+ T cells with target cells, by exerting a force, wherein said method comprises the steps of: a) providing PBMCs, on day 0; b) selecting CD8+ T cells from the PBMCs, wherein the selected population is at least 90% CD8+; c) seeding the CD8+ T cells in an appropriate cell culture medium, and activating cells for 2 days by providing an activation stimulus such as a CD3/CD28 activator in the medium and supplementing IL- 2 in the medium; d) transducing the activated CD8+ T cells with a retroviral vector, on day 2, while supplementing the medium with IL-2, and allowing the transduction to proceed for 72 hours; e) on day 5, optionally, removing the activation stimulus, determining transduction efficiency and/or enriching for transduced CD8+ T cells for providing transduced CD8+ T cells having a transduction efficiency of at least 80 percent; f) on day 5, further seeding and culturing the transduced CD8+ T cells in the presence of IL-2, wherein the culture medium is supplemented with IL-2 every 2-3 days; g) determining cellular avidity of the cultured transduced CD8+ T cells to target cells, by providing target cells attached to a surface, allowing the cultured transduced CD8+ T cells to interact therewith and subsequently exerting a force away from the attached target cells, wherein cellular avidity is determined on day 6, up to, and including, day 21 .

As said, and as shown in the examples, the enrichment step highly preferably comprises a positive selection using MACS. A positive selection means that the microbeads bind to the cells one wishes to isolate, a negative selection means that the cells that are undesired are bound by microbeads and thereby removed from cell sample with a strong magnetic field, as is all well understood in the art. Furthermore, the PBMCs that are provided are to be isolated from fresh blood and/or buffy coat. Suitable means and methods are known for such isolation and commercially available as well. Such isolation may be performed e.g. with a density gradient separation, or with SepMate™ as described in the examples. Furthermore, the CD8+ T cells may be obtained via MACS as well, e.g. by using positive selection. In yet another further embodiment, in the methods in accordance with the invention, the selected CD8+ T cells of step b) are subjected to cryopreservation, and subsequently thawed at a later time, and allowed to recover, prior to performing step c).

As said, cryopreserved cells may be advantageous as that may allow one to have better control of the conditions transduced CD8+ T cells are subjected to and/or plan experiments. In yet another embodiment, the transduced CD8+ T cells are subjected to cryopreservation, to thereby provide for frozen transduced CD8+ T cells that can be used for subsequent cellular avidity measurement when convenient. Such may be, as said, also highly useful when incorporating e.g. suitable control CD8+ T cells in cellular avidity measurements therewith providing highly advantageous control of experiments conducted.

Hence, in another embodiment, a method is provided for providing transduced CD8+ T cells, for determining cellular avidity of said transduced CD8+ T cells with target cells, by exerting a force, wherein said method comprises the steps of: a) providing PBMCs, on a day 0; b) selecting CD8+ T cells from the PBMCs, wherein the selected population is at least 90% CD8+; c) seeding the CD8+ T cells in an appropriate cell culture medium, and activating cells for 2 days by providing an activation stimulus in the medium and providing IL-2 in the medium; d) transducing the activated CD8+ T cells with a retroviral vector, on day 2 while supplementing the medium with IL-2, and allowing the transduction to proceed for 72 hours; e) on day 5, optionally, removing the activation stimulus, optionally determining transduction efficiency and/or optionally enriching for transduced CD8+ T cells for providing transduced CD8+ T cells having a transduction efficiency of at least 80 percent; and, f) cryopreserving the transduced CD8+ cells on day 5, or on day 5, further seeding and culturing the transduced CD8+ T cells in the presence of IL-2, wherein the culture medium is supplemented with IL-2 every 2-3 days, and cryopreserving at a day up to, and including, day 9 after providing the PBMCs; g) thawing the frozen cells, and further seeding and culturing the transduced CD8+ T cells in a medium which is supplemented with IL-2 every 2-3 days; h) determining cellular avidity of the cultured transduced CD8+ T cells to target cells, on day 2, day 3, or day 4 after step g) of thawing the frozen transduced CD8+ T cells, by providing target cells attached to a surface, allowing the cultured transduced CD8+ T cells to interact therewith and subsequently exerting a force away from the attached target cells; wherein, optionally, after step g) and, 24 hours, or less, prior to step h), determining transduction efficiency and/or optionally enriching for transduced CD8+ T cells for providing transduced CD8+ T cells having a transduction efficiency of at least 80 percent.

As described above for experiments without a cryopreservation step of transduced CD8+ T cells, determining transduction efficiency and/or optionally enriching for transduced CD8+ T cells is optional. This is because, as said, it may be contemplated that one uses well controlled transduction procedures which results in highly reproducible efficiencies. Nevertheless, as said, it may be preferred, as it allows for appropriate and comparable cell preparations, e.g. when different donors are used.

Means and methods for cryopreservation are well known in the art, and include freezing cells contained in cryopreservation medium in liquid nitrogen (such as DMSO and FCS (fetal calf serum) as described in the examples herein. Cryopreservation in step f) can be done on day 5, up to, and including day 9, i.e. on day 5, 6, 7, 8 or 9.

In yet another embodiment, instead of Interleukin 2, Interleukin 7 and Interleukin 15 are used as cytokines to supplement culture medium when culturing T cells. As shown in the examples, at least in short term cultures, e.g. of thawed cryopreserved T cells, no differences were observed with regard to cellular avidity. Hence, IL2 and IL7/IL15 (or IL-2 and IL-7/IL-15, may be used interchangeably, of course taking into account recommended concentrations. Hence, such interleukins may be supplemented to the medium every 2-3 days. For example IL-2 may be used in a range of 4-150 U/mL, as it was observed that in preliminary experiments of about a week this may provide for relatively consistent results. In a further embodiment, IL-2 is used in a supplemented concentration in the range of 25 - 150, or 50 - 125. It may be preferred to use IL-2 in a supplement concentration of about 90 U/mL to be supplemented every 2-3 days. It is understood that a supplement concentration means the concentration of IL-2 that is added to the culture medium, wherein of course the culture medium may still have IL-2 remaining from earlier supplementation. With regard to IL7/IL15, these may be used in recommended concentrations known in the art. IL7/IL15 may each be supplemented to the medium in a concentration in the range of 1 - 20 pg/mL, 2 - 15 pg/mL. or in the range of 3 - 8 pg/mL. IL7/IL15, or of about 5 pg/mL of IL7ZIL15.lt is understood that this means when a concentration of 5 pg/mL for each is selected, this means that the concentration supplemented is 5 pg/mL IL7 and 5 pg/mL IL15.

As said, suitable culture medium is known in the art. For example RPMI 1640 is a well-known and suitable medium for culturing T cells that is widely commercially available. To such medium, serum and L-glutamine or glutamax can be added, as may be generally recommended, e.g. the medium may comprise 10% serum such as foetal bovine serum, and cells can be cultured using standard conditions such as in a humidified chamber. Hence, in one embodiment, the cell culture medium is RPMI 1640, and/or wherein the cell culture medium comprises 10% foetal bovine serum and glutamax and wherein cells are cultured in a humidified chamber at a temperature 37°C with 5% CO2. With regard to recommended cell culture densities (i.e. number of cells I cm2), it is remarked that ranges of cell culture densities may be selected in the range of 0.1 to 2.0 million cells per cm2, and may be preferably in the range of 0.8 to 2 million cells per cm2. An appropriate amount of culture medium can be selected (such as 0.5 - 2 mL in a 24-well format.

