Apparatus and method for analyzing living cells

Abstract

Evaluation of immune cells and cancer cells is provided by assessing cytolysis of cancer cells by effector cells, comprising: a) providing a cell-substrate impedance monitoring device operably connected to an impedance analyzer, the device including a well for receiving cells and an electrode array in the bottom of the well, and further operably connected to an imaging unit; b) adding target cells characterized as cancer cells to the well; c) disposing a layer comprising extracellular matrix (ECM) over the target cells; d) adding effector cells on top of the ECM layer; and e) imaging the well and monitoring the cell-substrate impedance of the well to determine invasion of the effector cells through the ECM layer and the effectiveness of the effector cells in killing the target cells directly or via migration and invasion through the extracellular matrix.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 383,245, filed November 10, 2022, and U.S. Provisional Patent Application No. 63 / 580,658, filed September 5, 2023, the contents of which are incorporated herein by reference. [Background technology]

[0002] The tumor microenvironment (TME) of solid tumors poses a significant challenge to the immune response, and recapitulating the characteristics of the TME in assays may be essential to identifying and improving cellular immunotherapies. Genetically engineered cytotoxic natural killer (NK) and cytotoxic T (CD8 + T cells must extensively infiltrate solid tumors to perform their effector functions. During migration, lymphocytes must navigate acellular spaces with a structural scaffold that constitutes the extracellular matrix (ECM). The ECM can regulate a wide variety of cellular responses in both resident tumor cells and infiltrating lymphocytes, thereby determining their outcomes. Therefore, there is a need to develop methods and systems to evaluate both tumor cell and immune cell responses in the context of the ECM. Summary of the Invention

[0003] In one aspect, the present disclosure provides a method for assessing cytolysis of cancer cells by effector cells, the method comprising: a) providing a cell-substrate impedance monitoring device operably connected to an impedance analyzer, the device including a well for receiving cells and an electrode array at the bottom of the well, and operably connected to an imaging unit; b) adding target cells characterized as cancer cells to the well; c) disposing a layer comprising extracellular matrix (ECM) over the target cells; d) adding effector cells on top of the ECM layer; and e) imaging the well and monitoring the cell-substrate impedance of the well to determine effector cell invasion through the ECM layer and the effectiveness of the effector cells in killing the target cells directly or via migration and invasion through the extracellular matrix.

[0004] In some embodiments, the imaging unit further comprises an imaging device positioned adjacent to the well.

[0005] In some embodiments, the cancer cells are derived from a solid tumor. In some embodiments, the effector cells are immune cells. In some embodiments, the effector cells are natural killer (NK) cells. In some embodiments, the effector cells are T cells. In some embodiments, the T cells are CD8 + In some embodiments, the effector cells are T cells. In some embodiments, the effector cells express a chimeric antigen receptor (CAR). In some embodiments, the NK cells are chimeric antigen receptor (CAR) CAR-NK cells. In some embodiments, the T cells are chimeric antigen receptor (CAR) CAR-T cells.

[0006] In some embodiments, the cancer cells have aberrant FGFR signaling. In some embodiments, the method further comprises adding an FGFR inhibitor to the well. In some embodiments, the FGFR inhibitor comprises pemigatinib.

[0007] In another aspect, the present disclosure provides a system configured to carry out a method of assessing cytolysis of cancer cells by effector cells, as described herein.

[0008] In yet another aspect, the present disclosure provides a method comprising measuring cell-substrate impedance between target cells and effector cells separated by a layer of extracellular matrix (ECM) at various times during an assay, acquiring images of the effector and target cells at various times during the assay, and determining the effectiveness of the effector cells against the target cells based on changes in cell-substrate impedance and images over the course of the assay.

[0009] In some embodiments, the method further includes applying a first dye of a first color to the target cells, and applying a second dye of a second color different from the first color to the effector cells.

[0010] In some embodiments, the method further comprises varying the thickness of the ECM within multiple wells of the multiwell plate in which the target cells and effector cells are disposed.

[0011] In some embodiments, the method further comprises applying a pharmaceutical compound of interest to one or more target cells, ECM, and effector cells; comparing a baseline efficacy of the effector cells on target cells to which the pharmaceutical compound of interest has not been applied with an experimental efficacy of the effector cells on target cells to which the pharmaceutical compound of interest has been applied; and, in response to the experimental efficacy meeting an efficacy threshold, using the pharmaceutical compound of interest to treat or prevent a disorder associated with the target cells in a biological subject.

[0012] In some embodiments, the method further comprises applying a first pharmaceutical compound of interest to a first subset of one or more target cells, ECM, and effector cells; applying a second pharmaceutical compound of interest to a second subset of one or more target cells, ECM, and effector cells distinct from the first subset; comparing a first efficacy of the effector cells on the target cells to which the first pharmaceutical compound of interest has been applied with a second efficacy of the effector cells on the target cells to which the second pharmaceutical compound of interest has been applied; and, in response to the first pharmaceutical compound of interest having greater efficacy than the second pharmaceutical compound of interest, using the first pharmaceutical compound of interest for the treatment or prevention of a target cell-associated disorder in a biological subject.

[0013] In yet another aspect, the present disclosure provides a method of treating a solid tumor, comprising administering a therapeutically effective amount of an FGFR inhibitor and a chimeric antigen receptor (CAR) therapy to a subject in need thereof.

[0014] In some embodiments, the solid tumor has aberrant FGFR signaling. In some embodiments, the FGFR inhibitor is an inhibitor of one, two, three, or all of FGFR1, FGFR2, FGFR3, or FGFR4. In some embodiments, the FGFR inhibitor is an FGFR1 inhibitor. In some embodiments, the FGFR inhibitor is an FGFR2 inhibitor. In some embodiments, the FGFR inhibitor is an FGFR3 inhibitor. In some embodiments, the FGFR inhibitor is an FGFR4 inhibitor. In some embodiments, the FGFR inhibitor comprises pemigatinib.

[0015] In some embodiments, the FGFR inhibitor is administered before the CAR therapy is administered. In some embodiments, the FGFR inhibitor is administered simultaneously with the administration of the CAR therapy. In some embodiments, the FGFR inhibitor is administered after the CAR therapy is administered.

[0016] In some embodiments, the CAR therapy is CAR T cell (CAR-T) therapy, CAR-NK cell therapy, CAR macrophage therapy, or CAR-gd-T therapy. [Brief explanation of the drawings]

[0017] [Figure 1A] 1 shows the delayed killing of target cells with increasing invasion distance according to embodiments of the present disclosure. Addition of NK92 after 24 hours (h) (E:T 3:1) results in a decrease in cell impedance (CI) due to target cell killing. [Figure 1B] 1 shows delayed target cell killing with increasing invasion distance according to embodiments of the present disclosure. Target cell elimination by NK cytotoxicity is delayed in Matrigel, and elimination slows with increasing volume, which indicates increased invasion distance. Live cell imaging confirms the delayed elimination of target cells in the field of view. [Figure 2A-2B] Figures 2A and 2B show representative images demonstrating NK cytotoxicity with increasing volumes of Matrigel, according to embodiments of the present disclosure. eLive Green stains NK-92 cells (Figure 2A) but not healthy Michigan Cancer Foundation-7 (MCF-7) cells (Figure 2B). Addition of NK-92 cells to the Matrigel layer results in a gradual disappearance of MCF-7 (red) targets, along with an increase in green fluorescence, suggesting the accumulation of NK-92 and dead target cells. Morphological changes are evident at earlier time points (approximately 45-55 hours). [Figure 3A] 1 shows the delay in target cell killing by NK cells with inhibition of MMPs according to embodiments of the present disclosure. GM6001 delays but does not eliminate the decrease in impedance (CI) due to target cell killing. [Figure 3B] 1 shows the delayed killing of target cells by NK cells with inhibition of MMPs according to embodiments of the present disclosure. In the presence of MMP inhibitors, a delayed disappearance of the red fluorescent signal of MCF7 and an increase in green fluorescence (eLive) were observed. [Figure 3C]

