A METHOD AND SYSTEM FOR INTRODUCING ONE OR MORE EXOGENOUS SUBSTANCES INTO AN IMMUNE CELL

Abstract

The present invention relates to a method and system for introducing one or more exogenous substances into an immune cell. The method comprises the steps of: providing a membrane having a plurality of pores configured to allow the immune cell to pass through, wherein the plurality of pores has an average pore diameter which is no less than an average diameter of the immune cell nucleus and no more than an average diameter of the immune cell, passing a fluid containing the immune cell and the on e or more exogenous substances through the plurality of pores to induce a mechanical stress to the immune cell and facilitate introduction of the one or more exogenous substances into the immune cell, and recirculating the fluid containing the immune cell and the one or more exogenous substances such that the same immune cell passes through the membrane more than once. In some embodiments, the immune cell is T-cells and the exogenous substance is mRNA.

Claims

1. A method of introducing one or more exogenous substances into an immune cell, the method comprising, providing a membrane having a plurality of pores configured to allow the immune cell to pass through, wherein the plurality of pores has an average pore diameter which is no less than an average diameter of the immune cell nucleus, passing a fluid containing the immune cell and the one or more exogenous substances through the plurality of pores to induce a mechanical stress to the immune cell and facilitate introduction of the one or more exogenous substances into the immune cell, and recirculating the fluid containing the immune cell and the one or more exogenous substances such that the same immune cell passes through the membrane more than once.

2. The method according to claim 1, wherein the average pore diameter of the plurality of pores is no more than an average diameter of the immune cell.

3. The method according to claim 1, wherein the specific flow rate for passing the fluid containing the immune cell and the one or more exogenous substances through the plurality of pores is from 10 nL/s to 1000 nL/s.

4. The method according to claim 1, further comprising, prior to the step of passing the fluid, activating the immune cell from an inactivated to an activated state by exposing the immune cell to one or more activation agents.

5. The method according to claim 4, wherein the method is devoid of introducing one or more conditioning substances into the fluid after the activating step.

6. The method according to claim 1, wherein the immune cell has not been pretreated to alter cell membrane pliability, prior to passing through the membrane.

7. The method according to claim 1, further comprising, prior to passing the immune cell through the membrane, seeding the immune cell into the fluid at a density of no less than 1 × 10.sup.5 cells/ml and adding the one or more exogenous substances into the fluid.

8. The method according to claim 1, wherein the fluid containing the immune cell and the one or more exogenous substances has a volume of at least 20 mL.

9. The method according to claim 1, wherein the fluid containing the immune cell and the one or more exogenous substances is substantially free of serum when the fluid is passed through the plurality of pores.

10. The method according to claim 10,further comprising providing a time interval of at least 5 minutes to rest the immune cell between any two consecutive passes through the membrane.

11. The method according to claim 1, wherein the immune cell is a T cell.

12. The method according to claim 11, wherein the T cell transfected with the one or more exogenous substances is to be used for immunotherapy.

13. The method according to claim 1, wherein the one or more exogenous substances comprise a nucleic acid configured for expressing one or more exogenous genes for a therapeutic application.

14. The method according to claim 1, wherein, for each pass through the plurality of pores, the number of viable immune cells recovered after passing though the plurality of pores is at least 30% of the number of viable immune cells prior to passing through the plurality of pores.

15. The method according to claim 1, wherein, for each pass through the plurality of pores, the number of immune cells transfected with the one or more exogenous substances is at least 10% of the number of untransfected immune cells prior to passing through the plurality of pores.

16. The method according to claim 1, wherein the number of immune cells transfected with the one or more exogenous substances increases with each additional pass across the membrane.

17. A system for introducing one or more exogenous substances into an immune cell, the system comprising, a membrane having a plurality of pores configured to allow the immune cell to pass through, wherein the plurality of pores has an average pore diameter which is no less than an average diameter of the immune cell nucleus, and a pump configured to pass a fluid containing the immune cell and the one or more exogenous substances through the plurality of pores to induce a mechanical stress to the immune cell and facilitate introduction of the one or more exogenous substances into the immune cell, wherein the pump is further configured to recirculate the fluid containing the immune cell and the one or more exogenous substances such that the same immune cell passes through the membrane more than once.

