Genetically modified cell preparation apparatus and use thereof

Abstract

The present application relates to the biomedical field, and particularly relates to a gene modified cell preparation device and application thereof. The present application provides a device for bedside preparation of gene modified immune cells, wherein a gene modification system can complete gene modification of T cells in a short time, which can not only realize faster preparation of gene modified T cells, but also has better original cell phenotype due to short-time gene modification of the prepared gene modified T cells, and has better in-vivo persistence and safety, thereby realizing ultra-short-time gene modification of resting T cells, and providing a possibility for bedside CAR-T cell preparation.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to bedside cell preparation equipment. Background Technology

[0002] CAR-T immunotherapy utilizes genetic engineering to specifically modify a patient's own immune T cells, enabling them to recognize and kill tumor cells. The general treatment process involves: first, collecting peripheral blood mononuclear cells using an apheresis machine; then, purifying and sorting these cells to obtain T cells; activating the T cells using magnetic beads; further genetically modifying them by introducing the CAR-T gene; expanding the CAR-T cells; and finally, aliquoting and cryopreserving them according to specifications. After quality control, a sufficient quantity of CAR-T cells is reinfused into the patient. The entire production process, including cryogenic transportation, is time-consuming, and the complex and costly preparation of CAR-T cells limits its widespread application to patients who might benefit from this novel treatment technology.

[0003] US15486362 and CN111373030A integrate the activation, gene modification, and amplification processes in CAR-T preparation into a commercial production platform. They aim to support personalized medicine through a shift in manufacturing technology, saving labor time and production space. However, they do not solve the problems of CAR-T cells needing cryopreservation and transportation, and the long preparation cycle. Summary of the Invention

[0004] In view of this, the present invention provides a bedside cell preparation device, wherein the viral gene modification system can complete the gene modification of T cells in a short time, realize ultra-short-time gene modification of resting T cells, and provide the possibility for bedside CAR-T cell preparation.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a gene modification system, including an injection line, a multi-port valve 18 or 18c, a pump, a first line 56a, a second line 56b, a gene modification device 28, and a collection line;

[0007] The gene modification device includes a hollow fiber membrane assembly, comprising an outer wall 283 and a hollow fiber membrane bundle 284, wherein the hollow fiber membrane bundle is disposed within the outer wall, the space within the hollow fiber membrane bundle is the hollow fiber membrane inner space 285, and the space between the outer wall and the hollow fiber membrane bundle is the hollow fiber membrane outer space 286.

[0008] The outer wall is provided with a pair of inner interfaces 30a and 30b and at least one outer interface 32a or 32b. The two inner interfaces are respectively connected to both ends of the hollow fiber membrane bundle, so that the inner interfaces are connected to the inner space of the hollow fiber membrane and the outer interfaces are connected to the outer space of the hollow fiber membrane.

[0009] The first pipeline and the second pipeline are respectively connected to the two internal interfaces;

[0010] The sample inlet line, the first line, and the second line are connected by the multi-way valve;

[0011] The first pipeline and / or the second pipeline are equipped with at least one of the pumps;

[0012] The collection pipeline is connected to the multi-way valve 18 or 18c, or connected to the first pipeline or the second pipeline through the second multi-way valve 18d.

[0013] In some specific embodiments of the present invention, the pump of the above-mentioned gene modification system includes a first pump 22a and a second pump 22b, wherein the first pump is disposed in the first pipeline and the second pump is disposed in the second pipeline.

[0014] In some specific embodiments of the present invention, the above-described gene modification system further includes a bubble sensor and / or a pressure sensor.

[0015] In some specific embodiments of the present invention, the bubble sensor of the above-mentioned gene modification system includes a first bubble sensor 20a and a second bubble sensor 20b, wherein the first bubble sensor is disposed in the first pipeline and the second bubble sensor is disposed in the second pipeline.

[0016] In some specific embodiments of the present invention, the pressure sensor of the above-mentioned gene modification system includes a first pressure sensor 24a and a second pressure sensor 24b, wherein the first pressure sensor is disposed in the first pipeline and the second pressure sensor is disposed in the second pipeline.

[0017] In some specific embodiments of the present invention, the membrane area of ​​the hollow fiber membrane module of the above-described gene modification system is 75 cm². 2 -1.2m 2 .

[0018] The present invention also provides an apparatus for preparing genetically modified immune cells, including the above-described genetic modification system.

[0019] In some specific embodiments of the present invention, the sample inlet line of the above-mentioned device is connected to at least one of the cell container 1, the virus container 2, and the matrix container 4.

[0020] The matrix container 4 can be composed of matrix containers 4a / 4b / 4c or combinations thereof.

[0021] In some specific embodiments of the present invention, the sample inlet line of the above-mentioned device is connected to the mixing chamber 16, and the mixing chamber is connected to the cell container and the virus container respectively through the pipeline.

[0022] In some specific embodiments of the present invention, the mixing chamber of the above-described device is also connected to a matrix container via a pipeline.

[0023] In some specific embodiments of the present invention, the mixing chamber, the cell container, the virus container, and the matrix container of the above-mentioned device are connected by a third multi-port valve 18a.

[0024] In some specific embodiments of the present invention, the pipeline between the cell container and the third multi-way valve of the above-mentioned device is provided with a clamping valve 8a.

[0025] In some specific embodiments of the present invention, the pipeline between the virus container and the third multi-way valve of the above-mentioned device is provided with a clamping valve 8b.

[0026] In some specific embodiments of the present invention, the pipeline between the matrix container and the third multi-way valve of the above-mentioned device is provided with a pinch valve 8c.

[0027] In some specific embodiments of the present invention, the external interface of the above-mentioned device is connected to the waste liquid collection device 44a via a pipeline.