As is understood, also from the example section, the means and methods in accordance with the invention, with the meets and bounds as provided provide herein, allow to provide for highly advantageous reproducible results when determining cellular avidity between transduced CD8+ T cells and target cells. As is understood, the means and methods in accordance with the invention may not solely be performed with a single type of transduced cell or single type of target cells, the means and methods in accordance with the invention can be advantageously performed with further transduced CD8+ T cells and/or target cells. These further transduced CD8+ T cells can be T cells transduced with different retroviral vectors, e.g. encoding different CAR receptors and/or having different expression construct, whichever is of interest to be the subject of investigation. The further transduced CD8+ T cells can be also further donors, such that donor variation can be assessed. It is understood that conditions selected when performing the methods in accordance with the invention, that these are to be selected to be the same for each of the different transduced CD8+ T cells. Of course, in addition to further transduced CD8+ T cells, proper control cells for the transduced CD8+ cells are to be provided as well. A control for transduced CD8+ T cells which expresses a CAR, can be cells transduced with the same vector not expressing a CAR or a CAR not capable of binding the target cell. A control for transduced CD8+ T cells which expresses a CAR or the like, can be untransduced cells (of the same donor e.g.), such as described in the examples herein. This way cellular avidity as determined for these different cells can be compared.

Hence, in a further embodiment, in a method in accordance with the invention furthermore control transduced, or, untransduced CD8+ T cells are provided, wherein the method is in addition performed with the control transduced CD8+ T cells or untransduced CD8+ T cells, instead of the transduced CD8+ T cells, and cellular avidity of control transduced CD8+ T cells or untransduced CD8+ T cells with target cells is determined. In yet another further embodiment, the methods in accordance with the invention are performed with CD8+ T cells provided of different donors, wherein of each donor, the method is performed with transduced CD8+ cells and control cells thereof.

As said, the means and methods in accordance with the invention, and as shown in the examples as described herein, allow for an experimental design requiring cellular avidity measurement experiments to be conducted with a low number of biological replicates (e.g. with cells obtained from only two or three different donors), which already may allow to assess differences between experimental conditions to which the biological samples (or replicates) are subjected to. Hence, in a further embodiment, the methods in accordance with the inventions are conducted with 2 or 3 biological replicates, or more for a defined CAR, or condition of interest to study. Performing more experiments (more than in duplicate or triplicate) in accordance with the means and methods of the invention for cellular avidity measurements, may increase statistical power, but as shown in the example section, sufficient statistical power can already be obtained by performing experiments in duplicate or triplicate.

In any case, in accordance with the invention, the cellular avidities as determined of transduced CD8+ cells with target cells are compared with the cellular avidities of control cells thereof, with target cells. Of course, instead of providing control cells of transduced CD8+ T cells, one may also provide for control cells of the target cells, i.e. providing target cells without the target antigen of interest which are to be the targeted by the transduced CD8+ T cells. Cellular avidities of transduced CD8+ T cells with the target cells, and with the control target cells, are than compared. Comparing determined cellular avidities, i.e. the difference between e.g. cellular avidities, can be useful information e.g. when comparing different CARs.

Hence, in a further embodiment, in the methods in accordance with the invention, cellular avidities as determined of transduced CD8+ cells with target cells is compared with the cellular avidities as determined of control cells thereof, with target cells. In particular, such methods may include determining the cellular avidity of transduced CD8+ T cells with target cells and comparing it with the cellular avidity as determined of control transduced CD8+ T cells or untransduced CD8+ T cells with target cells. In another embodiment, further cellular avidities may be determined by performing the methods in accordance with the invention by further providing control target cells, and performing the method in addition with the control target cells instead of target cells and determining cellular avidity of transduced CD8+ T cells with the control target cells is determined. Subsequently of course, the determined cellular avidities are compared. Hence, in a further embodiment, the cellular avidity determined for transduced CD8+ T cells with target cells is compared with the cellular avidity determined for transduced CD8+ T cells with control target cells.

As is understood a prime objective of the methods in accordance with the invention is to provide for transduced T cells, such as CD8+ T cells. Such cells are, as said, of high interest in the development of cell therapies, e.g. under development for indications such as cancer. Transduction of T cells highly preferably is carried out with retroviral vectors, as these can provide for stable gene transfer and durable expression of a gene of interest. Highly suitable and advantageous gene of interests are chimeric antigen receptors, also referred to as CARs, which are well known in the art. One may also have the retroviral vector encode for a T-cell receptor of interest, e.g. of a gamma delta T cell receptor, or, an alpha beta TCR. A TCR of interest, or a CAR of interest, or any other suitable receptor can be expressed. Hence, in particular and highly useful embodiments, the retroviral vector as provided in the means and methods in accordance with the invention encodes a receptor, such as a CAR or a TCR, said receptor comprising a binding domain capable of binding to the target cell.

Although the means and methods in accordance with the invention are highly suitable to be performed with CD8+ T cells, in some embodiments it may be contemplated to use primary T cells instead. Primary T cells can be selected e.g. with CD3+ MACS as described herein. Hence, in such embodiments, when in such methods CD8+ T cells are provided, these can be understood to be replaced by primary T cells and the methods performed accordingly without the selection of CD8+ T cells. Hence, in a further embodiment, methods in accordance is performed with primary T cells instead of CD8+ T cells. Of course, it may be highly useful to monitor e.g. the percentages of CD4+ and CD8+ cells present in the provided primary T cells, as such information may be useful when interpreting results, as percentages may differ between donors and/or in time.

As shown in the example section, surprisingly it was found that when determining cellular avidity, positive MACS selection of cells may be performed shortly prior to determining cellular avidity between an effector cell, such as a transduced CD8+ cell (or control cell thereof), and a target cell. Such a positive MACS selection comprises at least MACS selection methods which involve binding agents conjugated to magnetic beads. Binding agents may be selected to bind e.g. with a receptor ora reporter or marker protein presented on the cell surface. It is understood that the MACS selection method relied upon should not rely e.g. on binding e.g. a CAR receptor which is involved in cellular avidity such that it would block the interaction of the CAR receptor with its target antigen on the target cell. For example, marker genes are known that are not linked e.g. to CAR constructs (e.g. non- limiting examples are a marker such as RQR8 as used in the examples herein (see Mosti et al. Gene Therapy 2021 ; Philip et al., Blood, 2014 Aug 21 ;124(8):1277-87), a FLAG-tag (Berahovich et al., 2017, Frontiers in Bioscience-Landmark, 22(10), pp.1644-1654), c-Myc tag (Repellin, et al., 2020. Advanced biosystems, 4(1), p.1900224), or mCherry (Choi, et al., 2019, Nature biotechnology, 37(9), pp.1049-1058.). Such a marker, or the like, presented on the cell surface may be highly useful for affinity based magnetic microbead sorting. As shown in the examples, MACS is compatible with cellular avidity measurements which involve exerting a force on effector cells, e.g. as with a Z-movi® device. Such is highly useful in the means and methods in accordance with the invention as described above. In addition, such is also useful in any means and methods for determining cellular avidity in which a force is exerted on cells which have been subjected to a positive selection using MACS.

Hence, in another embodiment, a method is provided for determining cellular avidity between effector cells and target cells, by providing target cells attached to a surface, allowing the effector cells to interact therewith, and by subsequently exerting a force away from the attached target cells, wherein 24 hours or less prior to determining cellular avidity, effector cells or target cells are subjected to a selection step, wherein said selection step comprises a positive selection using MACS. The positive selection may be performed less than 12 hours, less than 6 hours, less than 3 hours, less than 1 hour or less than 15 minutes, prior to measuring cellular avidity. Such a MACS selection step preferably provides for transduced effector cells having a transduction efficiency of at least 80 percent. Of course, such a selection step may not be restricted to effector cells. Nevertheless, these are in particular useful cells as this type of cells may be provided as a heterogenous cell population, e.g. as these may comprise different types of effector cells and/or may be in part transduced.