18. The system according to claim 17, wherein the average pore diameter of the plurality of pores is no more than an average diameter of the immune cell.

19. The system according to claim 17, further comprising a vessel arranged to form an enclosed system such that the membrane is housed within the enclosed system.

Description

BRIEF DESCRIPTION OF FIGURES

[0090] FIG. 1A is a schematic drawing of a deformation-based transfection showing a cell prior to passing through a pore of a membrane in an example embodiment.

[0091] FIG. 1B is a schematic drawing of the deformation-based transfection showing the cell while passing through the pore of the membrane in the example embodiment.

[0092] FIG. 1C is a schematic drawing of the deformation-based transfection showing the cell after passing through the pore of the membrane in the example embodiment.

[0093] FIG. 2 is a micrograph of a lymphocyte in an example embodiment.

[0094] FIG. 3A is a schematic drawing of a deformation-based transfection showing a membrane with a pore size smaller than the diameter of a cell nucleus in an example embodiment.

[0095] FIG. 3B is a schematic drawing of a deformation-based transfection showing a membrane with a pore size that is at least the diameter of a cell nucleus in an example embodiment.

[0096] FIG. 4A is a typical FACS (fluorescence-activated cell sorting) result for Jurkat cells transfected with eGFP mRNA, analyzed two days after passing through the presently disclosed system once (46% transfection efficiency) in an example embodiment.

[0097] FIG. 4B is a typical FACS result for Jurkat cells transfected with eGFP mRNA, analyzed two days after passing through the presently disclosed system twice (77% transfection efficiency) in an example embodiment.

[0098] FIG. 5A is a brightfield micrograph of cells after transfection in an example embodiment.

[0099] FIG. 5B is a fluorescence micrograph of cells after transfection in the example embodiment.

[0100] FIG. 6 is a chart showing cell transfection efficiency as determined by FACS for different number of passes of cells through a membrane in an example embodiment.

[0101] FIG. 7A is a chart showing cell recovery after passing through membranes in an example embodiment.

[0102] FIG. 7B is a chart showing cell recovery as a percentage of previous pass in the example embodiment.

[0103] FIG. 8A is a schematic diagram of a system for introducing one or more exogenous substances into a cell in an example embodiment.

[0104] FIG. 8B is a photograph of the system for introducing one or more exogenous substances into a cell in the example embodiment.

[0105] FIG. 9 is a graph showing changes in cell diameters of primary T-cells cultured in AIM/V and 501U/mL or 200IU/mL IL-2 with time in an example embodiment.

DETAILED DESCRIPTION OF FIGURES

[0106] Example embodiments of the disclosure will be better understood and readily apparent to one of ordinary skill in the art from the following discussions and if applicable, in conjunction with the figures. It should be appreciated that other modifications related to structural and chemical changes may be made without deviating from the scope of the invention. Example embodiments are not necessarily mutually exclusive as some may be combined with one or more embodiments to form new exemplary embodiments. The example embodiments should not be construed as limiting the scope of the disclosure.

[0107] FIG. 1A to FIG. 1C are schematic drawings of a deformation-based transfection. The inventors had previously demonstrated a device and method of transfection (PipFect) using shear stress for various cell lines. The putative mechanism for this transfection is that the cells undergo mechanical stress during the deformation, which temporarily disrupts its nuclear membrane, resulting in uptake of a payload.

[0108] Briefly, cells 100 were pre-treated with a drug to allow partial disassembly of the nuclear membrane, making them more pliable prior to passing through a pore 102 of a membrane 104 (FIG. 1A). This ensures that the cells can deform through the pores, which may be smaller than the nuclear diameter. The cells 100 were then mixed with a payload 106 of interest (typically plasmid DNA encoding a reporter gene) and passed through micron-sized pores to deform them (FIG. 1B), and directly into serum-containing media (FIG. 1C). After a stipulated amount of time, the cells were characterized by the expression of the reporter. Pretreatment with drugs allows the use of smaller pores to achieve higher stresses and improve transfection efficiency (FIG. 1C).

[0109] The inventors noted that immunotherapy e.g., T-cell therapy such as Chimeric Antigen Receptor-T cell (CAR-T) therapy and/or T-Cell Receptor (TCR) therapy is a possible application of the above technology. However, several aspects of PipFect are not compatible with cell therapy.