[0028] In some specific embodiments of the present invention, the pipeline between the external interface of the above-mentioned device and the waste liquid collection device is provided with a clamping valve 8g.

[0029] In some specific embodiments of the present invention, the waste liquid collection device of the above-mentioned equipment has an exhaust port, and the exhaust port is provided with an air filter 54a.

[0030] In some specific embodiments of the present invention, the collection pipeline of the above-mentioned device is connected to the cell collection device 46.

[0031] In some specific embodiments of the present invention, the collection pipeline of the above-mentioned device is connected to the virus removal device 42, and the virus removal device is connected to the cell collection device 46.

[0032] In some specific embodiments of the present invention, the virus removal device of the above-described equipment is connected to the waste liquid collection device 44a or the second waste liquid collection device 44b.

[0033] In some specific embodiments of the present invention, the virus removal device, the waste liquid collection device or the second waste liquid collection device, and the cell collection device of the above-mentioned equipment are connected through a fourth multi-way valve 18e.

[0034] In some specific embodiments of the present invention, a third pump 22c is provided on the pipeline between the virus removal device and the fourth multi-way valve of the above-mentioned device.

[0035] In some specific embodiments of the present invention, a clamp valve 8i and / or a bubble sensor 20c are provided on the pipeline between the fourth multi-way valve of the above-mentioned device and the cell collection device.

[0036] In some specific embodiments of the present invention, a clamping valve 8h is provided on the pipeline between the fourth multi-way valve of the above-mentioned device and the waste liquid collection device or the second waste liquid collection device.

[0037] In some specific embodiments of the present invention, the virus removal device of the above-described equipment is connected to the matrix container or the second matrix container 4c.

[0038] In some specific embodiments of the present invention, the virus removal device of the above-mentioned equipment is connected to the matrix container or the second matrix container and the collection pipeline through a fifth multi-way valve.

[0039] In some specific embodiments of the present invention, a fourth pump 22d is provided on the pipeline between the virus removal device and the fifth multi-way valve of the above-mentioned device.

[0040] In some specific embodiments of the present invention, a pinch valve 8e is provided on the pipeline between the fifth multi-way valve and the fourth pump of the above-mentioned device.

[0041] In some specific embodiments of the present invention, the above-mentioned device also includes indicator lights and a computer control terminal.

[0042] In some specific embodiments of the present invention, the cell collection device of the above-described device is directly connected to the patient or connected to the patient through a reinfusion device.

[0043] The present invention also provides a method for preparing CAR-T cells, based on the above-described gene modification system or the above-described equipment.

[0044] In some specific embodiments of the present invention, the above method includes: sample introduction, infection, and collection;

[0045] The infection process includes: the first tubing, the second tubing, and the gene modification device are fully circulated with a cell-virus suspension; the matrix is ​​simultaneously introduced into the first and second tubings via the injection tubing, causing the cell-virus suspension to convect within the hollow fiber membrane bundle. Cells and viruses in the cell-virus suspension are trapped and in contact within the hollow fiber membrane, while the matrix permeates from within the hollow fiber membrane to the outside space. The convection velocity is 1.5–15.4 μL / min / cm. 2 .

[0046] In some specific embodiments of the present invention, the convection rate of the above method is 1.5 μL / min / cm. 2 3.2 μL / min / cm 2 6.7 μL / min / cm 2 7.8 μL / min / cm 2 Or 15.4 μL / min / cm 2 .

[0047] In some specific embodiments of the present invention, the convection time of the above method is 10 minutes or more.

[0048] In some specific embodiments of the present invention, the sample injection of the above method includes: filling the first pipeline, the second pipeline, and the hollow fiber membrane bundle with the cell-virus suspension through the sample injection pipeline, blocking the external interface, so that the multi-way valve connects only the first pipeline and the second pipeline to form a loop, and allowing the cell-virus suspension containing cells and viruses to circulate unidirectionally in the loop to complete the sample injection.

[0049] In some specific embodiments of the present invention, the unidirectional circulating flow rate of the above method is 1.5–66.7 μL / min / cm. 2 .

[0050] In some specific embodiments of the present invention, the unidirectional circulating flow time of the above method is 3 to 5 minutes.

[0051] In some specific embodiments of the present invention, the cells described in the above method are a population containing T cells, including but not limited to activated or inactivated PBMC cells, leukocytes, T lymphocytes, etc., and these cells can be fresh or cryopreserved.

[0052] Cell populations containing T lymphocytes: These can be T lymphocytes, leukocytes or PBMCs containing T cells, T cell populations of leukocytes or PBMCs after CD3-positive sorting, mixed cells of single-positive cells after CD4-positive and CD8-positive sorting, or single-positive T cell populations of leukocytes or PBMCs after CD3-positive sorting (CD4-positive or CD8-positive). The above cell populations can be fresh or cryopreserved.

[0053] In some specific embodiments of the present invention, the number of cells in the cell-virus suspension described above is 1E6 cells / mL or 2E6 cells / mL.

[0054] In some specific embodiments of the present invention, the virus described in the above method is a lentivirus or an engineered viral particle.

[0055] In some specific embodiments of the present invention, the titer of the virus in the cell virus suspension is 1 MOI, 2 MOI or 3 MOI.

[0056] In some specific embodiments of the present invention, the volume of the virus in the cell virus suspension is 1.5 to 40 mL.

[0057] In some specific embodiments of the present invention, the cell-virus suspension described above comprises any one of the following:

[0058] (i) Blood;

[0059] (ii) Single-sample resting sample collection;

[0060] (iii) Isolated PBMC cells.

[0061] In some specific embodiments of the present invention, the matrix of the above method is PLA + 10% HSA or 1640 + 10% FBS.