It is understood that although in the means and methods in accordance with the invention as outlined above, and as described in the examples, instead of having the target cells attached to a surface, the transduced CD8+ T cells (or T cells, or effector cells) can be attached to the surface instead. The target cells are subsequently provided to interact with the attached CD8+ T cells and a force is applied away from the CD8+ T cells. Means and methods may be more complicated, and, repeated use, like with monolayers of target cells (as shown in the examples, with up to 6 runs per chip with a monolayer of target cells), can only be done in case of multiple different target cells that are to be tested. Hence, it is most often more economical and efficient to have the target cells attached. Nevertheless, it may be contemplated to have CD8+ target cells attached to the surface instead. Hence, in an alternative embodiment, in the methods in accordance with the invention for determining cellular avidity methods are provided wherein instead of the target cells, the transduced CD8+ T cells or effector cells are attached to a surface.

With regard to cellular avidity, it is understood that this relates to the strength of binding of effector cells such as CD8+ T cells (or control or reference 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 \0), 1929-1947) for a description of force inhomogeneities in acoustic force application). As is clear from the above, the type of force that is to be applied in accordance with the invention is a force capable of breaking cell-cell bonds, e.g. the force exerted causes CD8+ T cells and target cells bound to each other move away from each other to such an extent that a cell-cell bond may break or rupture.

As shown in the examples, a force that can be applied when target cells are attached to a surface is an acoustic force, such as ultrasonic forces as applied e.g. in a device as available from LUMICKS, wherein the force is away from attached cells (e.g. such as in the LUMICKS z-Movi® Cell Avidity Analyzer, e.g. as used by Larson et al., Nature 604 7906:1-8, April 13, 2022). However, the means and methods in accordance with the invention when exerting a force away from e.g. target cells attached to a surface as described herein is not be restricted to the use of acoustic force. Other types of forces that can be applied away from attached cells, such as centrifugal (or other acceleration) forces or a shear flow force can be applied as well. With such forces, similar results as depicted in the graphs and tables as shown in the examples herein may be provided, relying on the same inherent properties of cellular avidity between T cells and target cells. As long as a force can be applied and controlled on e.g. T cells that bind/interact with target cells attached to a surface (or vice versa) such a force can be contemplated in accordance with the invention. Hence, in a further embodiment, in the means and methods in accordance with the invention, a force is applied away from the target cells, wherein the force is acoustic force, a shear flow or a centrifugal 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 I 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 away from cells attached to a surface and collect the cells, detached and/or attached cells, on which defined forces have been exerted.

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 target cells or T cells. 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 (<D²) 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.

A differential force means that the force on one cell differs from the force on the other cell with regard to direction of the force and/orthe magnitude of the force, resulting in a net force allowing to break cell-cell bonds if the differential force exceeds the binding force. Hence, in the means and methods in accordance with the invention as described herein, in the step of applying a force on the CD8+ T cells away from the target cells (or vice versa), instead a differential force may be applied which does not require attachment of the target cells to a surface.

For example, when a target cell bound to an effector cell, a doublet, is forced through a nozzle, the closer to the throat of the nozzle the faster the flow. This means that the first cell to enter the nozzle is subjected to a stronger acceleration than the cell lagging behind and the cells experience a differential force resulting in a net force which can result in cell-cell bond rupture, provided the force is large enough. A differential force that can be applied includes a shear force, e.g. such as can be applied utilizing repeated pipetting (repeated upwards and downwards flow of the sample) or flow through a nozzle.

Other means and methods are known in the art with which shear forces can be applied to cells, e.g. flowing a cell suspension at a constant speed and bombarding these cells to a flat surface at a defined angle. Furthermore, forcing a cell suspension through a needle with a defined internal diameter and a defined force may provide for a well controllable shear force as well. The cell suspension may be subjected to several rounds of such process steps to ensure substantially all cell-cell bonds experience the maximum force that may be achieved with the process step. Such a process step allows for automation, enabling control and repeatability of the process, therewith controlling shear forces exerted. Suitable devices for breaking apart cell-cell bonds which are not synapses are known in the art (e.g. Zahniser et al., J Histochem Cytochem. 1979, 27 (1), 635-641). Also, by properly tuning the forces in a flow-cytometer normally used to measure cell deformations, such as e.g. described in Otto, et al. Nat Methods 12, 199-202 (2015) suitable forces can be applied. Tuning can be achieved e.g. by changing the nozzle size or geometry and I or the flow speeds used. Other suitable devices known in the art may include a vortex mixer, with which shear forces may be suitably applied as well. Accordingly, in one embodiment, the force applied involves a shear force. In yet another further embodiment not requiring attachment of cells, the differential force applied is an ultrasonic force. It is understood, as outline above, that such ultrasonic forces are not forces such as applied e.g. in a device as available from LUMICKS, wherein the force is away from attached cells (e.g. such as in the LUMICKS z-Movi® Cell Avidity Analyzer, e.g. as used by Larson et al., Nature 604 7906:1-8, April 13, 2022). It is also understood that the ultrasonic force is selected such that cells are not lysed. Hence, appropriate ultrasonic forces can be applied to cells such that cell-cell bonds can be ruptured, which more preferably includes breaking aspecific cell-cell bonds and less preferably breaks specific cell-cell bonds in which an immune synapse is formed. Examples of using ultrasonic forces to break (aspecific) cell-cell bonds, are known in the art (e.g. as described in Buddy et al., Biomaterials Science: An Introduction to Materials in Medicine, 3^rd edition, 2013, Chapter II.2.8, page 576; and Moore et al., Experimental Cell Research, Volume 65, Issue 1 , 1971 , Pages 228- 232).

Accordingly, it is understood that in some embodiments, the differential force to be applied does not require either of the target cells or effector cells, e.g. CD8+ T cells, to be or remain to be attached, and the differential force may be a force selected from the range of 1 pN - 10 nN. In another embodiment, in methods in accordance with the invention wherein the force that is applied is a differential force, neither the target cells northe effector cells, e.g. transduced or untransduced CD8+ T cells, are required to be attached to a surface, and the differential force is applied in the range of 1 pN - 10 nN, thereby providing cells substantially comprised of target cells, CD8+ T cells, and CD8+ T cells bound to target cells. Of course, when cells are not required to be attached, it is understood that cells may be differentially labelled, before and/or after the differential force is exerted in order to identify CD8+ T cells that have remained attached after the force has been exerted and differentiate these from CD8+ T cells that have detached from the target cells. Yet, having the target cells attached to a surface is a convenient way allowing to differentiate between detached CD8+ T cells and CD8+ T cells that remained attached, and at the same time allow for a convenient separation of these cells. Hence, the (differential force) to be applied advantageously requires either of the target cells or CD8+ T cells, e.g. transduced CD8+ T cells (and controls thereof) cells, to be attached, and the force applied is directed away from the attached cells, and the (differential) force may be a force selected from the range of 1 pN - 10 nN. It is understood that by selecting an appropriate force, aspecific cellcell bonds may be (more) selectively broken, while retaining specific cell-cell bonds between CD8+ T cells and target cells. For example, in case of CD8+ T cells, CD8+ T cells expressing a CAR which specifically interacted with a target antigen expressed by a target cells may be retained.