[0023] Figure 1 shows the delay in target cell killing by NK cells with inhibition of MMPs, according to embodiments of the present disclosure. The calculated cytolysis rate [(1-(treated / untreated)*100] after 72 hours shows that the rate of cytolysis slows with increasing amounts of Matrigel and matrix metalloproteinase (MMP) inhibitors. [Figure 4] 1 is a schematic diagram showing an example of an ECM invasion and cytotoxicity assay according to an embodiment of the present disclosure. [Figure 5A] Figure 1 shows that pemigatinib enhances the efficacy of EpCAM-CAR-T in squamous cell carcinoma, according to embodiments of the present disclosure. EpCAM expression on the cell surface of various tumor cells. [Figure 5B] 1 shows that pemigatinib enhances the efficacy of EpCAM-CAR-T in squamous cell carcinoma, according to embodiments of the present disclosure. Normalized Cell Index over time shows that FGFR1 inhibitors reduce proliferation of A431 cells. [Figure 5C]

[0023] Figure 1 shows that pemigatinib enhances the efficacy of EpCAM-CAR-T in squamous cell carcinoma, according to embodiments of the present disclosure. Normalized Cell Index over time shows that EpCAM CART targets A431 cells in a cytotoxicity assay. [Figure 5D]

[0023] Figure 1 shows that pemigatinib enhances the efficacy of EpCAM-CAR-T in squamous cell carcinoma, according to embodiments of the present disclosure. Normalized Cell Index over time shows that EpCAM CART (E:T ∼3.5) has no effect on A431 cells growing in Matrigel. [Figure 5E]

[0023] Figure 1 shows that pemigatinib enhances the efficacy of EpCAM-CAR-T in squamous cell carcinoma, according to embodiments of the present disclosure. Normalized cell index over time shows that CAR-T cells effectively control A431 cell proliferation in the presence of FGFRi, even though EpCAM CAR-T (E:T ∼3.5) is ineffective against A431 cells in Matrigel. [Figure 5F]

[0023] Figure 1 shows that pemigatinib enhances the efficacy of EpCAM-CAR-T in squamous cell carcinoma, according to embodiments of the present disclosure. Normalized Cell Index over time shows that the rate of cell lysis calculated from impedance readings and live cell imaging data confirm the increased potency of EpCAM CART in the presence of pemigatinib. [Figure 5G]

[0023] Figure 1 shows that pemigatinib enhances the efficacy of EpCAM-CAR-T in squamous cell carcinoma, according to embodiments of the present disclosure. Representative images taken at 100 hours demonstrate the increased potency of EpCAM CART in the presence of pemigatinib. [Figure 6A] 1 illustrates an experimental test setup according to an embodiment of the present disclosure, showing the setup of the instrument, well plate, and one of the wells compared to a Boyden chamber. [Figure 6B] 1 illustrates an experimental test setup according to an embodiment of the present disclosure. The advantages of this setup over traditional approaches to data collection are outlined along with the steps of the assay. [Figure 7A] 1 shows that NK-92 cytotoxicity is delayed as the penetration distance increases, according to embodiments of the present disclosure. Addition of NK-92 after 24 hours (E:T=3:1) results in a decrease in impedance (CI) due to target cytolysis. [Figure 7B] 1 shows that NK-92 cytotoxicity is delayed as the penetration distance increases, according to embodiments of the present disclosure. Addition of NK-92 after 24 hours (E:T=3:1) delays the decrease in impedance in Matrigel. [Figure 7C]

[0023] Figure 1 shows that NK-92 cytotoxicity is delayed as the invasion distance increases, according to embodiments of the present disclosure. Increasing the volume of Matrigel further delays cytolysis. Cytolysis rates were calculated with reference to untreated MCF-7 cells growing in Matrigel. [Figure 8A-8B]Figures 8A and 8B show that MMP inhibition delays target cell killing by NK cells, according to embodiments of the present disclosure. Figure 8A shows that after 24 hours, GFP-NK92 (E:T=3:1) reduces cell lysis and target cell killing reduces impedance (CI). Figure 8B shows that live cell imaging confirms reduced loss of target cells (red fluorescence) in the field of view in the presence of MMP inhibitors (2 μM and 10 μM). [Figure 9] Representative images of MCF-7 clustering and the gradual loss of red fluorescence associated with cytotoxicity. Some green GFP-NK92 cells (outlined in yellow in Figure 9) make multiple contacts with MCF7-red target cells within the cluster, causing cell death over the course of the assay. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present disclosure relates, at least in part, to a co-culture model for real-time assessment of immune cell infiltration and tumor cell killing.

[0019] The extracellular matrix (ECM) is a collective term for the diverse types of acellular structural components within tissues that play an important role in maintaining homeostasis. Without wishing to be bound by theory, in some embodiments, the ECM is considered to be a complex network composed of a series of multidomain macromolecules organized in a cell / tissue-specific manner. For example, major ECM components may include collagens, proteoglycans, elastin, and / or cell-associated glycoproteins, each with distinct physical and biochemical properties. In solid tumors, the ECM undergoes significant changes and may be involved in regulating immune cell function in the tumor microenvironment. Cytotoxic natural killer (NK) and cytotoxic T (CD8) cytokines, which are utilized in immunotherapy, are also involved in the regulation of immune cell function. + Lymphocytes, such as CD8 T cells, can be regulated by the ECM. As detailed herein, the Matrigel layer poses a challenge for NK cells, and the increased penetration distance slows the kinetics of tumor cell killing. Furthermore, the infiltration and killing of tumor targets is regulated by NK or CD8 T cells.+ It relies on the ability of T cells to remodel the matrix using matrix metalloproteinases.

[0020] To assess the efficacy of cells in attacking targets within the ECM, experiments or assays are performed in which increasing volumes of Matrigel (used to represent increased invasion distance) are layered on top of target tumor cells expressing a fluorescent protein. Immune cells are seeded on top of the solidified Matrigel layer, and a second marker of a different color than the fluorescent protein is added to the well. Immune cell invasion and function are assessed using impedance-based measurements of immune cell killing and live-cell imaging data collected with the XCELLIGENCE RTCA ESIGHT® Imaging and Sensing System, available from Agilent Tech., Inc., Santa Clara, CA, USA.

[0021] As MCF-7 target cells adhere and proliferate, impedance increases over time, but upon addition of NK-92 cells, impedance decreases due to target cell death. The total time required for impedance levels to decrease to the level observed upon NK-92 addition gradually increased from 46 hours at 50 mL / well to 72 hours at 110 mL / well. eLive Green stained NK cells and dead cells but not healthy MCF-7 targets. Consistent with the impedance data, the loss of red fluorescence and increase in green fluorescence of target cells gradually delayed as the volume of Matrigel increased. Similar kinetic delays were observed with the broad-spectrum MMP inhibitor GM6001 (e.g., 2 millimolar (mM) and 10 mM), suggesting that MMPs play a role in NK function. Interestingly, NK-92 cells induced significant morphological changes in MCF-7-red target cells before invading throughout the Matrigel, suggesting an early distal effect.

[0022] The results suggest a role for NK cell effector functions, potentially involving cytokines, in killing susceptible target cells, independent of their ability to infiltrate and / or degrade matrix. This assay also demonstrates the adaptability of the XCELLIGENCE RTCA ESIGHT® imaging and sensing system to study various interactions of ECM with immune cells.

[0023] Immune cells infiltrate the extracellular matrix (ECM) to perform effector functions, including killing target cells. Traditionally, invasion and cytotoxicity are assessed in separate endpoint assays. A potential drawback is that cytotoxicity is not easily assessed in the presence of ECM, which can modulate lymphocyte cellular responses. Furthermore, traditional assay systems are often cumbersome and require multiple steps. In some embodiments, the present disclosure provides an easy-to-setup assay (e.g., the 96-well format of the XCELLIGENCE RTCA ESIGHT® Imaging and Sensing System, which combines impedance and imaging functions to provide real-time readout of invasion and cytotoxicity). The methods and systems described herein are relevant to cancer immunotherapy. In some embodiments, the present disclosure provides a real-time assay for simultaneously assessing both immune cell infiltration and cytotoxicity. In some embodiments, the assay is used to systemically study the effect of ECM on immune cell-tumor cell interactions. In some embodiments, the methods and systems described herein avoid the drawbacks of complex setups, such as the use of microfluidic configurations. Other advantages will be apparent to those skilled in the art after reading the detailed description of this disclosure.