[0062] The bedside cell preparation device of the present invention has the following effects:

[0063] Existing technologies have significantly lower efficiency in T-cell gene modification in large systems, while the gene modification efficiency is significantly higher with the large-system gene modification device described in this application, which can be used for large-scale CAR-T cell preparation to meet more clinical needs.

[0064] Existing technologies have limitations in sample injection speed. A slightly higher injection speed can cause the cell-virus suspension to be ejected from the hollow fiber membrane bundle. After improvement, the gene modification device of this application adopts a cyclic injection method to avoid the equipment pipeline disconnection and ejection, and to extend the life of the gene modification device.

[0065] Using the device described in this application, gene modification of single-collected resting samples can achieve a gene modification rate of nearly 40% within 10 minutes, which is more than 30% higher than that of samples left to stand at room temperature for 10 minutes.

[0066] The sample for the device of this application can be either frozen PBMC extracted by density gradient method, fresh PBMC extracted by density gradient method, single sample, blood environment, T lymphocytes, white blood cells containing T cells, T cell populations after CD3 positive sorting of white blood cells or PBMCs, or mixed cells after CD4 positive and CD8 positive sorting of single positive cells, or single positive T cell populations after CD4 positive or CD8 positive sorting of white blood cells or PBMCs. All of these can achieve short-term and efficient gene modification, and various matrices are suitable for the gene modification device of this application.

[0067] In summary, the gene modification system of this application can complete the gene modification of T cells in less than 90 minutes or even less than 10 minutes. This not only enables faster preparation of gene-modified T cells, but also, because the gene-modified T cells retain a better original cell phenotype after short-term gene modification, they have better in vivo persistence and safety, realizing ultra-short-term gene modification of resting T cells and providing a possibility for bedside CAR-T cell preparation. Attached Figure Description

[0068] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0069] Figure 1 The gene modification system described in this application is shown, and the direction of liquid flow during cyclic injection is marked;

[0070] Figure 2 The gene modification system described in this application is shown, and the direction of fluid flow during convective infection is marked;

[0071] Figure 3 The viral gene modification system described in this application, which is mainly composed of a gene modification device, is illustrated in detail.

[0072] Figure 4The convection infection of the cell-virus suspension in the gene modification system described in this application and the direction of fluid flow are illustrated in detail.

[0073] Figure 5 The detailed structure of the gene modification device described in this application is shown;

[0074] Figure 6 , Figure 7 and Figure 8 The states of the cell-virus suspension before, at the beginning of, and during convection infection inside the hollow fiber membrane bundle of the gene modification device were described respectively.

[0075] Figure 9 A plan view of a bedside preparation device conceived from a viral gene modification system is shown, along with the internal architecture of the gene modification device.

[0076] Figure 10 A schematic diagram of a bedside preparation device conceived using a gene-editing system;

[0077] Figure 11 This is a schematic diagram illustrating an application scenario where the bedside preparation device described in this application is connected to a patient and a single-cell apheresis machine.

[0078] Figure 12 The efficiency (%) of CAR gene modification in PBMC cells under different systems and durations using existing technologies was measured.

[0079] Figure 13 The efficiency (%) of CAR gene modification in PBMC cells at different durations in a large system using the technology of this application was determined.

[0080] Figure 14 To detect the gene modification ability of engineered viral particles on T cells of a single sample in the gene modification system of this application for a short period (10 minutes);

[0081] Figure 15A To measure the recovery rate (%) of the final cells prepared at the bedside in this application under different convection velocities;

[0082] Figure 15B To demonstrate the gene modification capabilities of this application at the bedside preparation stage under different convection flow rates;

[0083] Figure 16A The proportion of genetically modified cells obtained using the genetic modification device of this application for cryopreserved PBMCs (sample 1);

[0084] Figure 16B The proportion of genetically modified cells obtained from fresh PBMCs (sample 2) using the genetic modification device of this application, ****P<0.001;

[0085] Figure 16C The proportion of genetically modified cells obtained from a blood sample (sample 3) using the genetic modification device of this application;

[0086] Figure 17 This demonstrates the adaptability of the gene modification system described in this application to different gene modification substrates;

[0087] In the diagram, 1-cell container (e.g., cell bag); 2-virus container; 4a, 4b, 4c-matrix container (e.g., matrix bag); 6a, 6b, 6c, 6d, 6e-hooks; 8a, 8b, 8c, 8d, 8e, 8f, 8g, 8h, 8i-pinch valves; 10-equipment housing; 12-pinch valve housing; 14a, 14b, 14c, 14d, 14e, 14f, 14g, 14h, 14i-pipeline interfaces; 16-mixing chamber; 18, 18a, 18b, 18c, 18d, 18e-multi-port valves; 20a, 20b, 20c-bubble sensors; 22a, 22b, 22c, 22d-pumps; 24a, 24b-pressure sensors; 26-rotatable fixture; 28-gene modification device (e.g., hollow fiber membrane module); 2 81a, 281b - End caps; 282a, 282b - Sealing rings; 283 - Outer wall; 284 - Hollow fiber membrane bundle; 285 - Inner space of hollow fiber membrane; 286 - Outer space of hollow fiber membrane; 287 - Membrane pores; 30a, 30b - Inner interface; 32a, 32b - Outer interface; 34 - Computer control terminal; 36 - Power on / off button; 38 - Display screen; 40 - Alarm; 42 - Virus removal device (e.g., centrifuge); 44, 44a, 44b - Waste liquid collection device (e.g., waste liquid bag); 46 - Cell collection device (e.g., cell collection bag); 48 - Return tubing interface; 50 - Storage space; 52 - Base; 54a, 54b - Air filters; 56a, 56b - Tubing; 100 - CAR-T bedside equipment; 110 - Blood collection device. Detailed Implementation

[0088] This invention discloses a point-of-care cell preparation device. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0089] 1) This application provides a short-term viral gene modification system.