In any case, suitable applied forces which are known in the art include e.g. a force in the range of 1 pN - 10 nN, which said force is a net force exerted on one cell relative to the other cell, of two cells bound to each other. Which means the force is exerted on the cell-cell bond. In another embodiment, the force exerted on one of the two cells relative to the other cell is at least 1 pN, at least 10 pN, at least 20 pN, at least 50 pN, at least 100 pN, or at least least 200 pN. In another embodiment, the force exerted is at most 10 nN, at most 5 nN, at most 3 nM, at most 2 nM, or at most 1 nN. In yet another embodiment, the force is selected from the range of 1 pN - 10 nN, from 10 pN - 10 nN, from 500 pN - 10 nN, or from 1 nN - 10 nN. In still a further embodiment, the force is selected from the range of 500 pN - 5 nN, from 500 pN - 4 pN, from 500 pN - 3 pN. In one embodiment, the force that is applied is in the range of 1 pN - 10 nN. In another embodiment, the force that is applied is in the range of 10 pN - 10 nN. For example, a suitable amount of force that can be exerted between cells (e.g. such as in the z-Movi® device) can be selected to be in the range of 200 pN - 3000 pN. Of course, these force ranges are known to be useful with cells attached to a surface, and the maximum force that may be selected may exceed 3000 pN as it may not be required to have the cells to remain attached to a surface in accordance with the invention.

Hence, in one embodiment, in the means and methods in accordance with the invention the applied force is an acoustic force, a shear flow force or an acceleration force. In yet another embodiment, in the means and methods in accordance with the invention instead of applying a force away from the attached cells, the applied force is a differential force which does not require attachment of cells to a surface. In still another embodiment, in the means and methods in accordance with the invention, it may be contemplated that the cells are not attached to a surface.

Embodiments

1 . Method for providing transduced CD8+ T cells, for determining cellular avidity of said transduced CD8+ T cells with target cells, by exerting a force, wherein said method comprises the steps of: a) providing PBMCs, on day 0; b) selecting CD8+ T cells from the PBMCs, wherein the selected population is at least 90% CD8+; c) seeding the CD8+ T cells in an appropriate cell culture medium, and activating cells for 2 days by providing an activation stimulus e.g. through stimulation with a CD3/CD28 activator and supplementing IL-2 in the medium; d) transducing the activated CD8+ T cells with a retroviral vector, on day 2, while supplementing the medium with IL-2, and allowing the transduction to proceed for 72 hours; e) on day 5, optionally, removing the activation stimulus, optionally determining transduction efficiency and/or optionally enriching for transduced CD8+ T cells for providing transduced CD8+ T cells having a transduction efficiency of at least 80 percent; f) on day 5, further seeding and culturing the transduced CD8+ T cells in the presence of IL-2, wherein the culture medium is supplemented with IL-2 every 2-3 days; g) determining cellular avidity of the cultured transduced CD8+ T cells to target cells, by providing target cells attached to a surface, allowing the cultured transduced CD8+ T cells to interact therewith and subsequently exerting a force away from the attached target cells, wherein cellular avidity is determined on day 6, up to, and including, day 21 ; wherein, optionally, after step f) and, 24 hours, or less, prior to step g), determining transduction efficiency and/or optionally enriching for transduced CD8+ T cells for providing transduced CD8+ T cells having a transduction efficiency of at least 80 percent. 2. Method in accordance with embodiment 1 , wherein said enrichment step comprises positive selection using MACS.

3. Method according to embodiment 1 or embodiment 2, wherein the provided PBMCs are isolated from fresh blood and/or buffy coat.

4. Method according to any of embodiments 1-3, wherein the isolation is performed using density gradient separation.

5. Method according to any one of embodiments 1-4, wherein the CD8+ T cells are obtained via MACS.

6. Method according to any one of embodiments 1 to 5, wherein the selected CD8+ T cells of step b) are subjected to cryopreservation, and subsequently thawed at a later time, and allowed to recover, prior to performing step c).

7. Method for providing transduced CD8+ T cells, for determining cellular avidity of said transduced CD8+ T cells with target cells, by exerting a force, wherein said method comprises the steps of: a) providing PBMCs, on a day 0; b) selecting CD8+ T cells from the PBMCs, wherein the selected population is at least 90% CD8+; c) seeding the CD8+ T cells in an appropriate cell culture medium, and activating cells for 2 days by providing an activation stimulus in the medium and providing IL-2 in the medium; d) transducing the activated CD8+ T cells with a retroviral vector, on day 2 while supplementing the medium with IL-2, and allowing the transduction to proceed for 72 hours; e) on day 5, optionally, removing the activation stimulus, optionally determining transduction efficiency and/or optionally enriching for transduced CD8+ T cells for providing transduced CD8+ T cells having a transduction efficiency of at least 80 percent; and, f) cryopreserving the transduced CD8+ cells on day 5, or on day 5, further seeding and culturing the transduced CD8+ T cells in the presence of IL-2, wherein the culture medium is supplemented with IL-2 every 2-3 days, and cryopreserving at a day up to, and including, day 9 after providing the PBMCs; g) thawing the frozen cells, and further seeding and culturing the transduced CD8+ T cells in a medium which is supplemented with IL-2 every 2-3 days; h) determining cellular avidity of the cultured transduced CD8+ T cells to target cells, on day 2, day 3, or day 4 after step g) of thawing the frozen transduced CD8+ T cells, by providing target cells attached to a surface, allowing the cultured transduced CD8+ T cells to interact therewith and subsequently exerting a force away from the attached target cells; wherein, optionally, after step g) and, 24 hours, or less, prior to step h), determining transduction efficiency and/or optionally enriching for transduced CD8+ T cells for providing transduced CD8+ T cells having a transduction efficiency of at least 80 percent.

8. Method in accordance with any of embodiments 1-7, wherein instead of IL-2, IL-7/IL-15 is used.

9. Method in accordance with any of embodiments 1-7, wherein the IL-2 is supplemented to a concentration of IL-2 in the range of 4 - 150 U/mL.

10. Method in accordance with any one of embodiments 1-9, wherein the activation stimulus that is provided is Human T-Activator CD3/CD28 Dynabeads.

1 1. Method in accordance with any one of embodiment 1-10, wherein transduction of the retroviral vector is performed using a fibronectin reagent, such as RetroNectin.

12. Method in accordance with any one of embodiments 1 -11 , wherein the cell culture medium is RPMI 1640, and/or wherein the cell culture medium comprises 10% foetal bovine serum and glutamax and wherein cells are cultured in a humidified chamber at a temperature 37°C with 5% CO₂.

13. Method in accordance with any one of embodiments 1 -12, wherein transduced CD8+ T cells are seeded in a culture plate at a concentration of 0.1 to 2.0 million cells per cm2.

14. Method in accordance with any one of embodiments 1 -13, wherein furthermore control transduced, or, untransduced CD8+ T cells are provided, wherein the method is in addition performed with the control transduced CD8+ T cells or untransduced CD8+ T cells, instead of the transduced CD8+ T cells, and cellular avidity of control transduced CD8+ T cells or untransduced CD8+ T cells with target cells is determined.

15. Method in accordance with embodiment 14, wherein the cellular avidity of transduced CD8+ T cells with target cells is compared with the cellular avidity of control transduced CD8+ T cells or untransduced CD8+ T cells with target cells.

16. Method in accordance with any one of embodiments 1-15, wherein furthermore control target cells are provided, and wherein the method is in addition performed with control target cells instead of target cells and cellular avidity of transduced CD8+ T cells with control target cells is determined. 17. Method in accordance with embodiment 16, wherein the cellular avidity determined for transduced CD8+ T cells with target cells is compared with the cellular avidity determined for transduced CD8+ T cells with control target cells.