[0024] The methods and systems described herein can be equipped to assess both the infiltration and killing of target tumor cells by immune cells (e.g., T cells, NK cells, macrophages) in the context of ECM (e.g., ECM of any solid tumor described herein). Traditionally, invasion / migration and cytotoxic function are assessed in separate assays. In some embodiments, the methods and systems described herein can simultaneously collect impedance-based readout of cytotoxicity and live-cell imaging data in real time, e.g., in a 96-well format.

[0025] NK cells are innate lymphoid cells that eliminate infected, stressed, or transformed cells by using a series of activating and inhibitory receptors to identify and distinguish "altered" cells from healthy cells. NK cells and their genetically engineered variants, along with chimeric antigen receptor (CAR) T cells, have become attractive options for adoptive immunotherapy.

[0026] Reasons for the lack of efficacy of adoptive cell therapy for solid tumors include, for example, limitations of tumor-specific antigens, the immunosuppressive environment of the tumor itself leading to immune cell exhaustion, physical infiltration of the tumor stroma by immune cells, and combinations thereof. Most in vitro assays for evaluating adoptive immune cells focus on activation, proliferation, and cytotoxicity, with few assays focusing on immune cell migration and extravasation to tumor sites. In some embodiments, the present disclosure provides real-time in vitro assays that model both immune cell migration / infiltration to tumor sites and the tendency of immune cells to kill tumor cells.

[0027] Recapitulating the tumor microenvironment within solid tumors will play a crucial role in developing and refining cellular immunotherapy strategies for multiple cancers. The ECM can be dysregulated in the TME, and the ECM has the ability to alter cellular responses. Therefore, it is necessary to evaluate both tumor and immune cell responses in the context of the ECM. In this regard, various components of the ECM can modulate NK cell function.

[0028] The ability of NK cells to invade through the extracellular space and reach target cells may depend on their ability to degrade the ECM. NK cells can express numerous MMPs, and in extravasation assays, NK-92 invasion was reduced in the presence of the MMP inhibitor GM6001, suggesting a role for MMPs in NK cell migration through Matrigel. Traditionally, invasion / migration potential and cytotoxic function are assessed separately in workflows or using complex setups.

[0029] ECM invasion and cytotoxicity assay In some embodiments, NK infiltration and function are assessed using impedance-based measurements of immune cell killing (normalized cell index, CI) and live cell imaging in the XCELLIGENCE RTCA ESIGHT® Imaging and Sensing System (available from Agilent Tech., Inc.).

[0030] The experimental procedure is described as follows. Briefly, various volumes of Matrigel (50–110 microliters (µL) / well, 6 milligrams (mg) / milliliter (mL)) representing the extent of invasion distance are layered on top of MCF-7 (available from the American Type Culture Collection (ATCC)) human breast adenocarcinoma cells stably expressing nuclear-localized red fluorescent protein (NF-7). NK-92 cells (Creative Bioarray, available from ATCC) are added on top of the solidified Matrigel layer at a 3:1 E:T ratio. eLive Green (available from Agilent Technologies, shown in green) is added at 1 µL / mL. To assess the role of matrix metalloproteinases (MMPs), a broad-spectrum MMP inhibitor (e.g., GM6001, available from Selleck Chemicals) is used. An example schematic is shown in Figure 4.

[0031] Certain terms are used throughout the description and claims to refer to particular features or components. As one of ordinary skill in the art will appreciate, the same feature or component may be referred to by different names by different people. This document does not intend to distinguish between components or features that differ in name but function the same. Furthermore, while the various examples provided herein recite specific analytical systems, reagents, media, therapeutic agents, cell lines, etc., for the purpose of illustrating the underlying inventive concepts in a practical sense, it is believed that the present disclosure will enable one of ordinary skill in the relevant art to substitute a variety of different elements suitable for their purposes without undue experimentation when conducting assays to determine the efficacy of, or interactions between, potential therapeutic agents and various cell lines, using different imaging and sensing systems, well plate sizes / shapes, software controlling associated devices, etc.

[0032] As used herein, the term "optimize" and variations thereof are used in the sense understood by data scientists to refer to actions taken for the continuous improvement of a system against a goal. An optimized value is understood to represent the "near-best" value for a given reward framework, which may fluctuate around a local or global maximum for the "best" value or set of values ​​and may change in response to changing goals or changing input conditions. Thus, an optimal solution for a first goal at a given time may not be optimal for a second goal at that time, or may not be optimal for the first goal at a later time.

[0033] As used herein, various compounds are referred to by the associated element abbreviations established by the International Union of Pure and Applied Chemistry (IUPAC) and are familiar to those skilled in the relevant art. Similarly, various units of measurement may be used herein and are referred to by the associated abbreviations established by the International System of Units (SI), which are familiar to those skilled in the relevant art.

[0034] As used herein, "about," "approximately," and "substantially" are understood to mean a number within a range of a referenced number, for example, from -10% to +10% of the referenced number, preferably from -5% to +5% of the referenced number, more preferably from -1% to +1% of the referenced number, and most preferably from -0.1% to +0.1% of the referenced number.

[0035] Furthermore, all numerical ranges herein should be understood to include all whole integers, whole numbers, or fractions within the range. Furthermore, these numerical ranges should be construed as supporting claims directed to any number or subset of numbers within that range. For example, a disclosure of 1 to 10 should be construed as supporting ranges of 1 to 8, 3 to 7, 1 to 9, 3.6 to 4.6, 3.5 to 9.9, etc.

[0036] As used in this disclosure, the phrase "at least one of" a list of items refers to any set of those items, including sets containing a single member, and all possible combinations thereof. For example, "at least one of A, B, or C" or "at least one of A, B, and C" is intended to include the set A, B, C, AB, BC, and ABC, where a set may include one or more instances of a given member (e.g., AA, AAA, AAB, AABBCCC, etc.) and any order thereof. For the avoidance of doubt, the phrase "at least one of A, B, and C" should not be construed to mean "at least one of A, at least one of B, and at least one of C."

[0037] As used in this disclosure, the term "determining" encompasses a variety of acts, including calculating, computing, processing, deriving, investigating, retrieving (e.g., via a table, database, or other data structure), ascertaining, receiving (e.g., receiving information), accessing (e.g., accessing data in a memory), obtaining, resolving, selecting, choosing, establishing, and the like.

[0038] Without further elaboration, it is believed that those skilled in the art can use the preceding description to make full use of the claimed invention. The embodiments and aspects disclosed herein are to be construed as merely illustrative and are not intended to limit the scope of the present disclosure in any way. It will be apparent to those skilled in the art that changes can be made to the details of the above-described embodiments without departing from the basic principles discussed. In other words, various modifications and improvements of the embodiments specifically disclosed in the above description are within the scope of the appended claims. For example, any suitable combination of features of the various embodiments described is contemplated.

[0039] In the claims, reference to an element in the singular does not mean "one and only one," but rather "one or more" or "at least one," unless specifically so recited. The term "some" means one or more, unless specifically recited otherwise. Claim elements are not to be construed under 35 U.S.C. § 112(f) unless the element is expressly recited using the phrase "means for" or "step for." All structural and functional equivalents to the elements of the various embodiments described in this disclosure that are known or later become known to those skilled in the relevant art are expressly incorporated herein by reference and are intended to be encompassed by the claims. Furthermore, nothing disclosed in this disclosure is intended to be made available to the public, regardless of whether such disclosure is expressly recited in the claims.