[0110] Firstly, to be used in actual clinical application, the addition of any component, such as the drugs used for ‘softening’ the nucleus, is to be avoided, since it will require excessive testing to get through regulatory approval.

[0111] Secondly, lymphocytes have much smaller cytoplasm volumes than other cells, such as HeLa, HEK, and CHO. As shown in FIG. 2, lymphocytes, such as Jurkats, have very small cytoplasmic volumes, relative to their nuclear volume. Without the drugs to ‘soften’ up the nucleus, passing these cells through the pores can result in nucleus rupture, and consequently, cell death. This presents a challenge, since it is unclear from the outset whether a suitable pore size and shear condition can be found, that achieves high transfection efficiency, and cell recovery.

[0112] Thirdly, the form factor of the previous method PipFect was a pipette tip, through which a small volume (typically about 100 .Math.L to about 200 .Math.L) of cell/payload mixture was pushed, using a micropipettor. Not only is this volume too small to be practical for cell therapy use (around about 20 mL), the micropipettor is also unable to provide good control over the flow rate, which is recognised to play a role in controlling the transfection efficiency.

[0113] Fourthly, the PipFect device also requires culture to be conducted on a plate, when it may be preferable to perform the culture inside a self-contained vessel.

[0114] Example embodiments of the method and system as disclosed herein seek to overcome the abovementioned problems associated with the PipFect device and method.

[0115] In particular, example embodiments of the method and system seek to optimize the pore size through which a cell passes. As shown in FIG. 3A, in the absence of drugs that cause partial disassembly of the nuclear membrane, the cell nucleus of a cell 300 remains stiff, and may not pass through a pore 302 of a membrane 304 easily, especially if the pore size is smaller than the diameter of the cell nucleus. The result is that the cells can either be physically filtered out of the media (which will result in clogging of the device), or pass through the pores, and be severely damaged, to the point of cell death, in the process of forced deformation. As shown in FIG. 3B, the presently disclosed method and system emphasize on both cell recovery and transfection efficiency without the use of drugs that cause partial disassembly of the nuclear membrane. As such, the pore size is relatively larger, and the diameters of the cell nucleus and the entire cell (including small volume of cytoplasm) may be used to place upper and lower limits on the pore size.

EXAMPLE

Transfection of Lymphocyte Cell Line

[0116] Various embodiments of the cell transfection technology disclosed herein are capable of transfecting cells obtained from a lymphocyte cell line e.g., Jurkat cells.

[0117] The typical process for nonviral gene transfection involves the use of electroporation, which requires the cells to be removed from the expansion/stimulation media and placed in a low conductivity buffer for treatment in a specialized instrument capable of pulsing the cells in an electric field.

[0118] Various embodiments of the cell transfection technology disclosed herein seeks to simplify the workflow for cell transfection, by removing these handling steps. In this example, the payload (in this case, messenger RNA, or mRNA) is added to the cell mixture, directly into a low-serum or serum-free media, and the mixture is passed through a filtration device (flow rate from about 50nL/s to about 100 nL/s through each pore). After a fixed interval (typically about 2 hours), the same cells are passed through the membrane again. It is noted that the efficiency increases significantly on the second pass (see FIG. 4B). This process can be repeated several times, though for Jurkat cells, the increase in transfection efficiency was found to be almost two-fold after the second pass, but only marginally on subsequent passes.

[0119] It will be appreciated that these parameters (i.e., interval, number of passes, pore diameter, etc) depend on the type, state, and number of cells treated. Nevertheless, since Jurkats are a T lymphocyte cell line, the parameters here may serve as a good starting point for other similar cells, especially for T-cell engineering in immunotherapy applications.

[0120] It will also be appreciated that control of the various parameters allows for good transfection, as well as cell recovery, which are typically mutually exclusive goals. The inventors accomplished these by choosing a pore size that is larger than the cell nucleus diameter, which would normally mean minimal stress on the cell. However, the inventors then subjected the cells to around 5 to 10 times the flow rate at the pores than previously attempted.

