WO2025050133A1

WO2025050133A1 - Isolation and expansion of natural killer cells using fluorescence-activated droplet sorting

Info

Publication number: WO2025050133A1
Application number: PCT/US2024/045065
Authority: WO
Inventors: Tania KONRY; Jose ESTEVAM; Rachel White
Original assignee: Northeastern University China; Northeastern University Boston
Current assignee: Northeastern University China; Northeastern University Boston
Priority date: 2023-09-01
Filing date: 2024-09-03
Publication date: 2025-03-06
Anticipated expiration: 2026-03-01

Abstract

A high-throughput microfluidics-based fluorescence activated droplet sorting (FADS) system provides activation, sorting, and feeder cell-free expansion of NK cells that are enriched for killing activity. The NK cells are activated and identified based on their ability to kill target cells, such as tumor cells, without prior sensitization. The diversity of endogenous NK cells can be overcome to allow selection of a highly effective population of cells for therapeutic use, such as in the treatment of cancer.

Description

TITLE

Isolation and Expansion of Natural Killer Cells

Using Fluorescence-Activated Droplet Sorting

STATEMENT REGARDING FEDERALLY SPONSORED

RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Number CBET- 1803872 awarded by the National Science Foundation. The Government has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 63/536,327, filed 1 September 2024, which is hereby incorporated by reference in its entirety.

BACKGROUND

NK cells are a type of innate immune cell that can recognize and kill cells displaying non-self antigens including cancer cells without previous exposure to cancer antigens, are of increasing interest in cancer immunotherapeutic research (Bae et al., 2014); they offer an improved safety profile compared to T-cells, more specific anti-tumor effects, as well as less associated off-target complications (Laskowski et aL, 2022; Guillerey et al., 2016; Vivier et al., 2012). In several types of hematological malignancies, for example, the presence and activity of tumor-infiltrating NK cells is correlated with a better prognosis and reduction in metastatic risk (Melaiu et aL, 2020; Nersesian et aL, 2021 ; Cozar et aL, 2021 ). However, the immunoinhibitory nature of the tumor microenvironment leads to a lower total number of NK cells in tumor tissue compared to healthy tissue, and it also suppresses NK cell effector function (Du et aL, 2021 ; Laskowski et aL, 2022). Therefore, NK cell immunotherapies to supplement patients’ own immune systems provide a promising therapeutic approach for cancer treatment. Nevertheless, clinical application of NK cell immunotherapies has been /limited due to the difficulty of expanding NK cells and poor functional activity of the resultant expanded NK cell population; this is believed to be due in large part to the heterogenous nature of these cells (Hodgins et aL, 2019).

Feeder-cell systems and feeder-free systems are currently available for NK cell expansion (Llames et aL, 2015; Johnson et aL, 2022; Gurney et aL, 2022). Feeder cell systems use viable, adherent cell lines, such as K562 cells, that have been growth arrested and genetically modified to express membrane-bound IL-21 to support the expansion of NK cells (Llames et aL, 2015; Ojo et aL, 2019). The feeder cells and the NK cells are co-cultured together and, in this environment, feeder cells assist NK cell proliferation by providing extracellular secretions of growth factors, removing toxic substances from the cell culture medium, as well as synthesizing matrix proteins (Llames et aL, 2015). The addition of cytokines to this co-culture allows for in-vitro NK cell activation, thus producing NK cells with cancer cell targeting and killing capabilities (Chu et aL, 2022). While this methodology allows for efficient expansion of NK cells; there are safety concerns regarding the clinical application of cancer cell-derived feeder cells, as well as difficulty in removing non-NK cells following expansion (Llames et aL, 2015; Johnson et aL, 2022; Ojo et aL, 2019). To address these limitations, feeder-free systems employing a combination of cytokines and antibodies have been developed, but their expansion efficiency is lower and the purity of the resulting NK cells range from 30% to 90% (Gurney et aL, 2022; Fang et aL, 2019). The reasons for the heterogeneity within an expanded cell system are complex, but differences in cell functionality appear to play a role (Adams et aL, 2021 ; Subedi et aL, 2022). These inconsistencies make it difficult to assess the clinical efficacy of cell therapy products and, because of this heterogeneity, outcomes observed in clinical trials can be highly variable (Liu et aL, 2021 ; Lamers-Kok et aL, 2022). Therefore, methods are needed to to reduce the heterogeneity of expanded NK cells and to improve the efficacy of their production for use as NK cell therapeutics.