[0040] Enumerated Embodiments 1. A method for assessing cytolysis of cancer cells by effector cells, comprising: providing a cell-substrate impedance monitoring device operably connected to an impedance analyzer, the device including a well for receiving cells and an electrode array at the bottom of the well, the device further operably connected to an imaging unit; adding target cells characterized as cancer cells to the wells; disposing a layer comprising an extracellular matrix (ECM) over the target cells; adding effector cells onto the ECM layer; imaging the well and monitoring the cell-substrate impedance of the well to determine the infiltration of the effector cells through the ECM layer and the effectiveness of the effector cells in killing the target cells directly or via migration and invasion through the extracellular matrix.

[0041] 2. The method of embodiment 1, further comprising an imaging device positioned adjacent to the well.

[0042] 3. The method of embodiment 1 or 2, wherein the cancer cells are from a solid tumor or a metastatic lesion thereof, and optionally the solid tumor is a carcinoma or cancer of the prostate, breast, lung and bronchus, colon and rectum, bladder, thyroid, kidney and renal pelvis, uterine corpus, oral cavity, or ovary.

[0043] 4. The method of any of embodiments 1-3, wherein the effector cells comprise immune cells (e.g., immune cells described herein), such as natural killer (NK) cells, T cells (e.g., CD8+ T cells, cytotoxic T cells), macrophages, or a combination thereof.

[0044] 5. The method of any of embodiments 1-3, wherein the effector cells comprise NK cells.

[0045] 6. The method of any of embodiments 1-3, wherein the effector cells comprise T cells.

[0046] 7. The method of any of embodiments 1-3, wherein the effector cells comprise immune cells, such as CAR-T cells, CAR-NK cells, CAR-macrophages, gamma-delta T cells (e.g., CAR-gd-T cells), or a combination thereof.

[0047] 8. The method of any of embodiments 1-7, wherein the effector cells are obtained from a subject.

[0048] 9. The method of embodiment 8, wherein the subject has cancer.

[0049] 10. The method of embodiment 9, wherein the cancer is a solid tumor.

[0050] 11. Measuring cell-substrate impedance between target cells and effector cells separated by a layer of extracellular matrix (ECM) at various times during the assay; acquiring images of the effector and target cells at various time points during the assay; determining the effectiveness of the effector cells against the target cells based on changes in cell-substrate impedance and images during the assay period.

[0051] 12. applying a first dye of a first color to the target cells; and 12. The method of embodiment 11, further comprising applying a second dye of a second color different from the first color to the effector cells.

[0052] 13. The method of embodiment 11 or 12, further comprising the step of varying the thickness of the ECM within multiple wells of the multi-well plate in which the target cells and effector cells are disposed.

[0053] 14. Applying a pharmaceutical compound of interest to one or more target cells, ECM, and effector cells; comparing the baseline efficacy of the effector cells against target cells not treated with the pharmaceutical compound of interest with the experimental efficacy of the effector cells against target cells treated with the pharmaceutical compound of interest; 14. The method of any of embodiments 11-13, further comprising the step of: using the pharmaceutical compound of interest for the treatment or prevention of a target cell-associated disorder in a biological subject in response to the experimental efficacy meeting an efficacy threshold.

[0054] 15. Applying a first pharmaceutical compound of interest to one or more target cells, ECM, and a first subset of effector cells; applying a second pharmaceutical compound of interest to a second subset of one or more target cells, ECM, and effector cells distinct from the first subset; comparing a first efficacy of the effector cells against target cells pulsed with a first pharmaceutical compound of interest to a second efficacy of the effector cells against target cells pulsed with a second pharmaceutical compound of interest; 14. The method of any of embodiments 11-13, further comprising: in response to the first pharmaceutical compound of interest having greater efficacy than the second pharmaceutical compound of interest, using the first pharmaceutical compound of interest for the treatment or prevention of a target cell-related disorder in a biological subject.

[0055] 16. The method of any of embodiments 11-15, wherein the target cells are cancer cells.

[0056] 17. The method of embodiment 16, wherein the cancer cells are from a solid tumor or a metastatic lesion thereof, and optionally the solid tumor is a carcinoma or a cancer of the prostate, breast, lung and bronchus, colon and rectum, bladder, thyroid, kidney and renal pelvis, uterine corpus, oral cavity, or ovary.

[0057] 18. The method of any of embodiments 11-17, wherein the effector cells comprise immune cells (e.g., immune cells described herein), such as natural killer (NK) cells, T cells (e.g., CD8+ T cells, cytotoxic T cells), macrophages, or a combination thereof.

[0058] 19. The method of any of embodiments 11-17, wherein the effector cells comprise NK cells.

[0059] 20. The method of any of embodiments 11-17, wherein the effector cells comprise T cells.

[0060] 21. The method of any of embodiments 11-17, wherein the effector cells comprise immune cells, such as CAR-T cells, CAR-NK cells, CAR-macrophages, gamma-delta T cells (e.g., CAR-gd-T cells), or a combination thereof.

[0061] 22. The method of any of embodiments 11-21, wherein the effector cells are obtained from a subject.

[0062] 23. The method of embodiment 22, wherein the subject has cancer.

[0063] 24. The method of embodiment 23, wherein the cancer is a solid tumor.

[0064] 25. A method for treating a solid tumor having or identified as having aberrant FGFR signaling, comprising administering to a subject in need thereof a therapeutically effective amount of an FGFR inhibitor (e.g., an inhibitor of FGFR1, FGFR2, FGFR3, FGFR4, or any combination thereof) and CAR therapy.

[0065] 26. The method of embodiment 25, wherein the FGFR inhibitor comprises pemigatinib.

[0066] 27. The method of embodiment 25 or 26, wherein the FGFR inhibitor is administered before the CAR therapy is administered.

[0067] 28. The method of any of embodiments 25-27, wherein the FGFR inhibitor is administered simultaneously with the administration of the CAR therapy.

[0068] 29. The method of any of embodiments 25-28, wherein the FGFR inhibitor is administered after the CAR therapy is administered.

[0069] 30. The method of any of embodiments 25-29, wherein the CAR therapy comprises immune cells expressing a chimeric antigen receptor comprising an antigen recognition domain, a hinge region, a transmembrane domain, and an intracellular cell signaling domain.

[0070] 31. The method of embodiment 30, wherein the antigen recognition domain binds to a tumor antigen, and optionally the solid tumor is a carcinoma or a cancer of the prostate, breast, lung and bronchus, colon and rectum, bladder, thyroid, kidney and renal pelvis, uterine corpus, oral cavity, or ovary.

[0071] 32. The method of any of embodiments 25-31, wherein the CAR therapy is CAR-T therapy.

[0072] 33. An FGFR inhibitor (e.g., an inhibitor of FGFR1, FGFR2, FGFR3, FGFR4, or any combination thereof) for use in a method of treating a solid tumor in a subject that has, or has been identified as having, aberrant FGFR signaling, in combination with CAR therapy.

[0073] 34. The FGFR inhibitor for use in embodiment 33, wherein the FGFR inhibitor comprises pemigatinib.

[0074] 35. The FGFR inhibitor for use in embodiment 33 or 34, wherein the FGFR inhibitor is administered before CAR therapy is administered.

[0075] 36. The FGFR inhibitor for use in any of embodiments 33-35, wherein the FGFR inhibitor is administered simultaneously with the administration of CAR therapy.

[0076] 37. The FGFR inhibitor for use in any of embodiments 33-36, wherein the FGFR inhibitor is administered after CAR therapy is administered.

[0077] 38. The FGFR inhibitor for use in any of embodiments 33-37, wherein the CAR therapy comprises immune cells expressing a chimeric antigen receptor comprising an antigen recognition domain, a hinge region, a transmembrane domain, and an intracellular cell signaling domain.

[0078] 39. The FGFR inhibitor for use in embodiment 38, wherein the antigen recognition domain binds to a tumor antigen, and optionally the solid tumor is a carcinoma or a cancer of the prostate, breast, lung and bronchus, colon and rectum, bladder, thyroid, kidney and renal pelvis, uterine corpus, oral cavity, or ovary.