[0090] This gene modification system is connected to the gene modification device via tubing to form a circulation loop. A pump at one end of the circulation loop pumps the virus-target cell mixture at a rate of 1.5–66.7 μL / min / cm². 2 The flow rate is from one end of the gene-editing device to the other, filling the entire gene-editing device's circulation loop, such as... Figure 1 As shown.

[0091] In some embodiments, a membrane area of ​​1.2 m² is used. 2 A hollow fiber membrane module with an in-column dead volume of 40 mL (also referred to as large volume in this application) is used, and the circulating injection flow rate of the virus and target cell mixture is 1.5–4.6 μL / min / cm. 2 In some embodiments, a membrane area of ​​75 cm² is used. 2 A hollow fiber membrane module with an in-column dead volume of 1.5 mL (also referred to as small volume in this application) is used, and the circulating injection flow rate of the virus and target cell mixture is 66.7 μL / min / cm. 2 In some embodiments, the so-called large system in this application can be a membrane area of ​​0.5m². 2 -10 m 2 For hollow fiber column components, the preferred large-scale system can be 0.5m. 2 -2.5m 2 Small systems can have a membrane area of ​​10 cm². 2 -0.1m 2 Hollow fiber column components, preferably with a small system size of 10cm. 2 -100cm 2 .

[0092] The virus and target cell mixture was circulated in the gene modification device's loop for 3–5 minutes, then the loop was disconnected. The substrate was then pumped at a rate of 1.5–15.4 μL / min / cm using pumps at both ends. 2 The flow rates are respectively flowing into the gene modification device from both ends in a convection manner, such as Figure 2 As shown.

[0093] The continuous perfusion time was 10 minutes to complete the viral gene modification of the target cells.

[0094] 2) A bedside preparation device for genetically modified immune cells

[0095] The main body of the device includes a cell infection section, a virus removal section, a sample collection device and tubing, indicator lights, and a computer control terminal. The cell infection section includes a mixing chamber, a viral gene modification system, a pressure sensor, and a pump; the virus removal section includes a virus remover and a pump. The final product after virus removal enters the sample collection device, which can be directly connected to the patient via a tubing system or connected to the patient via a reinfusion device. All parts are connected by connecting tubing such as capillaries. Figure 10 and Figure 11 As shown.

[0096] 3) A method for preparing CAR-T cells from ultrashort-time resting T cells.

[0097] Viral particles expressing activation stimuli such as anti-CD3 antibodies and CD86 proteins and containing the target CAR gene are mixed with resting T cells. Using the viral gene modification system described in this invention, ultra-short-term (within 10 minutes) gene modification of resting T cells can be achieved, providing the possibility for bedside CAR-T cell preparation.

[0098] In some embodiments, the short-term viral gene modification system provided in this application includes: a gene modification device 28, wherein a multi-way valve 18, bubble sensors 20a and 20b, pressure sensors 24a and 24b, and a pump 22a (e.g., Figure 9 (As shown). The infection mode and collection mode can be distinguished by selecting the on / off state of the multi-way valve, controlling the flow from pipe interface 14c (the pipe interface of the mixer, as shown). Figure 10 As shown, Figure 3 The sample input gene modification device 28 (with the matrix pathway interface at 14d) can complete sample introduction and viral infection under the action of pumps 22a and 22b. The gene modification device 28 has internal interfaces 30a and 30b, and external interfaces 32a and 32b (e.g., ...). Figure 10 (As shown).

[0099] In some embodiments, under the sample introduction mode: the cell virus suspension, driven by pump 22b, flows from the inner interface 30b to the inner interface 30a of the gene modification device 28. Pump 22a is turned on, and the multi-way valve 18 is set to form a circulating flow path between tubing 56a, tubing 56b, and the gene modification device, maintaining the flow for 3–5 minutes to establish a stable flow path and complete the cell introduction. The introduction rate is 1.5–66.7 μL / min / cm. 2 Flow rate (e.g.) Figure 3 (As shown). In some embodiments, cells, matrix, or cell-virus suspension may be simultaneously introduced into the gene modification device from inlet 30b and outlet 30a, respectively, for cell injection via convection at an injection rate of 1.5–66.7 μL / min / cm. 2The flow rate. The cell virus suspension is passed through tubing 56a, tubing 56b and gene modification device 28 until the tubing is full (e.g., Figure 4 (As shown).

[0100] In some embodiments, the cyclic injection process includes: first, opening the inlet and outlet, allowing the cell-virus suspension to enter the gene modification device and fill the entire device (including tubing and hollow fiber membrane assembly); then closing the inlet and outlet, allowing the cell-virus suspension to circulate within the tubing and hollow fiber membrane assembly for 3-5 minutes to ensure the suspension stably fills the entire gene modification device. The reason for using cyclic injection is to address the air and bubbles generated during the preparation process. Circulating for 3-5 minutes fills the tubing and hollow fiber membrane assembly, reducing the impact of bubbles; the air is expelled through the waste outlet by the liquid compression, and this process does not affect the circulation.

[0101] In some implementations, under virus infection mode: multi-port valve 18 closes the sample inlet line for the cell virus suspension, and opens the sample valve and access line of matrix 4b (e.g., Figure 9 As shown), under the drive of pumps 22a and 22b, the matrix 4b simultaneously perfuses the cell-virus suspension present in the tubes 56a and 56b and the gene-modification device 28 into the hollow fiber bundle inside the gene-modification device 28 in the convection direction through tubes 56a and 56b, respectively. At this time, the pumping speed directions of pumps 22a and 22b are opposite. By continuously perfusing the matrix for a set time, the cell infection process is completed (e.g., Figure 4 (As shown); the pump speed is set to 1.50~15.4μL / min / cm. 2 The continuous perfusion time is 10 minutes.