Current NK cell therapeutics include autologous NK cells, allogeneic NK cells, and CAR NK cells. Autologous therapies involve transforming and expanding the patient’s NK cells ex vivo and re-infusing them into the patient for treatment (Chu et aL, 2022). In contrast, allogeneic therapies use ex vivo expansion of donor NK cells for an “off-the-shelf’ approach that is generally more time efficient and cost effective (Franks et aL, 2020). CAR NK cells, which are engineered with chimeric antigen receptors (CAR) designed with antigen recognition domains for binding to specific antigens on target tumor cells and co-stimulatory domains for activation and proliferation, have shown success in treating hematological cancers such as leukemia and lymphoma (Moscarelli et al. 2022, Pan et al., 2022, Khawar et al., 2021 ). The current NK therapeutic approaches offer an improved safety profile compared to CAR T cell therapies, yet they have limitations in persistence and survival within the patient (Pan et aL, 2022, Khawar et al., 2021 ). Improved methods are needed to enhance NK cell proliferation, activation, and tumor cell targeting capabilities. Typical NK cell therapy dosing 5 to 50 million NK cells per kilogram of patient body weight for each dose, across a multiple-dose regimen, which poses significant challenges for existing expansion methods (Fang et aL, 2019, Lapteva et aL, 2014).

SUMMARY

The present technology provides a high-throughput microfluidics-based fluorescence activated droplet sorting (FADS) system to activate, sort, and expand NK cells that are enriched for killing activity. The NK cell “killers” are activated and identified based on their ability to kill target cells, such as tumor cells, without prior sensitization. The present technology can assist in overcoming NK cell diversity to allow selection of a highly effective population of cells for therapeutic use, such as in the treatment of cancer.

The present technology also can be summarized with the following list of features.

1. A method for enriching a population of natural killer (NK) cells with active NK cells, the method comprising:

(a) providing

(i) a microfluidic system configured for co-encapsulating single cells in aqueous microdroplets in an oil stream and sorting the microdroplets based on fluorescence of labeled cells within the microdroplets;

(ii) a population of single NK cells loaded with an intracellular Cabsensitive fluorescent indicator; and

(iii) a population of single target cells;

(b) co-encapsulating the single NK cells and single target cells at a ratio of about 1 :1 in aqueous microdroplets in an oil stream using the microfluidic system;

(c) allowing the NK cells and target cells to interact in the microdroplets, whereby some of the NK cells become activated by interaction with the co- encapsulated target cells and produce a fluorescence signal from the intracellular Ca²⁺-sensitive fluorescent indicator;

(d) sorting the microdroplets based on a level of said fluorescence signal in the activated NK cells, yielding a first group of microdroplets containing activated NK cells with fluorescence level at or above a selected threshold and a second group of microdroplets containing non-activated NK cells with fluorescence level below the selected threshold; and

(e) collecting the activated NK cells from the first group, thereby producing a population of NK cells that is enriched in active killer cells compared to the initially provided NK cells.

2. The method of feature 1 , wherein target cells co-encapsulated with NK cells are killed, and the population of enriched active NK cells produced in (e) is essentially free of living or intact target cells.

3. The method of feature 1 or 2, wherein the single target cells are tumor-derived cells.

4. The method of any of the preceding features, wherein the intracellular Cabsensitive fluorescent indicator is Fura-10.

5. The method of any of the preceding features, wherein analysis of cytotoxic activity of the enriched NK cells is performed using a lactate dehydrogenase (LDH) assay.

6. The method of any of the preceding features, further comprising

(f) expanding the enriched population of active NK cells by performing one or more rounds of proliferation of the enriched population of active NK cells in culture.

7. The method of feature 6, wherein the expansion in (f) is completed in 21 days or less.

8 The method of feature 6 or 7, wherein the expansion in (f) is carried out without the use of feeder cells.

9. The method of any of features 6-8, further comprising one or more of (i) analysis of cytotoxic activity of the expanded NK cells, (ii) re-evaluation of target cell killing activity of the expanded NK cells using another fluorescence-activated droplet sorting process, or (iii) transcriptomic analysis of the expanded NK cells.

10. The method of feature 9, wherein (ii) re-evaluation of target cell killing activity of the expanded NK cells is carried out by a fluorescence-activated droplet sorting process wherein (a) target cells are loaded with a fluorescence-based indicator of target cell viability, such as a fluorogenic substrate for caspase 3 or caspase 7, or (b) NK cells are loaded with an intracellular Ca²⁺-sensitive fluorescent indicator.

11. A method for enriching a population of NK cells in active NK cells, the method comprising the steps of:

(a) providing

(ii) a population of single NK cells; and

(iii) a population of single target cells loaded with a fluorescence-based indicator of target cell viability;

(c) allowing the NK cells and target cells to interact in the microdroplets, whereby target cells that become apoptotic by interaction with NK cells produce a fluorescence signal from said indicator;

(d) sorting the microdroplets based on their level of fluorescence in (c), yielding a first group of microdroplets with fluorescence level indicative of apoptotic or dead cells and a second group of microdroplets with fluorescence level indicative of non-apoptotic or live cells, wherein said first group of microdroplets contain apoptotic or dead target cells and active NK cells; and

(e) collecting active NK cells from the first group of microdroplets, thereby producing a population of NK cells that is enriched in active killer cells compared to the initially provided NK cells.