[0079] 40. The FGFR inhibitor for use in any of embodiments 33 to 39, wherein the CAR therapy is CAR-T therapy, CAR-NK cell therapy, CAR-macrophage therapy, or CAR-gd-T therapy. [Example]

[0080] [Example 1] Assessment of immune cell infiltration and cytotoxicity using impedance and imaging It was hypothesized that the Matrigel layer would pose a challenge for NK-92 cells, and that increasing their invasion distance would delay tumor cell killing. Furthermore, it has been suggested that invasion / migration through Matrigel is dependent on matrix metalloproteinases (MMPs), which cleave components of the ECM. Therefore, the effect of inhibiting MMP function was also evaluated.

[0081] The results demonstrate that the methods and systems described herein can be readily used to systemically study the effects of ECM components on tumor and immune cell interactions in the TME, depending on the needs of the user.

[0082] material and method cell The MCF-7 human breast adenocarcinoma cell line (available from ATCC, catalog number HTB-22) was transduced with eLenti Red (available from Agilent Technologies, catalog number 8711011) at a multiplicity of infection of 1 and cultured for 14 days in the presence of puromycin (2 micrograms (μg) / mL) to select for MCF-7-red cells stably expressing nuclear-localized red fluorescent protein (RFP). Both MCF-7 and MCF-7-red cells were maintained in Eagle's minimum essential medium (EMEM, available from ATCC, 30-2003) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (available from Sigma, catalog number 12106C-500ML) and 1% penicillin / streptomycin (available from Hyclone, catalog number SV30010).

[0083] NK-92 cells (available from Creative Bioarray, CSC-C0499, and ATCC) were grown in MyeloCult H5100 medium (available from Stemcell Technologies, catalog number 05150) supplemented with 30 mL of horse serum (available from Gibco, catalog number 16050-122), 600 IU / mL of rhIL-2 (available from Stemcell Technologies, catalog number 78036), and 1% penicillin / streptomycin (available from Hyclone, catalog number SV30010). All cell lines were maintained at 37°C in 5% carbon dioxide (CO2).

[0084] ECM invasion and cytotoxicity assays The ability of NK-92 cells to invade Matrigel and kill MCF-7-red target cells was assessed by simultaneous impedance and imaging readout on an XCELLIGENCE RTCA ESIGHT® Imaging and Sensing System (available from Agilent Tech., Inc.). Background impedance signals were measured in 50 μL of EMEM medium in the wells of an E-plate VIEW microplate (available from Agilent Technologies, catalog number 0030060101030). MCF-7-red target cells (30,000 in 100 μL) were added to each well. The plate was left at room temperature for 30 minutes to allow the cells to evenly distribute across the bottom. The plate was then transferred to the XCELLIGENCE RTCA ESIGHT® Imaging and Sensing System, and data acquisition began. Impedance readings were collected every 15 minutes, and photographs were taken every 60 minutes. Images were acquired in brightfield, red, and green fluorescent channels from four fields of view in each well. Exposure times were set to the default setting for brightfield and 150 milliseconds (ms) for the red and green channels. Matrigel (available from Corning, catalog numbers 356234 and 354234) was thawed overnight at 4°C, diluted with Dulbecco's modified Eagle's medium (DMEM), and supplemented with 10% FBS to a final total protein concentration of 6 mg / mL. After 24 hours, data collection was stopped. The medium in the wells was aspirated, and various volumes of Matrigel (e.g., 50, 75, and 100 μL, as shown here) were layered on top of the MCF-7-red cells. eLive Green (available from Agilent Technologies, catalog number 8711003) was added to the Matrigel at a final concentration of 1:1000. Wells without ECM were supplemented with EMEM (100 μL) containing eLive Green. The plate was incubated at 37°C and 5% CO2 for 1 hour to solidify the Matrigel. Impedance and imaging data were collected for 1 hour, and NK-92 cells were suspended in EMEM medium containing eLive Green and the cell number was adjusted to an E:T ratio of 3:1 in 100 μL.100 μL of NK-92 cell suspension was layered on top of the Matrigel, adjusting the total volume in all wells to 200 μL. The plate was returned to the cradle of the XCELLIGENCE RTCA ESIGHT® Imaging and Sensing System, and data acquisition resumed for 7 days. The percentage of cell lysis was calculated from the normalized cell impedance readings after 72 hours using the formula [(1-(treated / untreated)*100].

[0085] Kinetics of MMP inhibition and NK infiltration To assess the role of MMP-dependent ECM remodeling for NK-92 infiltration, the broad-spectrum MMP inhibitor Ilomostat (GM6001, available from Selleck Chemicals, catalog number S7157) was dissolved in Matrigel and medium at final concentrations of 2 micromolar (μM) and 10 μM.

[0086] result Target cell killing is delayed as the infiltration distance of NK cells increases As MCF-7-red target cells adhere, proliferate, and stabilize at confluence, impedance increases over time (Figure 1A). Addition of NK-92 cells results in a decrease in impedance as adherent target cells disappear during death. This disappearance of target cells takes longer in the presence of Matrigel and is delayed with increasing volumes, representing longer invasion distances (Figure 1A, data representing 50, 75, and 100 μL).

[0087] Image analysis demonstrates delayed invasion and death with increasing invasion distance Images were acquired at the focal plane of tumor cells adhered to the bottom of the wells of the E-view plate (Figure 1B). Consistent with the impedance readings, a gradual decrease in red fluorescence was detected after the addition of NK-92 cells, accompanied by the disappearance of MCF-7-red cells. In wells containing NK-92, green fluorescence increased over the course of the assay. Importantly, the kinetics of the disappearance of MCF-7 (red) cells and the increase in green staining (eLive Green) after NK addition was delayed with increasing Matrigel volume (invasion distance).

[0088] MMP inhibition delays target cell killing by NK cells MMPs have been suggested to play an important role in lymphocyte infiltration. As described herein, inhibition of MMPs slows infiltrating NK-92 cells and therefore slows target cell killing. Impedance readings (Figure 3A) revealed that Ilomostat (2 μM and 10 μM) delayed target cell killing. Image analysis (Figure 3B) confirmed that inhibition of MMP function delayed the disappearance of MCF-7 (red) and increased eLive Green-stained cells. The time it took for impedance to return to levels at the time of NK-92 addition (normalization time point) gradually increased from 46 h at 50 μL / well to 72 h at 110 μL / well after NK addition to the wells. The cell lysis rate at the 72-h time point of the assay (Figure 3C) highlights the increased invasion distance and delayed NK-mediated killing in the presence of MMP inhibitors.

[0089] The decrease in impedance reading due to target cell killing by infiltrating NK-92 cells slowed with increasing distance. Consistent with a role for MMPs in NK infiltration, broad-spectrum MMP inhibitors slowed the kinetics of target cell killing in the assay.

[0090] Live-cell imaging confirmed the increased invasion distance and delayed target cell killing in the presence of MMP inhibitors. Morphological changes in target cells were detected at approximately 45–55 h, suggesting that the distance effect is likely mediated by cytokines.

[0091] This assay demonstrates that the 96-well format of the XCELLIGENCE RTCA ESIGHT® Imaging and Sensing System can be adapted to systemically study immune and tumor cell interactions in the presence of ECM.

[0092] In this example, we assessed the ability of NK-92 cells to invade and kill tumor cells through various distances in Matrigel as a proof-of-concept to establish that this assay format allows for the simultaneous assessment of invasion and cytotoxicity in the presence of an ECM. Target cell killing is delayed as the NK cell invasion distance increases and in the presence of a broad-spectrum MMP inhibitor. Cytotoxicity results measured by impedance loss and live-cell imaging provide a proxy for the migration / invasive potential of NK-92 cells. Because the ECM can modulate the cellular responses of both infiltrating NK cells and resident tumor cells, this is useful for TME-focused research and the refinement of immunotherapy strategies. The platform described herein can be used to collect simultaneous real-time data on lymphocyte invasion of the extracellular matrix and target cell killing in a 96-well format.

[0093] [Example 2] Pemigatinib enhances the efficacy of EpCAM-CAR-T in squamous cell carcinoma Chimeric antigen receptor (CAR) T-cell therapy is generally more effective in hematological malignancies than in solid tumors. Given the complex tumor microenvironment, which plays a role in regulating lymphocyte responses and promoting tumor growth, combining it with other therapies may improve the response rate of CAR-T therapy in solid tumors.