[0102] In some embodiments, the gene-modifying device 28 is a hollow fiber membrane assembly, including a gene-modifying device inlet end 30b, a gene-modifying device outlet end 30a, a porous membrane bundle 284, end caps 281a / 281b at both ends, and sealing rings 282a / 282b. The porous membrane bundle is located inside the gene-modifying device and has many fine pores 287, allowing the matrix to pass through but preventing cells and viruses from passing through. The two ends of the micelle are sealed and fixed at the inlet and outlet of the gene-modifying device with sealing rings. The gene-modifying device inlet 30b and outlet 30a are connected to an external pipeline interface. The outer shell of the gene-modifying device has one or two waste liquid outlets 32a / 32b (e.g., Figure 5 (As shown).

[0103] The states of the cell-virus suspension inside the gene-modification device 28 before, at the beginning of, and during convective infection are as follows: Figure 6 , Figure 7 and Figure 8 As shown, Figure 6 The distribution of cells and viruses in the matrix solvent, with or without contact, when the cell suspension has just entered the gene modification device at 28 minutes; Figure 7 Driven by pumps 22a and 22b, the matrix 4b simultaneously passes through pipes 56a and 56b, respectively, to infuse the cell-virus suspension present in pipes 56a and 56b and the gene modification device 28 into the hollow fiber bundle inside the gene modification device 28 in the convection direction, thereby squeezing the cell-virus suspension inside the gene modification device 28 in opposite directions, causing the virus and cells to collide closely. Figure 8 When the matrix enters the gene modification device 28 in the direction of convection, infection begins, and the cell and virus come into close contact and fully interact, further promoting gene modification.

[0104] This device can be used to mix, infect, and even amplify viral vectors with specific immune cells. It is rotatable, allowing for both horizontal and vertical placement. During use, pre-mixed cells and viruses flow into the hollow fiber membrane bundle from the inlet. Driven by a pump at the same rate, the matrix is ​​propelled into the hollow fiber membrane bundle from both ends in opposing directions (convection), promoting the interaction between cells and viruses within the membrane bundle.

[0105] by Figure 5 Taking the internal structure of the gene modification device shown as an example, in the hollow fiber membrane bundle 284, cells (from cell container 1) and viruses (from virus container 2) are uniformly dispersed in the hollow fiber membrane space 285. As liquid flows in from both ends of the hollow fiber membrane bundle 284, the cells and viruses move towards the middle, and the matrix (from matrix container 4) flows out from the hollow fiber membrane pores 287.

[0106] In some implementations, under collection mode: pump 22a is turned off, multi-port valves 18d and 18e are opened, and clamp valve 8f is opened. Matrix 4b, under the action of pump 22b, flows from the inner interface 30b of the gene modification device 28 to the inner interface 30a to flush the hollow fiber membrane module. It then enters the sample collection device through the input gene modification device 28 and clamp valve 8f, completing sample collection. Clamp valve 8g is opened, and waste liquid flows out from the outer interface 32a through pipe 8g (e.g., ...). Figure 9 (As shown).

[0107] In some implementations, a bubble sensor can be installed to monitor whether air bubbles are mixed in with the liquid passing through the pipeline, and a pressure sensor can be installed to measure the pressure in the circuit.

[0108] The gene modification device 28 can be placed horizontally, vertically, or at any angle.

[0109] In some embodiments, the gene-modification device 28 or viral gene-modification system described in this application can be used in conjunction with clinical cell therapy, along with the patient and the apheresis machine. The apheresis machine collects the total amount of PBMCs required for the patient's treatment and stores it in a cell bag; then, the cell bag containing the collected PBMCs is transferred to the cell container 1 of this device, which serves as the cell container 1 for this protocol. After cell infection and virus removal, the final gene-modified T cell product is obtained. The entire process is carried out under completely closed tubing conditions, and the entire preparation and reinfusion process can be performed without interference between the device and the patient or apheresis machine, allowing all operations to be completed normally.

[0110] In some embodiments, the gene modification device 28 and the virus container 2 described in this application can be directly installed in the single-cell collection device. One end 30b of the gene modification device 28 is connected to the virus container 2, and one end of the virus container is connected to the separation pipe of the single-cell collection device. After the mononuclear cells separated by the single-cell collection device are mixed with the virus containing the target gene pre-stored in the virus container, the mixture flows through the inner interface 30b of the gene modification device 28 to the inner interface 30a. Through the gene modification device 28, the pump 22a is turned on, and the multi-way valve 18 is set so that the cell-virus suspension forms a circulating flow path between the pipes 56a, 56b and the gene modification device and is kept flowing for 3-5 minutes to form a stable flow path of 1.5-66.7 μL / min / cm. 2 The flow rate is used to complete cell injection. For viral gene modification mode, please refer to [reference needed]. Figure 3 and Figure 4The design involves opening the valve and passage of the matrix bag (sample). Driven by pumps 22a and 22b, the matrix simultaneously flows through pipes 56a and 56b, respectively, perfusing the cell-virus suspension present in pipes 56a and 56b and the gene modification device 28 into the hollow fiber membrane bundle within the gene modification device 28 in a convection direction. At this time, the pumping speeds of pumps 22a and 22b are opposite. The cell infection process is completed by continuously perfusing the matrix for a set duration. The passage is then adjusted, and the matrix, under the action of the pumps, flows from the inner interface 30b to the inner interface 30a of the gene modification device 28 to flush the gene modification device 28. The other end 30a of the gene modification device 28 is connected to the patient access pipe, flowing into the patient input end to complete the patient reinfusion. In some embodiments, the apheresis device can be an integrated device capable of separating blood components, such as a blood component separator, apheresis machine, apheresis system, or plasma collection machine. The gene modification device 28, virus container 2, and mixing chamber can be directly installed in the single-cell collection device. One end 30b of the gene modification device 28 is connected to the mixing chamber. After the system starts running, the mononuclear cells separated by the single-cell collection device and the virus containing the target gene pre-stored in the virus container are collected in the mixing chamber for mixing. After mixing, the mixture flows through the inner interface 30b of the gene modification device 28 to the inner interface 30a. Pump 22a is turned on, and multi-way valve 18 is set to form a circulating flow path between the tubes 56a, 56b, and the gene modification device, and maintain the flow for 3-5 minutes to form a stable flow path of 1.5-66.7 μL / min / cm. 2 The flow rate is adjusted to complete cell injection. The viral suspension fills the entire tubing without limitation; adjustments can be made based on actual needs without affecting the final result. In some embodiments, the time can be 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes; in some embodiments, 1-2 minutes is preferred.