12. The method of feature 11 , wherein target cells co-encapsulated with NK cells are killed, and the population of enriched active NK cells produced in (e) is essentially free of living or intact target cells.

13. The method of feature 11 or 12, wherein the single target cells are tumor- derived cells.

14. The method of any of features 11-13, wherein the fluorescence-based indicator of target cell viability is a fluorogenic caspase substrate which serves as an indicator of caspase 3 or caspase 7 activity.

15. The method of feature 14, wherein the fluorogenic caspase substrate is a peptide derivative of cresyl violet. 16. The method of any of features 11-15, further comprising testing cytotoxic activity of the enriched NK cells, such as by using a lactate dehydrogenase (LDH) assay.

17. The method of any of features 11-16, further comprising

18. The method of feature 17, wherein the expansion in (f) is completed in 21 days or less.

19. The method of feature 17 or 18, wherein the expansion in (f) is carried out without the use of feeder cells.

20. The method of any of features 17-19, further comprising one or more of (i) analysis of cytotoxic activity of the expanded NK cells, (ii) re-evaluation of target cell killing activity of the expanded NK cells using another fluorescence-activated droplet sorting process, or (iii) transcriptomic analysis of the expanded NK cells.

21. The method of feature 20, wherein (ii) re-evaluation of target cell killing activity of the expanded NK cells is carried out by a fluorescence-activated droplet sorting process wherein (a) target cells are loaded with a fluorescence-based indicator of target cell viability, such as a fluorogenic substrate for caspase 3 or caspase 7, or (b) NK cells are loaded with an intracellular Ca²⁺-sensitive fluorescent indicator.

22 A method of immunotherapy of a subject in need of NK cell supplementation, the method comprising:

(a) obtaining a population of enriched and expanded NK cells using the method of any of features 6-10 or 17-21; and

(b) administering at least a portion of the population of enriched and expanded NK cells to the subject.

23. The method of feature 22, wherein (a) comprises: obtaining a population of NK cells from the subject, and using the population of the subject’s NK cells as the initial population of single NK cells in the method of expansion of active NK cells.

24 The method of feature 22 or feature 23, wherein (a) comprises: obtaining a population of single target cells from the subject, and using the population of the subject’s target cells as the single target cells in the method of enriching and expanding NK cells. 25. The method of feature 22 or 23, wherein the single target cells are not derived from the subject.

26. The method of any of features 22-25, wherein the subject has cancer, and the method is used for cancer therapy of the subject.

27. A method of performing transcriptomic analysis on a population of active NK cells, the method comprising:

(a) sorting and enriching a population of NK cells according to the method of any of features 1-21 , thereby obtaining an enriched population of active NK cells;

(b) isolating mRNA from a pool of the enriched population of active NK cells;

(c) performing RNA sequencing on the isolated mRNA; and

(d) analyzing the sequenced RNA from (c) to determine a level of expression of one or more genes of the sorted and enriched population of active NK cells.

28. A kit comprising a microfluidic device and instructions for performing the method of any of the preceding features.

29. The kit of feature 28 further comprising one or more reagents, such as a fluorescent indicator for intracellular Ca²⁺, a fluorogenic caspase substrate, one or more LDH assay reagents, one or more reagents for NK cell expansion, one or more reagents for transcriptomics analysis, or a reagent for specifically detecting an NK cell biomarker.

30. A population of NK cells obtained using the method of any of features 1 -27 or the kit of feature 28 or 29.

31 . The population of NK cells of feature 30 comprising at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% active NK cells.

32. The population of NK cells of feature 30 or 31 containing a total of at least 20 million NK cells.

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 shows a schematic illustration of a process of activation of NK cells by co-encapsulation with target cells in microfluidic droplets, enrichment of activated NK cells by fluorescence activated droplet sorting, and expansion of the enriched NK cells in culture, followed by analysis of the enriched NK cell population.

Fig. 2 shows a flowchart of a process for activation, sorting, and expansion of active NK cells. DETAILED DESCRIPTION

The present technology applies a high-throughput microfluidics-based fluorescence activated droplet sorting (FADS) system to activate, sort, and expand NK cells that are enriched for killing activity. The NK cell “killers” are activated and identified based on their ability to kill target cells, such as tumor cells, without prior sensitization as required by T cell killers. NK cells are known to be phenotypically diverse, with some being effective at killing cells recognized as non-self and others being less effective or ineffective. The present technology can assist in overcoming NK cell diversity to allow selection of a highly effective population of cells for therapeutic use, such as in the treatment of cancer. The ability to pre-select those cells that are active killers makes it possible to then expand those cells and achieve a substantial number of active, more homogeneous NK cells for therapeutic use. A particular advantage of the present technology is the ability to expand active NK cells without the use of feeder cells, such that the final expanded population is free of foreign cells, cancer-derived cells, and mutated cells. The technology also offers an advantage over other feeder-free techniques in that the original population of NK cells is enriched by selection for actively killing cells prior to expansion. Further, because the cells are known to have a predominantly “active killer” phenotype by virtue of the activation and sorting process carried out on a cell-by-cell level, the analysis of the isolated cells can be extended to include transcriptomics, genomics, epigenetics and signal transduction or metabolic pathways at the level of a few cells or even single cells and to relate the findings to NK cell function. That is, the method can support analysis over the full “function-to-omics” spectrum.