[0094] Aberrant fibroblast growth factor (FGF) receptor (FGFR) signaling plays a critical role in the proliferation and survival of malignant epithelial cells. In this study, we investigate the use of FGF inhibitors in combination with CAR-T cells to target squamous cell carcinoma (SCC) cells in the presence of extracellular matrix (ECM). The ECM of solid tumors poses a challenge for lymphocyte infiltration and may serve as a reservoir of FGFs, which are involved in tumor survival and proliferation.

[0095] It was hypothesized that inhibition of FGFR signaling would result in reduced proliferation and increased killing of tumor targets, improving control of tumor growth by EpCAM-CAR-T cells.

[0096] material and method cell The A-431 human epidermoid carcinoma cell line (ATCC, catalog no. CRL-1555) was transduced with eLentiRed (Agilent Technologies, catalog no. 8711011) at a multiplicity of infection of 1 and cultured for 14 days in the presence of 2 μg / mL puromycin (InvivoGen, catalog no. ant-pr-1) to select for A431-red cells stably expressing nuclear-localized red fluorescent protein (RFP). A431-red cells were cultured in DMEM medium (Corning, catalog no. 10-013-CV) supplemented with 10% heat-inactivated FBS (Sigma, catalog no. 12106C-500ML) and 1% penicillin / streptomycin (Hyclone, catalog no. SV30010). This medium is referred to as c-DMEM in the procedure.

[0097] EpCAM-CAR-T cells were cultured in ImmunoCult™-XFT cell growth medium (Stemcell Technologies, catalog no. 10981) supplemented with 200 IU / ml rhIL-2 (Stemcell Technologies, catalog no. 78036). T cells were used for the assay four days after recovery.

[0098] A quick explanation of how Briefly, A431-red target tumor cells expressing nuclear-localized mKate2 (a red fluorescent protein) were overlaid with Matrigel (6 mg / mL) (50 μL / well). After 2 hours, epithelial cell adhesion molecule (EpCAM)-CAR-T cells were seeded onto the Matrigel layer. Tumor cell proliferation and CAR-T cytotoxicity were determined from real-time impedance and live-cell imaging data collected with the XCELLIGENCE RTCA ESIGHT® Imaging and Sensing System. The role of FGFR signaling was assessed using pemigatinib (1 μM, 5 μM, and 10 μM), an inhibitor of FGFR isoforms 1–3, sold under the trade name Pemazyre. The rate of cell lysis was calculated from normalized cell impedance and imaging (red fluorescence) data using the formula [(1-(treated / control)*100)].

[0099] ECM invasion and cytotoxicity assay The ability of EpCAM-CAR-T cells to invade Matrigel and kill A431-red target cells was assessed using impedance and imaging readout on the XCELLIGENCE RTCA ESIGHT® Imaging and Sensing System. Background impedance signals were measured in the wells of an E-plate VIEW microplate (Agilent Technologies, catalog number 0030060101030) using 50 μL of c-DMEM medium. A cell suspension of A431-red cells was prepared in c-DMEM at the desired cell number and dispensed at 100 μL per well. After tumor cell addition, the E-plate was left at room temperature for 30 minutes to ensure uniform cell distribution across the bottom. The plate was then returned to the cradle of the XCELLIGENCE RTCA ESIGHT® Imaging and Sensing System and data were acquired. Impedance readings were taken every 15 minutes, and images were taken every 60 minutes. Images were acquired in the brightfield and red fluorescent channels from four fields of view in each well. Exposure times were set to the default for brightfield and 150 ms for the red channel. Data were collected for 24 hours.

[0100] Matrigel (available from Corning, catalog number 354234) was thawed overnight at 4°C and kept on ice during preparation. Matrigel was prepared with pemigatinib (Selleckchem, catalog number S0088) at final concentrations of 1 μM, 5 μM, and 10 μM and dimethyl sulfoxide (D2650, Sigma-Aldrich) as a control. FBS (10% v / v) was added to the Matrigel, and the total protein concentration was adjusted to 6 mg / ml with DMEM.

[0101] Data collection was stopped, and the plate was removed from the cradle. The medium in the wells was aspirated, and Matrigel was layered on top of the A431-red cells. The plate was left at room temperature for 30 minutes and then incubated at 37°C / 5% CO2 for Matrigel polymerization. Impedance and imaging data were collected for 1 hour, during which time EpCAM-CAR-T cells were suspended in c-DMEM medium and the cell number adjusted to the desired E:T in 100 μL. The T cell suspension (100 μL) was carefully dispensed on top of the Matrigel. The plate was returned to the XCELLIGENCE RTCA ESIGHT® Imaging and Sensing System cradle, and data acquisition resumed.

[0102] Data analysis The software generated real-time plots of impedance data, including time (X-axis) and cell index (CI, Y-axis). The cell index was normalized to the time point before the addition of CAR-T cells. The normalized cell index (NCI(t)) was calculated by dividing the CI at time point t (CI(t)) by the CI at the normalized time point (CI(normalized)). Therefore, the NCI at the normalized time point was set to 1.0 by default. Image analysis was performed by default to generate red object counts per well. The cell lysis rate was plotted using the immunotherapy module of the software. The cell lysis rate was calculated from the NCI (impedance) using the formula [(1 - (CAR-T treatment / pemigatinib treatment) * 100]. The cell lysis rate was calculated from the red object counts / well (imaging) using the formula [(1 - (CAR-T treatment / pemigatinib treatment) * 100].

[0103] result Figures 5A-5G show that pemigatinib enhances the efficacy of EpCAM-CAR-T in squamous cell carcinoma, according to embodiments of the present disclosure, suggesting that pemigatinib can be effectively combined with CAR-T therapy for solid tumors with aberrant FGFR signaling.

[0104] Figure 5A plots EpCAM expression on the cell surface of various cells. Preliminary data from invasion-cytotoxicity assays suggest that EpCAM-CAR-T cells are less effective against A431 cells. Therefore, we hypothesize that FGFR signaling plays a role in A431 cell proliferation and survival in the presence of Matrigel / ECM, and that pemigatinib (Pemazil, Incyte) inhibits FGFR1-3 isoforms.

[0105] Figure 5B shows that the normalized cell index plotted over time demonstrates that FGFR1 inhibitors reduce A431 cell proliferation, and pemigatinib concentration-dependently slows A431 cell proliferation in native DMEM medium.

[0106] Figure 5C plots EpCAM-CAR-T targeting of A431 cells in a cytotoxicity assay (Figure 5C), demonstrating that EpCAM-CAR-T cells induce cytolysis of A431 cells cultured in DMEM medium (E:T 3.5). A431 cell lysis accelerates in the presence of pemigatinib (5 µM and 10 µM). This data suggests that reduced proliferation aids in efficient killing of target cells. E:T is calculated based on the CAR-expressing T cells in the transduced T cell population.

[0107] The plot in Figure 5D shows that EpCAM CART (E:T ∼3.5) has no effect on A431 cells growing in Matrigel, and EpCAM-CAR-T cells are unable to kill A431 cells (E:T 3.5) cultured in Matrigel in the absence of pemigatinib.

[0108] The plot in Figure 5E shows that CAR-T cells effectively controlled A431 cell proliferation in the presence of FGFRi (even though EpCAM CAR-T cells (E:T approximately 3.5) had no effect on A431 cells in Matrigel), and that pemigatinib concentration-dependently reduced A431 cell proliferation in the presence of Matrigel and improved the outcome of cell lysis by EpCAM-CAR-T cells (E:T 3.5).

[0109] The plots in Figure 5F show that the cytolysis rate calculated from impedance readings and live-cell imaging data confirms the increased potency of EpCAM-CAR-T cells in the presence of pemigatinib (top panel shows cytolysis rate plotted from normalized cell index (NCI) and bottom panel shows normalized red fluorescence counts / well). These results indicate that pemigatinib concentration-dependently reduces A431 cell proliferation and improves cytolysis by EpCAM-CAR-T cells (E:T 3.5).