18. Method in accordance with any one of embodiments 1 -17, wherein the retroviral vector encodes a receptor, such as a CAR or a TCR, said receptor comprising a binding domain capable of binding to the target cell.

19. Method according to any one of embodiments 1 to 19, wherein the method is performed with primary T cells instead of CD8+ T cells.

20. Method for determining cellular avidity between effector cells and target cells, by providing target cells attached to a surface, allowing the effector cells to interact therewith, and by subsequently exerting a force away from the attached target cells, wherein 24 hours or less prior to determining cellular avidity, effector cells or target cells are subjected to a selection step, wherein said selection step comprises a positive selection using MACS.

21 . Method according to embodiment 20, wherein the selection step provides for transduced effector cells having a transduction efficiency of at least 80 percent.

22. Method in accordance with any one of embodiments 1 -21 , wherein determining cellular avidity comprises:

- determining the number of transduced CD8+ T cells or effector cells that have moved away and/or remain bound after applying the force.

23. Method according to embodiment 22, wherein based on the determined number(s), a cellular avidity score is provided.

24. Method for determining cellular avidity in accordance with any one of embodiments 1 -23, wherein instead of the target cells, the transduced CD8+ T cells or effector cells are attached to a surface.

25. Method in accordance with any one of embodiments 1 -24, wherein the applied force is a force ramp, preferably a linear force ramp.

26. Method in accordance with any one of embodiments 1 -25, wherein the applied force is an acoustic force, a shear flow force or an acceleration force. 27. Method in accordance with any one of embodiments 1 -25, wherein instead of applying a force away from the attached cells, the applied force is a differential force which does not require attachment of cells to a surface.

28. Method in accordance with embodiment 27, wherein cells are not attached to a surface.

Examples

Materials and Methods

T cell isolation from fresh buffy coat

Peripheral blood mononuclear cells (PBMCs) are isolated from fresh blood or buffy coats obtained from human donors. In the experiments as described herein, SepMate™-50 tubes are used in accordance with the manufacturer’s instructions (Stemcell, SepMate™-50 (IVD), Catalog #85450). Isolated PBMCs are sorted for desired cell population (typically pan T cell (for negative selection); and CD3+, CD8+ or CD4+ T cells (for positive selection) by magnetic separation using relevant microbeads (Miltenyi Biotec) in accordance with instructions of the manufacturer (see, human Pan T Cell Isolation Kit, Order no. 130-096-535; human CD3 MicroBeads, Order no.130-050-101 ; human CD8 MicroBeads, Order no. 130-045-201 and human CD4 MicroBeads, Order no. 130-045-101 all from Miltenyi Biotec). It was observed that more consistent results with regard to measuring cellular avidity were obtained when using CD8+ cells as opposed to e.g. when pan T cells were used of different donors. Highly comparable results were obtained with either the positive (i.e. CD3+ microbeads) or negative selection of T cells (i.e. Pan T cell kit).

Target cell lines

Nalm6 and Raji cell lines, both human B cell derived cancer cell lines, expressing CD19, are used as target cells and were obtained from the ATCC.

Retroviral vectors

Retroviral vectors are used in this study express chimeric antigen receptors. In the experiments herein, second-generation CAR constructs are used with binding domains derived from anti-CD19 antibodies.

Effector cells - T cell stimulation and subsequent transduction

Isolated T cells are counted/plated and activated. Suitable activation is by using Dynabeads™ Human T-Activator CD3/CD28 forT Cell Expansion and Activation (ThermoFisher Scientific, Catalog number: 1 1161 D) and supplemented with 90U/mL IL2. The cells are incubated for 48 hours, i.e. 2 days, at 37°C 5% CO2. Following the 48 hour incubation, retroviral transduction of the activated T cells is performed. Retroviral transduction is performed with RetroNectin® in accordance with manufacturer’s instructions (TaKaRa, Cat. # T100A/B). 5 days after initial T cell stimulation, i.e. 3 days (or 72 hours) after transduction, transduced T cells are harvested and replated. When activation involves beads, these can be removed as well. After activation with Dynabeads, these are removed using DynaMag™ (ThermoFisher Scientific) magnet. Harvested T cells are spun at 400xG for 6 minutes and resuspended in fresh T cell medium (typically RPMI 1640 supplemented with Glutamax and 10% FCS, optionally Penicillin/Streptomycin) supplemented with 90U/mL IL2.

Effector Cells - Purification and Culture

Transduced T cells are thus provided. The transduced T cells may be enriched to provide for T cells of which at least 80% is transduced. Enrichment may be performed with appropriate means. Transduced cells have an RQR8 sort-suicide marker gene expressed on the cell surface and enrichment is performed using CD34 magnetic microbeads (i.e. reference 130-046-702, from Miltenyi Biotec) which specifically binds with the RQR8 marker. Transduction efficiency can also be determined and/or conditions selected such that T cells with at least 80% transduction efficiency are provided without an enrichment step.

The transduced T cells are used for avidity measurements as outlined below. Alternatively, cells are subjected to cryopreservation for later use, at least 5e6 cells/ml are suspended cryopreservation medium (90% FCS, 10% DMSO) in I mL/vial aliquots. In the experiments below, cells are always sorted on day 3 post-transduction (prior to freezing and in case experiments continue without freezing). Transduction efficiency is always at least 20%. Vials with frozen T cells are stored in liquid nitrogen for up to one year.

T cells are cultured in RPMI 1640 medium supplemented with Glutamax, 10% FCS, and, optionally with Penicillin/Streptomycin), and supplemented every 2-3 days with with 90U/mL IL2, also referred to as RPMI complete.

Thawing frozen T cells

Vial(s) needed for an experiment are thawed before an avidity measurement experiment. The cells are centrifuged at 400xG for 6 minutes, resuspended in T cell medium supplemented with 90U/mL IL2 and cultured at 37°C 5% CO2 before performing avidity experiment.

Cellular avidity measurement

Z-Movi® chips (obtained and as available from LUMICKS, with channel dimensions of 7x2x0.1 mm and made of glass) are coated the day before performing the experiment with Poly-L-Lysine (Reference P4707, Sigma-Aldrich, Poly-L-lysine 0.01 % solution, sterile-filtered, CAS Number: 25988-63-0). The next day target cells, e.g. Nalm6 or Raji cells, are seeded in the chip in serum- free culture medium, incubated for 0.5 h at 37°C, and the medium is replaced with RPMI complete medium (Ref.). The target cells are incubated in the chip for at least 1 .5 h before starting the cellular avidity experiment. Effector cells, i.e. the T cells (transduced or untransduced), are stained with CellTrace™ Far Red dye (Thermo Fisher Scientific, Cat. no. C34564 or C34572) at 1 pM for 15 minutes in PBS at 37°C, then the cells are resuspended at 1 x 10e7 cells/mL in complete medium and used for the avidity experiments. The T cells are subsequently introduced in the target cell- seeded flow cell, incubated for 5 minutes, and then a force ramp is applied for 2.5 minutes using Z- Movi® operated with the Oceon V1 .2.8 or later. A chip provided with target cells is typically used for multiple runs. Cells remaining attached to the target cells after each run are masked thereby allowing multiple cellular avidity measurements. The number of runs, i.e. cellular avidity measurements, is generally in range of 2-10, and is commonly 6, and the quality of a monolayer of cells is monitored when conducting runs. The run order for a chip seeded with target cells can be random or can be predefined. Of biological replicate samples (as outlined herein of different donors, or any biological sample) technical replicate measurements are performed. A datapoint of a biological replicate sample represents an average of technical repeats. Of a biological replicate sample, at least three technical repeat measurements were performed. A technical repeat measurement is performed on the same day, with the same cells and preferably on different chips that are also prepared on the same day. Statistical analysis is performed with the obtained biological replicate datapoints. For all statistical test, ordinary one-way or 2-way ANOVA is used. The indication “ns” means there is no statistically significant difference and *, **, ***, and **** indicates increasing statistically significant differences (ns is p>0.05; * is 0.01 <p<0.05; ** is 0.05<p< 0.01 ; *** is 0.01 <p< 0.001 ; and lastly **** is p<0.0001 , which corresponds with the GraphPad automatically selected settings of the software used for graph generation).