[0111] In some embodiments, the device 100 for bedside preparation of genetically modified immune cells includes a cell container 1, a virus container 2, matrix containers 4a / 4b / 4c, a mixing chamber 16, a gene modification device 28, a virus removal device 42, a cell collection device 46, a flow path system connected to the above devices, and multi-way valves 18a / 18b / 18c / 18d / 18e and pumps 22a / 22b / 22c / 22d (e.g., for adjusting flow path changes) for altered flow paths. Figure 9 (As shown).

[0112] In some embodiments, the apparatus 100 for preparing genetically modified immune cells further includes bubble sensors 20a / 20b / 20c to monitor whether the fluid in the pipeline is fully filled; pressure sensors 24a / 24b to monitor the pressure in this section of the pipeline in real time; waste collection devices 44a / 44b for collecting waste liquid during the gene modification and virus removal stages, respectively; and air filters 54a / 54b for discharging gases generated during the preparation process while ensuring that the cells are not contaminated by gases from the external environment. It also includes clamp valves 8a / 8b / 8c / 8d / 8e / 8f / 8g / 8h / 8i for controlling the opening and closing of the flow paths of the cell container 1, virus container 2, matrix container 4a / 4b / 4c, and waste collection devices 44a / 44b (e.g., ...). Figure 9 (As shown). The gene modification device 28 is the aforementioned viral gene modification system. Cell container 1, virus container 2, and matrix container 4a / 4b / 4c can be various shapes and materials of storage cavities, such as bag-structured cell collection bags, cell culture bags, matrix bags, etc., such as cuboid, cube, and polygonal containers made of materials such as metal, glass, and plastic; in some embodiments, cell container 1 can be a tubing directly from a blood collection device such as an apheresis machine, regulated by clamp valves and multi-port valves. Cell container 1 can be a sample bag for directly transferring cells from the patient to the CAR-T bedside preparation device; matrix container 4a / 4b / 4c can be a matrix bag for storing matrix, which can be used for different matrix input ports, and can add matrix to a mixing bag to dilute the cell suspension, or add it to the gene modification device to promote the infection process.

[0113] In some embodiments, the above-described bedside apparatus 100 for preparing genetically modified immune cells is as follows: Figure 10 The device shown has a housing 10, hooks 6 for securing cell bags, virus containers, and matrix bags (two or more hooks can be provided depending on the components, such as hooks 6a / 6b / 6c / 6d / 6e), and a main body. The main body includes a cell infection section, a virus removal section, disposable sterile bags and tubing, indicator lights, and a computer control terminal. 14a to 14i can be connected in any way that supports the normal operation of the device, and can include reagent bags, waste bags, various interfaces, etc., but does not include other components that may affect the overall preparation process. In some embodiments, genetically modified cells are output from 14d and then enter the virus removal device (e.g., 14f) from 14f. Figure 10 (As shown).

[0114] In some embodiments, the apparatus 100 for the bedside preparation of genetically modified immune cells described above is as follows: Figure 10The main body shown includes a clamp valve housing 12 with multiple clamp valves 8a-8e, a mixing chamber 16, a gene modification device 28, pressure sensors 24a / 24b at both ends of the gene modification device, bubble sensors 20a / 20b / 20c, pumps 22a / 22b, a computer control terminal 34, a virus removal device 42, a waste liquid bag 44, a cell collection bag 46, a base 52, and air filters 54a / 54b. The disposable sterile bag 1 and tubing are used in conjunction with the cell infection and virus removal sections, typically serving as containers for starting cells, viruses, and substrates, for mixing cells and viruses, sample transfer, and collection of samples and waste liquids. It also includes air filters and a user interface. The computer control terminal 34 has a power button and a power button 36, a large color touchscreen display 38, and an alarm 40 (e.g., ...). Figure 10 (As shown). Gene modification device 28 is the aforementioned viral gene modification system.

[0115] The device described in this application also includes a cell bag 1 and a virus container 2 for storing cells and viruses respectively, a collection bag 46 for storing biological product cells, a mixing chamber 16 for mixing cells and vectors, matrix bags 4a / 4b / 4c for storing matrix, and a waste liquid bag 44. The device is connected to a gene modification device via connecting tubes. The flow path is switched using multi-way valves 18a / 18b / 18c / 18d / 18e, pinch valves 8a / 8b / 8c / 8d / 8e, and peristaltic pumps 22a / 22b (e.g., ...). Figure 9 (As shown). In some embodiments, the device of this application further includes a rotatable mounting bracket 26.