[0110] Figure 5G shows a representative image taken at 100 hours of the assay from which the data plotted in Figures 5B-5F were derived, supporting the hypothesis made with respect to Figure 5A.

[0111] To select A431-red cells stably expressing nuclear-localized red fluorescent protein (RFP), the exemplary assay and its analysis shown in Figures 5A-5F were performed using the A431 human epidermoid carcinoma cell line transduced with eLentiRed at a multiplicity of infection of 1 and cultured for 14 days in the presence of 2 μg / mL puromycin. A431-red cells were cultured in DMEM medium (sometimes referred to as c-DMEM) supplemented with 10% heat-inactivated FBS and 1% penicillin / streptomycin.

[0112] As demonstrated in assays using the methodology described herein, pharmaceutical compounds with improved therapeutic and preventive properties have been discovered. These methodologies can be used to identify other pharmaceutical compounds with improved therapeutic and preventive properties for treating various conditions in biological subjects. As shown herein, pemigatinib can be effectively combined with CAR-T therapy to treat solid tumors with aberrant FGFR signaling. This conclusion is based, at least in part, on results showing that A431 cells express EpCAM and that EpCAM-CAR-T cells effectively eliminate these tumor cells in cytotoxicity assays. CAR-T is ineffective in the presence of Matrigel (effector:target, E:T = 4:1). Because FGF signaling can promote the survival and proliferation of squamous cell carcinoma cells, the effects of pemigatinib, a potent FGFR inhibitor, were tested. Pemigatinib reduced A431 cell proliferation as the concentrations tested increased. Assessment of CAR-T killing revealed that cell lysis rates increased from baseline (approximately 6%) to approximately 20% in the presence of 5 μM pemigatinib and approximately 40% in the presence of 10 μM pemigatinib. Complete elimination of A431 cells in the invasion assay was achieved at a very high E:T ratio of 20:1 and 1 μM pemigatinib.

[0113] [Example 3] Real-time Co-culture Assay for Immune Cell Infiltration and Cytotoxicity Using the XCELLIGENCE RTCA ESIGHT Imaging and Sensing System Immune cells migrate from blood vessels, infiltrate tissues, and perform effector functions that play pivotal roles in tumor immunosurveillance. These capabilities are also exploited by genetically engineered cytotoxic natural killer (NK) and CAR (chimeric antigen receptor) T cells used in cancer immunotherapy.

[0114] Although cellular immunotherapy has demonstrated efficacy in hematological cancers, clinical responses are needed to improve in solid tumors. The complex tumor microenvironment (TME) in solid tumors regulates lymphocyte recruitment and function. A key challenge faced by lymphocytes in solid tumors is navigating the acellular space filled with the structural scaffolding of the extracellular matrix (ECM) during migration / invasion. This ECM is heterogeneous, and its components can regulate diverse cellular responses in both resident tumor cells and infiltrating lymphocytes.

[0115] Traditionally, invasion, migration, and cytotoxicity have been evaluated in various endpoint assays using transwell assay systems to predict in vivo function. The Boyden chamber is a conventional transwell arrangement widely used to evaluate migration and invasion. It consists of a cylindrical cell culture insert with a porous membrane embedded within the well of a standard cell culture plate. A cell suspension is added to the inner chamber of the insert and induced to migrate out through pores of various sizes (typically 3-12 µm) using chemoattractants in the outer chamber. The inner well may be coated with an ECM to assess invasion. Invaded / migrated cells are imaged and / or collected for quantification at predetermined time points. The Boyden chamber may also be modified to evaluate effector cell migration and cytotoxicity in endpoint assays.

[0116] This disclosure describes a novel real-time co-culture assay using the XCELLIGENCE RTCA ESIGHT® Imaging and Sensing System to examine immune cell infiltration and cytotoxicity, as shown in Figures 6A and 6B, without the need for collection of embedded cells for endpoint readout. In this setup, an ECM layer directly contacts target tumor cells, specifically allowing infiltrating lymphocytes to perform their corresponding cytotoxic functions, modulating the responses of both tumor targets and infiltrating lymphocytes. In a transwell-based approach, cytotoxicity of migrated immune cells is performed in an external chamber free of ECM.

[0117] This example demonstrates the evaluation of NK cell invasion and killing of target tumor cells as a proof-of-concept assay system. NK cells are innate lymphoid cells that kill infected, stressed, or transformed cells by using a series of activating and inhibitory receptors to identify and distinguish "altered" cells from healthy cells. This example uses NK-92 cells as a model to study invasion and cytotoxicity. The allogeneic NK derived NK-92 cell line is the first NK-based cellular immunotherapy to be granted Investigational New Drug status by the U.S. Food and Drug Administration.

[0118] It has been hypothesized that the Matrigel layer presents a challenge for NK-92 cells, and that extending their invasion distance delays tumor cell killing. NK cells also rely on proteases from the matrix metalloproteinase (MMP) family to degrade various components in the ECM during the invasion phase.

[0119] This example demonstrates that, depending on the needs of the user, the 96-well format can easily be used to systemically study the effects of various ECM components on tumor and immune cell interactions in the TME.

[0120] material and method cell The MCF-7 human breast adenocarcinoma cell line was transduced with eLentiRed at a multiplicity of infection of 1 and cultured for 14 days in the presence of 2 μg / mL puromycin to select for MCF7-red cells stably expressing nuclear-localized red fluorescent protein (RFP). MCF7 and MCF7-red cells were cultured in EMEM medium supplemented with 10% heat-inactivated FBS and 1% penicillin / streptomycin.

[0121] NK-92 cells were cultured in MyeloCult H5100 medium supplemented with 30 ml of horse serum, 600 IU / ml of rhIL-2, and 1% penicillin / streptomycin.

[0122] eGFP-NK92 cells were grown in X-VIVO15 supplemented with 5% human serum, 500 IU / ml rhIL-2, and 0.5 μg / ml puromycin.

[0123] ECM invasion and cytotoxicity assay The ability of NK-92 cells to invade Matrigel and kill MCF7-red target cells was assessed by simultaneous impedance and imaging readout on the XCELLIGENCE RTCA ESIGHT® Imaging and Sensing System, as outlined in Figure 6A. Background impedance signals were measured in 50 μL of EMEM medium in the wells of an E-plate VIEW microplate. MCF7-red target cells (30,000 cells in 100 μL) were added to the wells and placed at room temperature for 30 minutes to ensure uniform distribution across the bottom. The plate was then placed back into the cradle of the XCELLIGENCE RTCA ESIGHT® Imaging and Sensing System, and data were acquired. Impedance readings were taken every 15 minutes, and images were captured every 60 minutes. Images were acquired in the brightfield, red, and green fluorescent channels from four fields of view in each well. Exposure times were set to the default settings for brightfield, 150 ms for the red channel, and 300 ms for the green channel, respectively. Matrigel was thawed overnight at 4°C, diluted with DMEM, and supplemented with 10% FBS to a final total protein concentration of 6 mg / ml. After 24 hours, data collection was discontinued. The medium in the wells was aspirated, and various volumes of Matrigel (50, 75, and 100 μL, data shown herein) were layered on top of the MCF7-red cells and incubated at room temperature for 30 minutes, followed by another 30 minutes at 37°C / 5% CO2 to allow the Matrigel to polymerize. Impedance and imaging data were collected for 1 hour, during which time NK-92 cells were suspended in EMEM medium and adjusted to a cell number of 3:1 E:T in 100 μL. The NK-92 cell suspension (100 μL) was layered on top of the Matrigel, bringing the total volume in all wells to 200 μL. The plate was then returned to the cradle, and data acquisition resumed. The percentage of cell lysis was calculated from the normalized cell impedance readings using the formula [(1-(treated / untreated)*100].

[0124] Kinetics of MMP inhibition and NK infiltration To assess the role of MMP-dependent NK-92 infiltration, the broad-spectrum MMP inhibitor, Ilomostat, was dissolved in Matrigel and medium at final concentrations of 2 μM and 10 μM. The percentage of cell lysis was calculated from normalized cell impedance readings and red object count data using the formula [1 - (NK92 + MMPi / MMPi treatment) * 100].