Experiment 1: comparing IL2 vs IL7/IL15

In this experiment, interleukin 2 (IL2) was compared with interleukins 7 and 15 (IL7/IL15).

Cells, culture conditions and avidity measurements were generally as outlined above. Briefly, in this experiment, provided frozen primary (CD3+) T cells (transduced and untransduced) were thawed, and subsequently cultured for 72h in the presence of interleukins. In this experiment, either 90U/mL of IL2, or, 5pg/mL IL7 and 5pg/mL IL15 was used to supplement culture medium. Avidity measurements were performed on day 3 (72 hours) after thawing using either a Raji or Nalm6 monolayer with transduced, or untransduced cells of three different donors.

Chips were prepared on separate days with either a Raji or Nalm6 monolayer of cells. The prepared primary T (CD3+) cells were run on each chip in mixed order. For each day, transduced and untransduced T cells of a different donor were provided. Results are shown in Figure 1 . As can be observed, with both 90U/ml IL2 and 5ug/mL IL7/IL15, a highly similar cellular avidity measurement result was obtained after 72h. Hence, supplementation of culture medium with 5ug/mL IL7 and 5ug/mL IL15, instead of 90U/mL IL2, for up to at least 72h does not appear to affect cellular avidity measurements.

Based on these results, in short term culture experiments of several days, at least up to 72 hours, either IL2 or a mixture of IL7/IL15 at recommended concentrations can be used to culture primary T cells, and provide for highly comparable cellular avidity measurements and can provide for statistical power already with three data points.

Experiment 2: time post-thaw and timing IL2 addition

The time post-thaw may affect cell avidity measurements, as cells may need some time to recover. Hence, at different culturing times after thawing, cellular avidity measurements were performed. In addition, timing of addition to the cell culture medium of IL2 post-thaw and effect on cellular avidity measurement was assessed.

Cells, culture conditions and cellular avidity measurements were generally as outlined above. Briefly, as target cells, Nalm6 cells were provided. Cryopreserved untransduced (UNT) and transduced (CAR) primary T-cells (CD3+) were provided as effector cells. The CD3+ cells were used immediately after thawing for cellular avidity measurements (DO) or at 1 , 2, 3, or 4 days after thawing (i.e. D1- D4). A first set of experiments was conducted with 4 donors, and IL2 (90 U/mL) was added immediately to the culture medium (see Figure 2A), and a second set of experiments was conducted with 3 donors, and IL2 was added late, i.e. 24 hours after thawing.

Chips were prepared with Nalm6 cells as a monolayer. The prepared cells were run on chips in mixed order. For each day, transduced and untransduced cells of a different donor were provided.

Results are shown in Figure 2. 72h culture time post thaw results in the best separation when comparing transduced vs. untransduced cellular avidity measurements. Culturing 48h and 96h after thawing also result in nice separation. At 24h an increase in background binding of UNT effector cells was observed. Delaying supplementation of IL2 until 24h after thaw, apparently, decreases T cell recovery and negatively impacts avidity measurements because the cells expanded worse and generally speaking did not obtain statistically significant results apart from one day.

Based on these results, the best time for a cellular avidity measurement is about 72 hours post-thaw. A culture time of 48h and 96h also provides for good cellular avidity measurements. Culturing time, or resting time, post-thaw should always be kept the same within replicates of the same experiment. It is recommended to add IL2 to the cell culture medium as soon as possible after thawing, as delaying IL2 supplementation may results in delayed T cell recovery post thaw.

Experiment 3: magnetic microbeads

Sorting with magnetic microbeads is often performed to enrich for cell populations. As cells are covered with microbeads, the presence thereof at the surface of the cells may affect cellular avidity measurements which utilize exerting a force on the cells. In this experiment cells were labelled with beads targeting epitopes at the cell surface selected not to impact the cell-cell interaction that is the subject of the cellular avidity measurements (i.e. a bead bound to the cell was not to (sterically) hinder cell-cell interaction). Effects of magnetic beads bound to the CAR molecule or TCR complex was not investigated.

Cells, culture conditions and avidity measurements were generally as outlined above. Briefly, as target cells, Nalm6 cells were provided Chips were prepared days with Nalm6 cells as a monolayer. Cryopreserved cells, i.e. untransduced (UNT) and retroviral vector transduced (CAR) CD3+ cells, were provided and thawed and allowed to recover. Effector cells were labelled with the CD4 and CD8 magnetic microbeads (as described above, from Miltenyi Biotec) following manufacturer's guidelines and used for cellular avidity measurement straight away in parallel with non-labelled T cells. Three different donors were used. On three separate days, three runs were performed for each donor in mixed order (UNT or CAR, and, Regular or Beads conditions).

The results show that labelling with magnetic beads targeting human CD4 and/or human CD8 did not affect cellular avidity measurements (see Fig. 3).

Hence, cellular avidity measurements may be performed directly after sorting with magnetic microbeads. As shown, targeting CD4 and CD8 is suitable for magnetic bead sorting and compatible with cellular avidity measurement. Marker genes not linked e.g. to the CAR constructs (and which are presented on the cell surface may also be selected for affinity based magnetic bead sorting, which can be conducted as the data shows immediately prior to cellular avidity measurements utilizing a force, e.g. such as with a Z-movi® device.

Experiment 4: time post-transduction

In this experiment, time in culture post-transduction of T cells was investigated, using retroviral vectors, and its effect on cellular avidity measurements. Cells, culture conditions and avidity measurements were generally as outlined above. Briefly, isolated and stimulated primary T cells (CD3+) and CD8+ cells were transduced (CAR) and untransduced (UNT) as described above with a retroviral vector with a marker gene. After transduction, transduced cells were sorted using the marker gene on day 3 post-transduction (72 hours post-transduction). Cellular avidity measurements were performed immediately thereafter, and the subsequent 4 days (i.e. on day 3, and days 4, 5, 6 and 7). IL2 supplementation was every 48 hours.

Chips were prepared days with Nalm6 cells as a monolayer. Untransduced (UNT) and retroviral vector transduced (CAR) cells were provided. Three different donors were used. On each day, runs were performed for each donor with the following run order UNT, CAR, UNT, CAR, UNT, CAR.

The results (shown in Figure 4), show that good separation between transduced (CAR) and untransduced effector cells (UNT) was obtained on all days. Best separation was obtained on days 5, 6 and 7 post-transduction with retroviral vectors, as judged by best separation - the biggest difference between negative and positive sample..

Based on the results, it is recommended that days 5 (120 hours), 6 (144 hours) and 7 (168 hours) are the best culture times for cellular avidity measurement post T cell transduction (i.e. days 7, 8 and 9 post isolation/selection/stimulation). Culturing times should be constant between replicates when conducting experiments. IL2 is to be added to the cell culture medium on day 3 post-transduction (at 72 hours post-transduction) and then supplemented about every 48 hours.

Experiment 5: time in culture

Longer times in ex vivo culture may affect T cell health and activity. Hence, in this experiment the effects of longer culturing times on cellular avidity measurements were studied.