Fig. 1 presents an overview of an embodiment of a process for activation, enrichment by function, and expansion of NK cells. Initially, a heterogeneous population of primary NK cells obtained from a donor undergo fluorescence-activated droplet sorting (FADS) process to produce a first population of activated killer cells, which effectively target and kill co-encapsulated target cells, such as K562 tumor cells, and a second population of non-kil ler cells which do not. In preparation for FADS, NK cells are co-encapsulated with target cells at an approximately 1 :1 ratio in aqueous microdroplets 101 in an oil stream in microfluidic device 102. In one variant of the method, the NK cells are preloaded with a fluorescent indicator for intracellular calcium ion concentration, such as Fura-10 or another Ca²⁺-sensitive fluorescent dye. In another variant of the method, the target cell is preloaded with a fluorescent indicator capable of signaling the death of the target cell, e.g., by apoptosis or membrane permeabilization. Droplets containing co-encapsulated cells can be collected from the device into container 103, which is moved to an incubator for several hours to allow cell-cell interactions and target cell killing to take place. After the incubation period, the droplets are introduced into second microfluidic device 104, which contains window 105 for illuminating individual cells with light for exciting the fluorescent indicator as individual cells stream towards a sorting junction under control by electrodes 106. Detection of fluorescent emission above a pre-selected threshold triggers the electrodes to provide a high voltage AC signal that causes selected droplets to become charged through dielectrophoresis and move into sorting or collection channel 107 and then to a collection vessel. Droplets that do not exhibit fluorescence emission above the threshold continue down non-sorting collection channel 108. Sorted droplets are then broken and their NK cells transferred to a cell culture container with culture medium. In the depicted embodiment, cells are first allowed to proliferate in 96-well plate 109 for 10 days and then transferred to 6-well plate 110 for an additional 11 days of further proliferation.

Following FADS and expansion, the enriched and expanded effective killer cell population can be optionally characterized using one or more of LDH assay 111 (for quantification of target cell killing activity), transcriptom ic analysis 113 to elucidate upregulated and downregulated genes associated with the population, and another round of FADS 112 to re-assess their target cell cytolytic capabilities following expansion.

Fig. 2 illustrates the activation, enrichment, and expansion in flowchart format.

In an embodiment, FADS technology is used to sort a population of NK cells into distinct subpopulations based on changes in intracellular calcium signaling, as calcium signaling is an indicator of NK cell activation. In this procedure, NK cells are labeled with an intracellular calcium-binding fluorescent dye, co-encapsulated with K562 target tumor cells within droplets, and sorted based on measured calcium response using FADS. NK cells that produce a strong intracellular calcium signal indicate a high affinity for the target cells and are expected to demonstrate a strong cytotoxic response when co-encapsulated with target cells.

In another embodiment, NK cells are sorted based on their ability to initiate apoptosis in target cells, such as K562 cells. NK cells and K562 cells labeled with a fluorogenic substrate for caspase 3/7, used as a cell death indicator, are co- encapsulated within droplets and incubated in appropriate cell culture conditions to allow sufficient time for NK cells to kill K562 cells. Following incubation, the coencapsulations are separated using FADS based on whether the target tumor cell within the droplet was killed by the effector NK cell. This results in two populations of NK cells; a group of “killers” which are able to target and kill K562 cells, and a group of “non-killers” which are unable to do so. Both the calcium release and cell deathbased sorting approaches can be used to generate a homogenous subpopulation of NK cells with enhanced cytotoxicity.

FADS technology utilizes a microfluidic device with an on-chip module for generating aqueous microdroplets in an oil stream, an array of docking sites for incubating and observing the microdroplets (also referred to herein as “droplets”), and a sorting junction for sorting individual droplets based on a level of fluorescence within the droplet. The droplets generated contain co-encapsulated target and effector cells, in a pre-determined desired ratio, such as a 1 :1 ratio, for example. Prior to droplet generation, one cell type is labeled with a fluorescent indicator, whose level of fluorescence emission is which is set as a sorting parameter based on the sensitivity of the indicator (for example, cell death or intracellular calcium ion concentration). When sorting droplets, fluorescence intensity is measured as the droplets pass through a laser detection spot where they are excited by a laser with the appropriate wavelength for excitation of the type of fluorescent dye used. When the fluorescence emission intensity of a droplet has a value above a selected threshold value, the droplet registers as a “positive” droplet in a program used for data acquisition. This triggers a high-voltage AC pulse at the sorting junction, and a subsequent AC electric field results in a dielectrophoretic force that pulls the “positive” droplet into the collection channel. The droplets without fluorescence above the threshold do not trigger an AC pulse and therefore flow into the lower resistance waste channel.