[0125] result Target cell killing is delayed as the infiltration distance of NK cells increases As MCF7-red target cells adhere and proliferate, reaching confluence as shown in Figure 7A, impedance increases and stabilizes over time. Addition of Matrigel (50 μL) modulates the impedance signature of MCF-7 cells and, importantly, delays cell lysis by NK-92 cells added after 24 h. As shown in Figure 7B, which shows representative data from 50, 75, and 100 μL, the decrease in impedance due to cell lysis is further delayed as the volume of Matrigel increases. As shown in Figure 7C, the KT60 (time to achieve 60% killing relative to control) was 67, 76, and 89 h, respectively.

[0126] MMP inhibition delays target cell killing by NK cells Because cell lysis is delayed as the NK cell invasion distance increases, MMPs are hypothesized to play a critical role in lymphocyte infiltration. Consistent with the function of MMPs in invasion, cell lysis rates calculated from normalized impedance readings (Figure 8A) and live cell imaging (e.g., red fluorescence) (Figure 8B) confirmed both delayed and reduced target cell killing by GFP-NK92 cells in the presence of Ilomostat (2 μM and 10 μM). Image analysis, performed simultaneously with impedance measurements, confirmed the kinetics of cell killing.

[0127] Image analysis demonstrates delayed invasion and death with increasing invasion distance As shown in Figure 9, representative images of MCF-7 clusters reveal increased aggregation and cell death in response to GFP-NK92. Despite increased tumor cell death, little GFP-NK92 was detected in the imaged area. Interestingly, highly active NK cells with multiple contacts with various MCF-7red targets were detected within the clusters, suggesting sequential killing.

[0128] Recapitulating the characteristics of the tumor microenvironment within solid tumors will play a crucial role in developing and improving cellular immunotherapy strategies. Dysregulation of the ECM in the tumor microenvironment of solid tumors has been extensively studied for its ability to modulate diverse cellular responses. In this regard, efficient in vitro assays to evaluate tumor and immune cell responses in the presence of ECM are needed.

[0129] The ability of NK and other immune cells to invade the extracellular space and reach target cells depends on their ability to degrade the ECM. Various components of the ECM can modulate NK cell function. Therefore, as described herein, the ability of NK-92 cells to invade a Matrigel layer and kill target cells merits evaluation. Cytotoxicity results measured by impedance loss and live-cell imaging serve as surrogates for the migration / invasion potential of NK-92 cells. This assay format allows for the simultaneous evaluation of invasion and cytotoxicity in the presence of ECM by demonstrating that varying the invasion distance through Matrigel can delay target cell killing.

[0130] NK cells can express multiple MMPs, and extravasation assays have shown that NK-92 invasion into Matrigel is reduced in the presence of the MMP inhibitor GM6001. Typically, studies assess invasion / migration potential and cytotoxic function separately using complex workflows, as is the case with studies evaluating the effects of MMP inhibitors. Alternatively, some studies have assessed both migration and cytotoxicity using complex and cumbersome experimental setups. As described herein, live-cell imaging and real-time impedance readouts demonstrate the involvement of MMPs in NK-92 invasion-cytotoxicity assays. Consistent with the role of MMPs in facilitating invasion, target cell killing is delayed by broad-spectrum MMP inhibitors.

[0131] Although imaging and impedance data were consistent in the invasion-cytotoxicity assay, relying solely on imaging data can be challenging. Tumor cells respond differently to Matrigel, forming irregular, disorganized clumps as MCF-7 target cells undergo cell death, as highlighted in the representative images. Surprisingly, rather than discovering a cluster of infiltrating NK cells, only a small number of NK cells were detected within and around the MCF clusters. Nevertheless, images and videos collected in these assays show highly active GFP-NK92 cells making multiple contacts with various targets within the cluster, triggering cell death of those they contact over the course of the assay.

[0132] The novel real-time co-culture assay described in this disclosure demonstrates the utility of the XCELLIGENCE RTCA ESIGHT® imaging and sensing system for the simultaneous assessment of lymphocyte infiltration and cytotoxic function, which is essential for immunotherapy in solid tumors.

Claims

1. 1. A method for assessing cytolysis of cancer cells by effector cells, comprising: providing a cell-substrate impedance monitoring device operably connected to an impedance analyzer, the device including a well for receiving cells and an electrode array at the bottom of the well, the device further operably connected to an imaging unit; adding target cells characterized as cancer cells to the wells; disposing a layer comprising extracellular matrix (ECM) over the target cells; adding effector cells onto the ECM layer; imaging the well and monitoring the cell-substrate impedance of the well to determine the infiltration of the effector cells through the ECM layer and the effectiveness of the effector cells in killing the target cells directly or via migration and invasion through the extracellular matrix; A method comprising:

2. The method of claim 1 , further comprising an imaging device positioned adjacent to the well.

3. The method of claim 1 or 2, wherein the cancer cells are derived from a solid tumor.

4. The method of any one of claims 1 to 3, wherein the effector cells are natural killer (NK) cells.

5. The method of claim 1 , wherein the effector cells are T cells.

6. measuring cell-substrate impedance between target cells and effector cells separated by a layer of extracellular matrix (ECM) at various times during the assay; acquiring images of the effector cells and the target cells at various time points during the assay; determining the effectiveness of the effector cells against the target cells based on changes in the cell-substrate impedance and the image during the assay period.

7. applying a first dye of a first color to the target cells; and 7. The method of claim 6, further comprising applying a second dye of a second color different from the first color to the effector cells.

8. 8. The method of claim 6 or 7, further comprising varying the thickness of the ECM within multiple wells of a multi-well plate in which the target cells and the effector cells are disposed.

9. applying a pharmaceutical compound of interest to one or more of said target cells, said ECM, and said effector cells; comparing a baseline efficacy of the effector cells against the target cells without application of the pharmaceutical compound of interest to an experimental efficacy of the effector cells against the target cells with application of the pharmaceutical compound of interest; 9. The method of any one of claims 6 to 8, further comprising the step of using the pharmaceutical compound of interest for the treatment or prevention of a disorder associated with the target cell in a biological subject in response to the experimental efficacy meeting an efficacy threshold.

10. applying a first pharmaceutical compound of interest to one or more of said target cells, said ECM, and a first subset of said effector cells; applying a second pharmaceutical compound of interest to a second subset of one or more of the target cells, the ECM, and the effector cells, distinct from the first subset; comparing a first efficacy of the effector cells against the target cells pulsed with the first pharmaceutical compound of interest to a second efficacy of the effector cells against the target cells pulsed with the second pharmaceutical compound of interest; 9. The method of any one of claims 6 to 8, further comprising the step of: using the first pharmaceutical compound of interest for the treatment or prevention of a disorder associated with the target cell in a biological subject in response to the first pharmaceutical compound of interest having greater efficacy than the second pharmaceutical compound of interest.

11. 11. The method of any one of claims 6 to 10, wherein the target cells are characterized as cancer cells.

12. 12. The method of claim 11, wherein the cancer cells are from a solid tumor.

13. 13. The method of any one of claims 6 to 12, wherein the effector cells are natural killer (NK) cells.

14. The method of any one of claims 6 to 12, wherein the effector cells are T cells.

15. 1. An FGFR inhibitor for use in a method for treating a solid tumor in a subject in which FGFR signaling is aberrant, in combination with CAR therapy.

16. 16. The FGFR inhibitor for use according to claim 15, wherein the FGFR inhibitor comprises pemigatinib.

17. 17. The FGFR inhibitor for use according to claim 15 or 16, wherein the FGFR inhibitor is administered before the CAR therapy is administered.

18. 17. The FGFR inhibitor for use according to claim 15 or 16, wherein said FGFR inhibitor is administered simultaneously with the administration of said CAR therapy.

19. 17. The FGFR inhibitor for use according to claim 15 or 16, wherein the FGFR inhibitor is administered after the CAR therapy has been administered.

20. The FGFR inhibitor for use according to any one of claims 15 to 19, wherein the CAR therapy is CAR-T therapy.

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