Cells, culture conditions and avidity measurements were generally as outlined above. Briefly, freshly isolated T cells 8_were stimulated with CD3/CD28 Dynabeads™. The T cells, obtained from 2 donors, were transduced with retroviral vectors expressing CD19-targeting CARs. Such transduced T cells were known to have on Nalm6 and Raji monolayers a cellular avidity separation of >15% between negative and positive samples, i.e. the difference between the percentage of cells remaining attached after a cellular avidity run. Following retroviral transduction, the T cells were cultured and used for cellular avidity measurement for four weeks. At days 6, 9, 13, 14, 16, 20, 21 , 23, 27, 28 and 30 cellular avidity was measured on prepared Chips with Nalm6 cells. On a chip the run order was either UNT, CAR, UNT, CAR, UNT, CAR or UNT, UNT, UNT, CAR, CAR, CAR. The T cells were supplemented with IL2 every 48-72h. Samples with smaller separation windows may be affected by time in culture at an earlier time point.

Results show that over time in culture the untransduced sample showed a trend in increased background binding. 14 days after isolation and stimulation the background binding increased >15% relative to initial background measured on day 6. The best cellular avidity window (i.e. CAR - UNT) was seen at 9 days after isolation and stimulation of the T cells.

Based on these results, T cells can be best used for cellular avidity measurement around day 9 after isolation and stimulation, which is in line with the results obtained in experiment 4 which showed days 7, 8 and 9 were best. Different experiments should be measured at the same time point(s) in culture.

Experiment 6: IL2 supplementation prior to cellular avidity measurement

Primary T cells recover better post thaw if they are supplemented with IL2 immediately. Typically primary T cells are supplemented with IL2 every 48-72h. This means that depending on timing of cellular avidity measurement, IL2 supplementation can be shortly or somewhat longer before a measurement. In this experiment, timing of IL2 supplementation and its potential effect on cellular avidity measurement was studied.

Cells, culture conditions and avidity measurements were generally as outlined above. Thawed effector cells, either untransduced or transduced (CAR1 or CAR2) were supplemented with 90U/mL IL2 on the day of the thaw (DO) and divided into two groups. After 48h, one of the groups was supplemented further with 90U/mL IL2 (D0+D2). Following an additional 24h (i.e. 72h post thaw) cellular avidity measurements were performed on chips prepared with Nalm6 cells. The run order on the chips was mixed and 3 different donors were used in this experiment.

Results are shown in Figure 6. Results indicate that adding IL2 shortly before measurements did not affect cellular avidity results.

Based on these results, when conducting experiments, primary T cells can be optionally supplemented with IL2 the day before a cellular avidity experiment, or not. This result is consistent with the recommendation of IL2 supplementation every 2-3 days in cellular avidity measurement experiments.

Experiment 7: cell density 1 - cells/cm2 In this experiment, seeding of T cells at different cell densities (cells/cm2) and its effect on cellular avidity measurements was investigated. T cells provided were cryopreserved T cells that were thawed prior to seeding.

Cells, culture conditions and avidity measurements were generally as outlined above. Provided cryopreserved transduced (CAR) and untransduced T cells were thawed and resuspended at a concentration of 1.5 x 10e6 cell/mL of live cells. 1 mL of the prepared cell suspension was subsequently seeded per well in plates of different formats: 6-well, 12-well, 24-well and 48-well plates (0.15, 0.4, 0.8 and 2.0 million T cells per cm2 respectively) after thaw. Culture medium was supplemented with IL2. After 72h culture/recovery time post-thaw, the cells were used for a cellular avidity measurement. Chips were prepared with Nalm6 cells. The run order was mixed UNT (run 1- 3) and mixed CAR (run 4-6).

Figure 7 shows the results, wherein averages were plotted (3-4 technical repeats) from cellular avidity measurements from 3-4 separate experiments. It appears cell density (cell number per cm2) can affect avidity measurements. The best cellular avidity separation was achieved by culturing 1 mL of 1 ,5M/mL T cells in 48-well plate.

Cell density (cell number/cm2) can affect cellular avidity measurements. The best separation was observed for cells cultured in 48-well plates (corresponding to 2.0 million T cells per cm2 respectively) after thaw. Less well separation was found for 24 and 12 well plates, and lesser for a 6-well plate.

Experiment 8: cell density 2 - cells/mL

In this experiment one cell density was selected (cells / cm2) and cell concentration was varied. Effects of these conditions on cellular avidity measurements were studied.

Cells, culture conditions and avidity measurements were generally as outlined above. Provided cryopreserved transduced (CAR) and untransduced T cells were thawed. Cells were thawed and plated at 1.5e6 live cells per well in 24-well plate in different volumes: 0.5, 1.0 or 2.0mL of culture medium supplemented with 90U/mL IL2. After 72h in culture, the cells were used for a cellular avidity measurement. Chips were prepared with Nalm6 cells. The run order was mixed UNT (Run 1-3) and mixed CAR (run 4-6).

In Figure 8 results are plotted. Results indicates that cell concentration (cell number per mL) did not significantly affect avidity measurements as highly similar results were obtained with the different volumes. While cell density (cell number/cm2) (see experiment 7) was shown to have an effect on cellular avidity measurements, i.e. results differentiated between the different cell densities, in this experiment, cell concentration (cell number/mL) did not significantly affect the measurement.

Cellular Avidity Measurement Optimized Effector Cell Culture Protocol

This protocol is a recommended protocol for T cell isolation and culture. Cell handling variables have been optimized for cellular avidity measurement reproducibility and avidity plateau separation. Figure 9 shows a schematic of the recommended protocol.

T cell isolation from fresh blood or buffy coat:

1 . Isolate peripheral blood mononuclear cells (PBMCs) from fresh blood/buffy coats, eg. using SepMate™-50 (IVD) (Stemcell) tubes following manufacturer’s protocol.

2. Sort for CD8+ T cells, eg. by magnetic separation using human CD8 MicroBeads (Miltenyi Biotec) following manufacturer’s protocol. Having mixed CD4/CD8 T cell populations at variable ratios dependent on the donor can affect avidity measurements and data reproducibility. Ensure that the isolated CD8+ population is at least 90% pure.

3. Count T cells and plate at 1 ,0e6/mL and proceed directly to stimulation or freeze the cells for future use.

T cell stimulation and transduction:

4. Activate the T cells using Dynabeads™ Human T-Activator CD3/CD28 for T Cell Expansion and Activation (ThermoFisher Scientific). a. Supplement media with 5uL Dynabeads per 1 million T cells, and 90U/mL IL2. Incubate/culture 48 hours at 37°C 5% CO2.

5. Following the 48h incubation, proceed with the retroviral transduction of the activated T cells using RetroNectin® (TaKaRa) following manufacturer’s protocol. Always use processed viral supernatant to avoid contamination with virus-producing cells. Supplement the transduction reactions with 90U/mL IL2 and incubate 72 hours at 37°C 5% CO2.

6. 5 days after initial T cell stimulation (3 days after transduction), harvest and replate transduced T cells. a. Remove Dynabeads using DynaMag™ (ThermoFisher Scientific) magnet. b. Spin down harvested T cells at 400xG for 6 minutes and resuspend in fresh T cell medium (typically RPM1 1640 supplemented with Glutamax and 10% FCS, optionally Penicillin/Streptomycin) at 1 ,0e6/mL. c. Supplement media with 90U/mL IL2.

7. Check transduction efficiency and MFI using flow cytometry. Ensure comparable transduction efficiency and expression levels between samples. Purification and Culture

8. Enrich/purify transduced T cells to at least 80% transduced. This can be achieved by staining for transduction marker or molecule with an antibody conjugated to PE or APC, followed by magnetic separation using Anti-PE or Anti-APC MicroBeads (Miltenyi Biotec) following manufacturer’s guidelines. Ensure all samples have similar transduction/purification efficiencies. a. If transduction efficiencies are not matched, dilute higher efficiency populations to the lower transduction efficiency by supplementing with negative control cells.