The resultant population of enriched NK cell “killers” can be expanded using a feeder cell free culture medium, such as the CelIXVivo Human NK Cell Expansion Kit™ (R&D Systems). Other feeder cell-free media are known and several are commercially available for use in NK cell expansion. Typically such media contain mixtures of growth factors and cytokines selected to promote the proliferation of a given cell type. In the CelIXVivo Human NK Cell Expansion Kit™, the protocol expands cells extracted from droplets starting from about 2,000 cells/mL, which is compatible with the starting concentration of cells sorted using FADS. In an embodiment of the present technology, instead of utilizing a 75-cm² flask as outlined by the kit protocol, the protocol was adapted to expand NK cells in a 96-well plate, which enables expansion with lower starting concentrations. Additionally, the concentrations of the Cell Expander kit’s components in the media can be adapted based on the starting concentration of NK cells. A final cell count of 77,000 cells can be obtained from a starting count of 2,000 cells over a 3 week expansion period. The cytotoxicity of the expanded activated human NK cells against K562 cancer cells can be assessed using a commercial LDH kit and compared with the population of pre-sorted NK cells. This methodology allows for the expansion of exclusively highly active NK cells, which generates a population of homogeneous, highly active NK cells.

Further, the two populations of NK cells, “killers” and “non-killers”, generated from FADS can be analyzed by RNA sequencing. The “killers” and “non-killers” were sequenced using bulk RNA and single cell RNA sequencing following sorting. This allowed for the identification of genes that are upregulated or downregulated in one population versus the other. Further, this enables elucidation of specific molecular signatures, cellular pathways, and mRNA transcripts that result in enhanced tumor cell targeting and killing capabilities.

The following protocol can be used for sorting based on a fluorescent marker for the intracellular Ca²⁺ release that accompanies activation of NK cells by a target cell.

1. K562 cells, or other target cells, and primary NK cells are prepared in appropriate cell concentrations to achieve the desired 1 :1 co-encapsulation ratio within droplets.

2. NK cells are stained with Fura-10 AM, or another calcium release indicator.

3. Co-encapsulation of cells within droplets on the microfluidic device is achieved, and then the droplets are incubated briefly to allow NK cell activation.

4. Droplets are sorted using FADS, which separates the droplets into 2 distinct populations based on measured calcium response.

5. The separated populations then undergo cytotoxicity evaluation using a commercial LDH kit, as well as sequencing analysis.

The following alternative protocol can be used when sorting based on cell death of target cells.

1. K562 cells and primary NK cells are prepared in appropriate cell concentrations to achieve the desired 1 :1 co-encapsulation ratio. 2. K562 cells are stained with a fluorescent indicator for caspase 3/7 activity, a cell death (apoptosis) indicator.

3. Using a microfluidic device, K562 cells and primary NK cells are coencapsulated within aqueous droplets in oil. Droplets are dispensed into a conical mini-centrifuge tube containing mineral oil, which helps maintain droplet shape during incubation and allows for gas exchange.

4. Prepared droplets are incubated at 37°C for 16 hours to allow sufficient time for NK cells to target and kill K562 cells.

5. Following incubation, droplets are injected into a sorting device and sorted using FADS. The sorter separates the droplets into 2 distinct populations; NK cells that effectively killed target tumor cells (“killers”) and those that did not kill target tumor cells (“non-killers”).

6. These separated populations then undergo cytotoxicity evaluation using a commercial LDH kit, as well as expansion (part 2) or sequencing analysis.

The following protocol can be used for expansion of sorted “active killer” populations of NK cells:

1 . The day before droplet sorting, a 96-well plate is prepared using the protocol of the CelIXVivo Human NK Cell Expansion Kit™.

2. Following one of the sorting protocols described above, the sorted droplets containing NK cell killers are broken to release the NK cells from within the droplets.

3. The NK cells are then sedimented and resuspended in 200 microliters for a modified CelIXVivo Human NK Cell Expansion Kit. protocol. The concentration of the Cell Expander components for the media are adjusted based on the starting concentration of NK cells.

4. Using a modified version of the CelIXVivo Human NK Cell Expansion Kit, the NK cells are expanded over a 20-day period to yield sufficient cell counts for cytotoxicity assessment. During the 20-day period, media exchanges are performed in accordance with the protocol.