[0116] During operation, T cells 1 from apheresis, PBMC separation, or other sources are placed on hook 6a, virus 2 on hook 6b, and substrates 4a-4c on hooks 6c-6e respectively. They then enter the mixing chamber 16, the virus gene modifier 28, or the virus removal device 42 through clamp valves 8a-8e. The mixing chamber 16 uses shaking, paddle stirring, or magnetic stirring to thoroughly mix the cells and viruses in the substrate according to set conditions (e.g.,...). Figure 11 (As shown).

[0117] Driven by pump 22b, the cell-virus solution mixed in mixing chamber 16 is introduced into hollow fiber membrane module 28 via pipeline interface 14c. This module includes a multi-port valve 18c, bubble sensors 20a and 20b, pressure sensors 24a and 24b, and pump 22a. The multi-port valve allows selection between infection and collection modes. In infection mode, samples from pipeline interface 14c are introduced into gene modification device 28, where pumps 22a and 22b complete the injection and virus infection. In collection mode, matrix 4b from pipeline interface 14d is introduced into gene modification device 28, where pump 22b completes sample collection. Bubble sensor 20 monitors for air bubbles in the liquid flowing through the pipeline. Pressure sensor 24 measures the pressure within the circuit. A rotatable bracket 26 secures the hollow fiber membrane module 28 to the device housing 10. The bracket 26 can be rotated to position the gene modification device vertically or at any angle. The hollow fiber membrane module 28 has internal interfaces 30a and 30b, and external interfaces 32a and 32b. During sample introduction and collection, the sample or matrix 4b from the mixing chamber 16 flows from the internal interface 30b to the internal interface 30a of the hollow fiber membrane module 28 under the drive of the pump 22b, and the waste liquid flows out from the external interface 32a. During infection, the matrix 4b flows into the hollow fiber membrane module from the internal interface 30a and the internal interface 30b respectively under the drive of the pumps 22a and 22b, and the waste liquid flows out from the external interface 32a.

[0118] like Figure 10 and Figure 11 As shown, the virus removal device 42 is equipped with tubing interfaces 14f and 14g, which can be used as cell collection and washing containers. After infection, the sample enters the virus removal device 42 through tubing interface 14f. The virus removal device can be a centrifuge, centrifuge bag, or other centrifugation equipment. Matrix 4c is added, and the sample is washed multiple times. Finally, a small volume is resuspended to collect the cell product, which enters the cell collection bag 46 through tubing interface 14i. Waste liquid is discharged into the waste liquid bag 44. The bubble sensor 20c monitors the presence of air bubbles in the final cell product liquid. The final product can be returned via the return tubing interface 48 or temporarily stored in the storage space 50.

[0119] The gene-editing device and the virus remover are connected by tubing to multiple intermediate bags and pumps. At the end of the virus remover, a collection bag for collecting cells is connected. The entire device also includes multiple waste bags for collecting waste from each processing step.

[0120] In some embodiments, the bedside apparatus 100 for preparing genetically modified immune cells can be connected to a blood collection device, which is connected to the patient. The bedside apparatus 100 for preparing genetically modified immune cells reinfuses the prepared genetically modified T cells into the patient through the reinfusion tubing interface 48.

[0121] In some embodiments, the method for preparing CAR-T cells from ultrashort-time resting T cells includes: mixing viral particles containing the target CAR gene and expressing anti-CD3 antibody and CD86 protein on their surface with resting T cells. Using the gene modification system described in this invention, ultrashort-time (within 10 minutes) gene modification of resting T cells can be achieved, making point-of-care CAR-T cell preparation possible.

[0122] The following are the basic concepts involved in this application:

[0123] Gene modification refers to the expression of exogenous or endogenous genes in target cells, including but not limited to the transduction of target genes into target cells such as T lymphocytes, PBMCs, apheresis cells, and target cells in blood samples through viruses such as lentiviruses or retroviruses, engineered viral particles, and nanocarriers.

[0124] Gene modification device: A tool / device / system used to achieve or enhance the genetic modification of target cells by gene transduction vectors, viruses or engineered viral particles.

[0125] Genetically modified T cells: The genetically modified T cells described in this invention refer to T cells that express exogenous or endogenous genes, including T lymphocytes modified with target genes, such as CAR-T cells (T cells modified with chimeric antigen receptor CAR), TCR-T cells (T cells modified by introducing the T cell receptor (TCR) gene that can specifically recognize tumor antigens into T cells), STAR-T cells (synthetic T cell receptor and antigen receptor T cells, T cells modified with a fusion protein similar to the T cell receptor), and other genetically modified T cells used in adoptive cell therapy.

[0126] Adoptive cell therapy (ACT) refers to a treatment method that uses cells from the body's own immune system, which are cultured and modified externally before being reinfused into the body to eliminate diseases.

[0127] PBMC (Peripheral blood mononuclear cell, referred to as mononuclear cell in this example) are cells in peripheral blood that have a single nucleus, including lymphocytes and monocytes.

[0128] A cell population or cell group refers to a composition containing one or more cells. In some embodiments, each cell in the "cell population" has one or more identical characteristics, such as having the same nuclear typing, having similar functions, being within a similar size range, having one or more identical cell markers (i.e., the one or more identical cell markers are positive), or not having one or more specific cell markers (i.e., the one or more specific cell markers are negative). The "not having" or "negative" may not necessarily mean the absolute absence of the marker. Those skilled in the art can readily compare cells to positive and / or negative controls, and / or set predetermined thresholds, and classify cells as "not having" or negative for the marker when they have expression levels below a predetermined threshold or below a predetermined threshold using conventional detection methods (e.g., using flow cytometry). For example, in some embodiments, the cell population is a T cell population. In some embodiments, the cell population is PBMCs. In some embodiments, the population is a CD4 single-positive cell population isolated from PBMCs; in some embodiments, the population is a CD8 single-positive cell population isolated from PBMCs; in some embodiments, the population is a mixed population of CD4 single-positive cells and CD8 single-positive cells isolated from PBMCs.