[0121] The inventors showed that it is possible to achieve these goals of transfection efficiency and cell recovery through judicious choice of the membrane pore size (e.g., 12 microns) and density (e.g., 1E4/cm.sup.2). The inventors were also able to demonstrate that cells passed through the membrane multiple times, without introducing additional mRNA, can yield higher transfection efficiency. FIG. 4A and FIG. 4B show typical transfection results, in which Jurkat cells are transfected with eGFP green fluorescence protein, analyzed by FACS. FIG. 5A and FIG. 5B show that the transfected cells were highly-fluorescent in the micrographs. The brightfield (FIG. 5A) and fluorescence (FIG. 5B) images show that the transfection efficiency is very high in the sample. Quantitation by FACS indicate around 77% transfection efficiency.

[0122] Transfection efficiency generally improves with the number of cells in the mixture (see FIG. 6). Furthermore, while transfection efficiency improved significantly between one and two passes through the filter, there was less improvement with subsequent passes. The improvement achieved by increasing from one to two passes is significantly greater than from two to three passes. It was also found that if the cells are left in OptiMEM after transfection, without topping up with RPMI 1640 media containing serum, the efficiency is further improved to 77% (see last two columns of FIG. 6).

[0123] Although multiple passes improve transfection, they also reduce the number of cells recovered. As shown in FIG. 7A, more cells are lost with each pass through the membrane. However, the proportion of cells lost with each pass appears to be similar (see FIG. 7B). The cell recovery appears to be quite similar regardless of the cell concentration used.

[0124] The manufactured tips have some variability in the open aperture. This is because the membranes are manually glued onto the holders, and the glue can impinge on the aperture. This variability can be seen in the cell recovery results (FIG. 7B, series labeled “4e5/mL #2”). It is believed that by changing the assembly method, using pre-cut membranes clamped in fabricated holders, this variability can be greatly reduced.

[0125] The inventors have also tested re-circulating the media and buffer in an enclosed vessel, using a peristaltic pump. FIG. 8A and FIG. 8B show a system 800 for introducing one or more exogenous substances into a cell in an example embodiment. The system 800 comprises a membrane device 802 according to embodiments as described herein coupled to a vessel 804 via a tubing assembly 806. In the example embodiment, the system 800 is a self-contained, closed loop circulating system. The vessel 804 is configured to contain a suspension of cells in a suitable media and buffer. The vessel 804 comprises an outlet for allowing fluid e.g., cell suspension to leave the vessel and an inlet for allowing fluid to enter the vessel via the tubing assembly 806. A pump e.g., peristaltic pump 808 is provided to circulate the cell suspension through the membrane device 802 and the tubing assembly 806. A portion of the tubing assembly 806 is disposed within a head of the peristaltic pump 808 such that a rotor of the peristaltic pump 808 can operate against the tubing assembly 806 for pumping the cell suspension through the tubing assembly 806 and the membrane device 802. In the example embodiment, the cell suspension may be recirculated through the system 800 such that each cell in the cell suspension passes through the membrane device 802 at least once.

[0126] The inventors have verified that the flow rates needed can be achieved using such a system. The applicability of the principles derived herein may be translated to a larger, recirculating system.

[0127] In summary, the inventors have demonstrated a method to perform transfection that result in high transfection (more than 75% transfection efficiency), as well as cell recovery (about 70% of cells recovered after two passes). This simple to use method can be performed in a closed, re-circulating system that requires minimum handling, and uses only cheap instrumentation, such as a peristaltic pump, to accomplish the desired gene transfection.

Transfection of Primary T-Cells

[0128] Various embodiments of the cell transfection technology disclosed herein are capable of transfecting cells obtained from a primary cell line e.g., primary T-cells. Unlike Jurkats (about 11-13 microns), primary T-cells have very small cell diameters when first harvested (about 6-7 microns). However, the cells typically receive various treatments in culture prior to transfection, and the inventors closely followed established protocols.

[0129] Firstly, primary T-cells have to undergo activation for 3 days, prior to expansion in vitro, and this is part of the protocol for preparation of immune cells for immunotherapy. It will be appreciated that this process has profound effects on the cell diameters. FIG. 9 is a graph showing changes in the cell size of primary T-cells cultured in AIM/V and 501U/mL or 200IU/mL IL-2 over time. As shown, there is gradual decrease in cell diameter with time. Cells were activated on Day 0 with CD3/CD28. The inventors followed reported protocols and transfected the cells 4 days after activation. It will be appreciated that this protocol is subjected to change, depending on the specific protocol used.