5. Following expansion, the expanded population then undergoes cytotoxicity evaluation using a commercial LDH kit.

The following protocol can be used for transcriptom ic analysis of sorted NK cell populations: 1. Following sorting of an NK cell population as described above, the RNA from separate populations is extracted and submitted for sequencing. Following the calcium release protocol above, both the calcium negative and the calcium positive populations undergo RNA extraction and bulk sequencing. Following target cell death protocol above, both the killer and the non-killer populations undergo RNA extraction and bulk sequencing. In addition to sequencing cells obtained directly after sorting (i .e. , without expansion), NK cells also underwent RNA extraction and sequencing following the expansion protocol described above.

2. With the sequencing results, a standard bioinformatic analysis pipeline can be employed (QC, mapping/alignment, statistical analysis) to identify differentially expressed mRNA transcripts.

3. Additionally, following cell-death based sorting, both the killer and non-killer populations are resuspended in HypoThermosol FRS and RPMI + 10% FBS to maintain cell integrity and allow for transportation to the sequencing facility.

4. Using the results of scRNA-seq, individual NK cell populations are determined and evaluated to better understand which NK cell populations contribute to NK cell induced tumor cell death.

The present technology uniquely applies FADS to isolation a sub-population of active NK cells (i.e . , NK cells with proven ability to kill target cells, such as tumor cells) from a heterogeneous population of NK cells. This allows for the generation and expansion of a population of NK cells that effectively targets and kills cancer cells. Another unique aspect of the present technology is the use of feeder cell-free expansion following FADS-based enrichment of activated NK cells, leading to an expanded population of active NK cells for use in therapy of cancer, for example, without the concerns raised by the use of feeder cells that may themselves be cancer cells or at risk of causing harm such as promoting the growth of cancer cells within a treated subject. Expansion of a population of NK cells enriched in active killers leads to the generation of a more homogenous, highly active population of NK cells. Further, isolation of NK cells using FADS allows for genomic sequencing of the population of cells capable of effectively targeting and killing cancer cells, as well as sequencing of the population of cells unable to target and kill cancer cells. This allows for transcriptom ic analysis of the genes that are upregulated or downregulated in the distinct populations, and the association of changes in expression of certain genes with NK cell function. The present technology can be used to develop improved NK cell therapeutics, as well as for identification of biomarkers associated with successful NK cell activation. Such biomarkers may be proteins as well as transcriptomic and genomic biomarkers associated with highly active and cytotoxic NK cells, as well as identification of pathways that control NK cell activation and cytotoxicity. Knowledge of these factors can help design improved methodology to activate, enrich, and expand NK cells for enhanced targeting, cytotoxicity, and cancer cell targeting capabilities. Such knowledge also can lead to the development of CAR-expressing NK cells or other forms of genetically engineered NK cells with superior therapeutic properties.

EXAMPLES

Example 1. Fluorescence-Activated Droplet Sorting.

Microfluidic devices were fabricated from PDMS using standard soft lithography techniques. Each inlet of the device was connected to an individual syringe containing a cell suspension in media or oil-based fluid through Tygon Micro Bore PVC Tubing of the following dimensions: 0.010” ID, 0.030” OD, 0.010”wall thickness (Small Parts Inc., FL, USA). The device was treated with Aquapel glass treatment (Aquapel, Pittsburg, PA, USA) for 15 min, then flushed with air immediately before use. The syringes were operated by individually programmable syringe pumps (Harvard Apparatus, USA). The oil and aqueous flow rates were generally maintained at a ratio of 4: 1 to obtain optimal droplet sizes. The oil phase consisted of Fluorinert® FC-40 (Sigma, St. Louis, MO, USA) supplemented with 2% w/w surfactant (008-FluoroSurfactant, Ran Biotechnologies, Severely, MA, USA).

Cell images in droplets were captured using a Zeiss Axio Observer.ZI microscope (Zeiss, Germany) equipped with a Hamamatsu digital camera C10600 Orca-R2, 10x-40x objectives and standard FITC/DAPI/TRITC filters. The microfluidic device containing cell-encapsulated droplets was maintained in a humidified microscopic stage-top incubator at 37°C and 5% CO2 for the duration of the experiment. All time-lapse images were obtained by automated software control. The array was scanned to identify locations containing 1 :1 effector: target ratio and the specific x-, y-, and z-positions were programmed in the Zen imaging program (Zeiss). Images of these locations were obtained every 30 minutes for a total period of 48 h. Image analysis was done using Microsoft Office Excel. Contact periods were defined as cells forming visible conjugates. All periods of association and dissociation were counted for each cell and represented as percentage of total cells analyzed. NK- mediated cytolysis of target cells was characterized by loss of calcein AM fluorescence from the target cells. Target cell death was further verified by membrane rupture and blebbing. Killing time for target cell death was defined as the time elapsed from the initiation of contact to loss of fluorescence and morphological changes (as described above). All statistical analysis was performed using non-parametric T-test; p value < 0.05 was considered statistically significant.