9. For best avidity results, perform avidity measurements on day 7 to 9 post initial T cell stimulation. Avidity measurements can be performed between days 6 and 21 post stimulation. Always use the same time point for experimental replicates.

10. If freezing cells for later use, freeze cells at day 5 to 9 post initial T cell stimulation. Always use the same time point for effector cell freezing for experimental replicates. a. Freeze cells. Effector T cells should be frozen in at minimum 5e6/mL,

1 mL/vial aliquots in freezing medium (90% FCS, 10% DMSO). Frozen T cells should be stored in liquid nitrogen no longer than a year.

Thawing frozen T cells:

1 . Thaw the number of vial(s) needed for each experiment condition 72 hours before planned avidity measurement. Be mindful of replicates and a negative control in your experimental plan. a. 1 vial of 5 million cells is sufficient to collect 12 avidity measurements.

2. Prepare a labelled tube containing 10mL pre-warmed T cell medium per vial to be thawed. Retrieve the vials designated for thawing from liquid nitrogen and thaw the cells quickly in 37°C water bath. Do not thaw more than two vials at a time. As soon as the cells are thawed, they should be transferred into the corresponding tubes containing pre-warmed medium. It is essential to do this quickly as DMSO is toxic to the cells.

3. Spin down the cells at 400xG for 6 minutes and resuspend with 5mL T cell medium. Count the recovered effector cells and assess their viability. Resuspend at 1 ,5e6/mL supplemented with 90U/mL IL2 and culture 37°C 5% CO2 before performing avidity experiment.

4. For best results, perform avidity measurements on day 3 post T cell thaw. Avidity measurements are also possible on days 2 and 4 post thaw. Always use the same time point for avidity measurements post thaw for experimental replicates.

Claims

1 . Method for providing transduced CD8+ T cells, for determining cellular avidity of said transduced CD8+ T cells with target cells, by exerting a force, wherein said method comprises the steps of: a) providing PBMCs, on day 0; b) selecting CD8+ T cells from the PBMCs, wherein the selected population is at least 90% CD8+; c) seeding the CD8+ T cells in an appropriate cell culture medium, and activating cells for 2 days by providing an activation stimulus such as a CD3/CD28 activator in the medium and supplementing IL-2 in the medium; d) transducing the activated CD8+ T cells with a retroviral vector, on day 2, while supplementing the medium with IL-2, and allowing the transduction to proceed for 72 hours; e) on day 5, optionally, removing the activation stimulus, optionally determining transduction efficiency and/or optionally enriching for transduced CD8+ T cells for providing transduced CD8+ T cells having a transduction efficiency of at least 80 percent; f) on day 5, further seeding and culturing the transduced CD8+ T cells in the presence of IL-2, wherein the culture medium is supplemented with IL-2 every 2-3 days; g) determining cellular avidity of the cultured transduced CD8+ T cells to target cells, by providing target cells attached to a surface, allowing the cultured transduced CD8+ T cells to interact therewith and subsequently exerting a force away from the attached target cells, wherein cellular avidity is determined on day 6, up to, and including, day 21 ; wherein, optionally, after step f) and, 24 hours, or less, prior to step g), determining transduction efficiency and/or optionally enriching for transduced CD8+ T cells for providing transduced CD8+ T cells having a transduction efficiency of at least 80 percent.

2. Method in accordance with claim 1 , wherein in step g) cellular avidity is determined on day 7, 8 or 9.

3. Method for providing transduced CD8+ T cells, for determining cellular avidity of said transduced CD8+ T cells with target cells, by exerting a force, wherein said method comprises the steps of: a) providing PBMCs, on a day 0; b) selecting CD8+ T cells from the PBMCs, wherein the selected population is at least 90% CD8+; c) seeding the CD8+ T cells in an appropriate cell culture medium, and activating cells for 2 days by providing an activation stimulus such as a CD3/CD28 activator in the medium and providing IL-2 in the medium; d) transducing the activated CD8+ T cells with a retroviral vector, on day 2 while supplementing the medium with IL-2, and allowing the transduction to proceed for 72 hours; e) on day 5, optionally, removing the activation stimulus, optionally determining transduction efficiency and/or optionally enriching for transduced CD8+ T cells for providing transduced CD8+ T cells having a transduction efficiency of at least 80 percent; and, f) cryopreserving the transduced CD8+ cells on day 5, or on day 5, further seeding and culturing the transduced CD8+ T cells in the presence of IL-2, wherein the culture medium is supplemented with IL-2 every 2-3 days, and cryopreserving at a day up to, and including, day 9 after providing the PBMCs; g) thawing the frozen cells, and further seeding and culturing the transduced CD8+ T cells in a medium which is supplemented with IL-2 every 2-3 days; h) determining cellular avidity of the cultured transduced CD8+ T cells to target cells, on day 2, day

3. or day 4 after step g) of thawing the frozen transduced CD8+ T cells, by providing target cells attached to a surface, allowing the cultured transduced CD8+ T cells to interact therewith and subsequently exerting a force away from the attached target cells; wherein, optionally, after step g) and, 24 hours, or less, prior to step h), determining transduction efficiency and/or optionally enriching for transduced CD8+ T cells for providing transduced CD8+ T cells having a transduction efficiency of at least 80 percent.

4. Method in accordance with claim 3, wherein in step h) cellular avidity is determined on day 3 after the step g) of thawing.

5. Method in accordance with any one of claims 1-4, wherein said enrichment step comprises positive selection using MACS.

6. Method in accordance with claim 5, wherein said enrichment step is performed 24 hours or less prior to determining cellular avidity.

7. Method in accordance with any of claims 1-6, wherein instead of IL-2, IL-7/IL-15 is used.

8. Method in accordance with any one of claims 1-7, wherein furthermore control transduced, or, untransduced CD8+ T cells are provided, wherein the method is in addition performed with the control transduced CD8+ T cells or untransduced CD8+ T cells, instead of the transduced CD8+ T cells, and cellular avidity of control transduced CD8+ T cells or untransduced CD8+ T cells with target cells is determined.

9. Method in accordance with claim 8, wherein the cellular avidity of transduced CD8+ T cells with target cells is compared with the cellular avidity of control transduced CD8+ T cells or untransduced CD8+ T cells with target cells.

10. Method in accordance with any one of claims 1 -9, wherein the retroviral vector encodes a receptor, such as a CAR or a TCR, said receptor comprising a binding domain capable of binding to the target cell.

1 1 . Method for determining cellular avidity between effector cells and target cells, by providing target cells attached to a surface, allowing the effector cells to interact therewith, and by subsequently exerting a force away from the attached target cells, wherein 24 hours or less prior to determining cellular avidity, effector cells or target cells are subjected to a selection step, wherein said selection step comprises a positive selection using MACS.

12. Method for determining cellular avidity in accordance with any one of claims 1-11 , wherein instead of the target cells, the transduced CD8+ T cells or effector cells are attached to a surface.

13. The method in accordance with any one of claims 1-12, wherein the applied force is a force ramp, preferably a linear force ramp, and/or wherein the applied force is an acoustic force, a shear flow force or an acceleration force.

14. The method in accordance with any one of claims 1-13, wherein instead of applying a force away from the attached cells, the applied force is a differential force which does not require attachment of cells to a surface.

15. The method in accordance with claim 14, wherein cells are not attached to a surface.