[0130] Secondly, since the cells have a diameter of around 11 microns, a membrane with 10-micron pores was used. This pore size is slightly larger than the cell nucleus diameter (about 9 microns), but slightly smaller than the cell diameter. The same experiment was performed with cells that have diameters of 8 and 9 microns.

[0131] Table 1 provides a summary of the pore size, culture media conditions and transfection efficiency achieved using embodiments of the device and method disclosed herein.

TABLE-US-00001 Summary of pore size, culture media conditions and transfection efficiency achieved using the presently disclosed device and method. Size Media Efficiency 11 AIM-V, 2% AB Serum, IL-2* 0% 11 OPTIMEM 49.9% 11 AIM-V, IL-2 34.3% 9 OPTIMEM 14.9% 9 AIM-V, IL-2 20.8% 8 OPTIMEM 3.4% 8 AIM-V 2.3% * This is the media condition the cells are cultured under in a clinical protocol.

[0132] Further optimization to the protocol may be carried out while still allowing the primary T-cells to be efficiently transfected. One principle adhered to in this process is that while existing components to which the cells are exposed may be removed, new molecules will not be introduced into the workflow, so as to avoid having to perform extensive safety studies. Using the media that the cells are known to have been cultured in (AIM-V, 2% AB Serum, IL-2) for clinical applications, the inventors noted that there was almost no transfection. However, this is mostly attributed to the presence of serum, which is known to degrade mRNA, which is the payload. By removing the serum, much better transfection efficiency was achieved.

[0133] In summary, the inventors observed that relative to the pore size, the smaller the cells, the worse the transfection efficiency. This is expected, since the stresses to which the cells are subjected increases with larger cell sizes. However, the trade-off for cell production is that the cell numbers recovered is lower when the diameter is too large with respect to the pore size. Therefore, the two factors of transfection efficiency and cell recovery should be balanced.

APPLICATIONS

[0134] Embodiments of the methods disclosed herein provide a method and system of introducing one or more exogenous substances into an immune cell. In various embodiments, the method and system are applicable to immune cells, for the purpose of engineering them for immunotherapy.

[0135] In various embodiments, the cell transfection technology disclosed herein is an improvement of previous work done on cell transfection. It was previously demonstrated the transfection of cells using a combination of drugs and mechanical shearing, can achieve reasonably good results for many cell lines. However, the drugs used, culture conditions, and the workflow are not compatible with cell therapy standards. By referring to parameters reported in the literature for preparation of patient immune cells used in immunotherapy e.g., T-cell therapy such as Chimeric Antigen Receptor-T cell (CAR-T) therapy and/or T-cell receptor (TCR) therapy, the inventors developed a new protocol that works very well with the immune cell line (e.g., Jurkat), with transfection efficiency exceeding 75%, and which requires minimal handling of the cells. Furthermore, unlike the previous work, cell recovery and survival are actively pursued in the presently disclosed cell transfection technology. The inventors have also carefully tested a range of conditions, including cell concentration, membrane pore size, pore density, and flow rate.

[0136] Advantageously, embodiments of the method and system disclosed herein are designed to be compatible with existing workflow for such preparations for clinical applications, including the use of appropriate media, flow conditions, and number of times cells are passed through a recirculating device (that is, a self-contained vessel in which the cells are circulated), in order to achieve high transfection without introducing new materials. It will be appreciated that these high levels of transfection may be achieved by embodiments of the method and system disclosed herein.

[0137] Advantageously, various embodiments of the method and system utilize a membrane-based system that is self-contained, and designed for high-throughput, high efficiency, and low-cost preparation of cells. Various embodiments of the device are capable of achieving high transfection, which is uniquely challenging for lymphocytes which have small cytoplasmic volumes, by reducing the pore size but limiting it to at least the nucleus diameter, by increasing cell numbers, and increasing the number of passes through the membrane device.

[0138] It will be appreciated by a person skilled in the art that other variations and/or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the disclosure as broadly described. For example, in the description herein, features of different exemplary embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, included etc. or the like across different exemplary embodiments. The present embodiments are, therefore, to be considered in all respects to be illustrative and not restrictive.