NK cells were isolated from human peripheral blood mononuclear cells by immunomagnetic negative selection using a Stemcell Technologies™ Easy Sep™ Human NK Cell Isolation Kit. K562 myelogenous leukemia cells were obtained from American Type Culture Collection. NK and K562 target cell suspensions were loaded in separate syringes at an initial concentration of 3 million/mL. The syringes were immediately loaded on the same programable pump, and the oil-to-aqueous flow rates were generally maintained at a ratio of 2:1 to obtain optimal droplet sizes. Following sorting, droplets were collected in a 2mL conical vial containing 1 mL of mineral oil for 1.5 hrs and then incubated at 37°C and 5% CO2 in a humidified atmosphere until further use.

Example 2. Transcriptomic Analysis of NK Cells.

Total RNA from the FADS sorted droplets was extracted using RNeasy kits (Qiagen, Germantown, MD, USA) in accordance with the manufacturer’s instructions and stored at -80°C in RNase-free water prior to being subjected to bulk RNA sequencing (RNA-seq). RNA was extracted from pooled droplet-sorted NK cells without expansion.

FADS-sorted RNA samples were subjected to the ultra-low input mRNA non- directional sequencing analysis pipeline (Novogene, Beijing, China). Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using dUTP for directional library. Samples were ready after end repair, A-tailing, adapter ligation, size selection, amplification and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries were pooled and sequenced. A difference between active “killer” NK cells and non- killer NK cells was considered signifcant when the adjusted p-value (p-adj) was < 0.05 and Iog2-fold change was > 1.

Example 3. Feeder-Free Expansion of NK Cells.

The R&D Systems CellxVivo Human NK Cell Expansion Kit was adapted for low starting cell counts consistent with the number of cells obtained using the FADS approach. Following FADS, the individual droplets were broken and residual oil from the sorting process was separated from the cells and media through use of a PTFE filter. This process selectively isolated the cells and media so that they could be transferred to a 96-well round bottom expansion plate, but was sufficiently gentle to prevent cell death.

Following transfer to the plate, the cells were then cultured using a modified version of the R&D Systems CellxVivo Human NK Cell Expansion Kit™ media, with media components adjusted based on the starting cell count. This was intended to ensure that NK cells received adequate nutrients and cytokines necessary for expansion, without providing excessive amounts. This adjustment was calculated based on the starting cell count and the volume of the wells in the expansion plate. In the R&D Systems CellxVivo Human NK Cell Expansion Kit™, media components are added based on the starting cell count of 40,000 cells per milliliter, or 40 cells per microliter. Therefore, volume of media components added to the media was determined by a conversion factor comparing the kit-standard 40 cell per microliter concentration to the concentration of NK cells to be expanded (total number of isolated NK cells/175 microliter well volume). The expansion plate was incubated at 37°C and 5% CO2, with partial media exchanges every 2 days to replace spent media.

Following 10 days of incubation, the cell concentration in the expansion plate was determined, and cells were transferred to a 6-well plate. The media for this plate was prepared again using a conversion factor comparing the kit-standard 40 cell per microliter concentration to the concentration of NK cells to be expanded (total number of isolated NK cells/3 milliliter well volume). This plate was then incubated at 37°C and 5% CO2, undergoing partial media exchanges every 2 days to replace spent media for another 11 days (a total of 21 days for the entire process). Using this approach, a starting population of 9,000 cells was expanded to 444,000 cells after three weeks. From larger starting cell counts, 13,500 and 19,000 cells, this methodology yielded final cell counts of 915,000 cells and 2,910,000 cells, respectively. Example 4. Evaluation of Cytotoxicity of Expanded NK Cells Using LDH Assay.

A Promega CytoTox 96 LDH³³³³ kit was optimized for efficacy at small starting cell counts. This approach was used to quantify the cytotoxicity of the expanded “killer” subpopulation of NK cells and compare the efficacy of this group with that of the initial heterogenous population, as well as with the population of pre-expanded “killer” cells following sorting. In the LDH plate assay, effector NK cells were co-encapsulated in a 1 :1 ratio with target K562 cells, then incubated for 16 hours to mimic the incubation period of droplets in FADS. Table 1 shows the results.

Table 1 . Results of LDH Plate Assay for NK Cell Cytotoxic Activity

As used herein, "consisting essentially of" allows the inclusion of materials or steps that do not materially affect the basic and novel characteristics of the claim. Any recitation herein of the term "comprising", particularly in a description of components of a composition or in a description of elements of a device, can be exchanged with "consisting essentially of" or "consisting of".

While the present invention has been described in conjunction with certain preferred embodiments, one of ordinary skill, after reading the foregoing specification, will be able to effect various changes, substitutions of equivalents, and other alterations to the compositions and methods set forth herein.

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Claims

CLAIMS What is claimed is

(a) providing

(iii) a population of single target cells;

(c) allowing the NK cells and target cells to interact in the microdroplets, whereby some of the NK cells become activated by interaction with the coencapsulated target cells and produce a fluorescence signal from the intracellular Ca²⁺-sensitive fluorescent indicator;

2. The method of claim 1 , wherein target cells co-encapsulated with NK cells are killed, and the population of enriched active NK cells produced in (e) is essentially free of living or intact target cells.

3. The method of claim 1 , wherein the single target cells are tumor-derived cells.

4. The method of claim 1 , wherein the intracellular Ca²⁺-sensitive fluorescent indicator is Fura-10.

5. The method of claim 1 , wherein analysis of cytotoxic activity of the enriched NK cells is performed using a lactate dehydrogenase (LDH) assay.

6. The method of claim 1 , further comprising

7. The method of claim 6, wherein the expansion in (f) is completed in 21 days or less.

8 The method of claim 6, wherein the expansion in (f) is carried out without the use of feeder cells.

9. The method of claim 6, further comprising one or more of (i) analysis of cytotoxic activity of the expanded NK cells, (ii) re-evaluation of target cell killing activity of the expanded NK cells using another fluorescence-activated droplet sorting process, or (iii) transcriptom ic analysis of the expanded NK cells.

10. The method of claim 9, wherein (ii) re-evaluation of target cell killing activity of the expanded NK cells is carried out by a fluorescence-activated droplet sorting process wherein (a) target cells are loaded with a fluorescence-based indicator of target cell viability, such as a fluorogenic substrate for caspase 3 or caspase 7, or (b) NK cells are loaded with an intracellular Ca²⁺-sensitive fluorescent indicator.

(a) providing

(i) a microfluidic system configured for co-encapsulating single cells in aqueous microdroplets in an oil stream and sorting the microdroplets based on fluorescence of labeled cells within the microdroplets; (ii) a population of single NK cells; and

12. The method of claim 11 , wherein target cells co-encapsulated with NK cells are killed, and the population of enriched active NK cells produced in (e) is essentially free of living or intact target cells.

13. The method of claim 11 , wherein the single target cells are tumor-derived cells.

14. The method of claim 11 , wherein the fluorescence-based indicator of target cell viability is a fluorogenic caspase substrate which serves as an indicator of caspase 3 or caspase 7 activity.

15. The method of claim 14, wherein the fluorogenic caspase substrate is a peptide derivative of cresyl violet.

16. The method of claim 11 , further comprising testing cytotoxic activity of the enriched NK cells, such as by using a lactate dehydrogenase (LDH) assay.

17. The method of claim 11 , further comprising

18. The method of claim 17, wherein the expansion in (f) is completed in 21 days or less.

19. The method of claim 17, wherein the expansion in (f) is carried out without the use of feeder cells.

20. The method of claim 17, further comprising one or more of (i) analysis of cytotoxic activity of the expanded NK cells, (ii) re-evaluation of target cell killing activity of the expanded NK cells using another fluorescence-activated droplet sorting process, or (iii) transcriptom ic analysis of the expanded NK cells.

21 . The method of claim 20, wherein (ii) re-evaluation of target cell killing activity of the expanded NK cells is carried out by a fluorescence-activated droplet sorting process wherein (a) target cells are loaded with a fluorescence-based indicator of target cell viability, such as a fluorogenic substrate for caspase 3 or caspase 7, or (b) NK cells are loaded with an intracellular Ca²⁺-sensitive fluorescent indicator.

(a) obtaining a population of enriched and expanded NK cells using the method of 6; and

23. The method of claim 22, wherein (a) comprises: obtaining a population of NK cells from the subject, and using the population of the subject’s NK cells as the initial population of single NK cells in the method of expansion of active NK cells.

24 The method of claim 22, wherein (a) comprises: obtaining a population of single target cells from the subject, and using the population of the subject’s target cells as the single target cells in the method of enriching and expanding NK cells.

25. The method of claim 22, wherein the single target cells are not derived from the subject.

26. The method of claim 22, wherein the subject has cancer, and the method is used for cancer therapy of the subject.

(a) sorting and enriching a population of NK cells according to the method of claim 1 , thereby obtaining an enriched population of active NK cells;

(b) isolating mRNA from a pool of the enriched population of active NK cells;

(c) performing RNA sequencing on the isolated mRNA; and

28. A kit comprising a microfluidic device and instructions for performing the method of any of the preceding claims.

29. The kit of claim 28 further comprising one or more reagents, such as a fluorescent indicator for intracellular Ca²⁺, a fluorogenic caspase substrate, one or more LDH assay reagents, one or more reagents for NK cell expansion, one or more reagents for transcriptom ics analysis, or a reagent for specifically detecting an NK cell biomarker.

30. A population of NK cells obtained using the method of claim 1 .

31 . The population of NK cells of claim 30 comprising at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% active NK cells.

32. The population of NK cells of claim 30 containing a total of at least 20 million NK cells.