[0129] The terms "T lymphocytes" and "T cells" are interchangeable in this application. T lymphocytes are a type of lymphocyte. A cell population containing T lymphocytes can be T lymphocytes, leukocytes or PBMCs containing T cells, a population of T cells after CD3-positive sorting of leukocytes or PBMCs, a mixture of single-positive cells after CD4-positive and CD8-positive sorting, or a population of single-positive T cells after CD3-positive sorting of leukocytes or PBMCs containing CD4-positive or CD8-positive cells; the above cell populations can be fresh or cryopreserved.

[0130] The terms “subject,” “individual,” or “patient” are used interchangeably herein. For therapeutic purposes, “individual” refers to any animal classified as a mammal, including humans, livestock, and farm animals, as well as zoo, farm, or pet animals such as dogs, horses, cats, and cattle. In some embodiments, “individual” refers to a human individual.

[0131] Chimeric antigen receptors (CARs): A CAR is a group of engineered peptides or proteins that, when present in immune effector cells, bind to specific antigens on target cells and generate intracellular signals upon recognition of the antigen, activating downstream pathways in the cell containing the receptor to initiate the killing action of the immune effector cells on the target cells. CARs typically include at least one extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain. The extracellular antigen-binding domain specifically recognizes antigens. Non-limiting examples include single-chain variable fragments (scFv) derived from antibodies, fragment antigen-binding regions (Fab) selected from libraries, single-domain fragments or natural ligands that bind to their homologous receptors, artificially designed target-specific recognition domains such as fibronectin type III (FN3) domain combinations, and designed ankyrin repeat proteins (DARPins) that recognize specific targets. The "signaling gene modification domain" typically contains an immune receptor tyrosine-based activation motif (ITAM), whose basic composition is YXXL / V. Here, Y represents tyrosine, L / V refers to leucine or valine, and X can be any amino acid. When the receptor binds to its corresponding ligand, the tyrosine residue in the ITMA linked to it can be phosphorylated by a protein tyrosine kinase PTK connected to the cell membrane, thereby recruiting other free intracellular protein kinases or adaptor proteins to transduce activation signals into the cell. In some embodiments, the "signaling gene modification domain" is selected as an intracellular signaling gene modification domain of TCRζ (CD3ζ) or FcεRIγ. As used herein, the "co-stimulatory domain," also known as the "co-stimulatory signaling domain," is primarily used to provide co-stimulatory signals to enhance the capabilities of immune cells, including, for example, enhancing the proliferation, survival, and / or development of memory cells. In some embodiments, the "co-stimulatory domain" is selected from intracellular domains of CD28, 4-1BB (CD137), OX40 (CD134), etc. As used herein, the "transmembrane domain," also known as a "transmembrane region," refers to a thermodynamically stable protein structural region anchored within the cell membrane. Transmembrane domains can be obtained from natural proteins, such as transmembrane domains derived from the T cell receptor (TCR). In some embodiments, the transmembrane domain is selected from transmembrane domains of CD4, CD8α, CD28, and CD3ζ.

[0132] In some embodiments of this application, the extracellular antigen-binding region of the "chimeric antigen receptor" or "CAR" structure can recognize target molecules expressed on the surface of solid tumor or hematologic malignancy cells / tissues. These target molecules include, but are not limited to, CD19, CD20, CD22, CD33, CLL-1 (CLEC12A), CD7, CD5, CD70, CD123, CEACAM5, CEACAM6, CEACAM7, Mesothelin, MUC1, CLDN18.2, and CDH17. Trop2, BCMA, NKG2D, PDL1, EGFR, EGFRVIII, PSCA, PSMA, MUC16, CD133, GD2, IL13R2, B7H3, Her2, CD30, SLAMF7, CD38, GPC3, WT1, AFP, FOLR1, c-Met, LeY (Lewis-Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), MAGE-A1 (melanoma-associated antigen 1), or TAG-72, etc.

[0133] "Chimeric antigen receptors" or "CARs" can have various structures, such as those containing secretible or membrane-expressed cytokines or antibody gene sequences; and those containing structures that can be regulated for activation or inactivation, including: suicide switches such as inducible caspase-9 (iCasp9), thymidine kinase (HSV-TK) and suicide epitopes in herpes simplex virus, truncated EGFR (EGFRt), and Fas-FasL apoptosis structures; and induced CAR structures such as: peptide neo-epitope (PNE), fluorescein (FITC), 10 amino acids (5B9 tag), FITC-HM-3 bifunctional molecule (FHBM) and scFv, leucine ZipFv linked to antibody, streptavidin 2 (mSA2) biotin-binding domain, VIPER CAR inducible structures, and biotin-biding immune receptors. The receptor (BBIR) system; the "logic gate" regulatory system that binds to the SynNotch receptor, etc.

[0134] Virus: The virus described in this application may be a virus with an envelope component. The enveloped viral vector particle according to the present invention comprises at least the following components: (i) an envelope component (in this application, "enveloping" and "emvesicle" are used interchangeably), which consists of a phospholipid bilayer bound to an envelope protein, wherein the envelope protein comprises at least the chimeric or modified glycoproteins defined above, the envelope surrounding (ii) a core component of structural proteins assembling virus-like particles, the core itself surrounding (iii) a genomic component typically composed of ribonucleic acid (RNA) and (iv) an enzyme component. The biological material may be present within the envelope, the core, and / or the genomic component. In some embodiments, the virus with the envelope component may be a retrovirus, such as a lentivirus, gamma retrovirus, etc.; in some embodiments, the virus with the envelope component may be a recombinant adenovirus (Ad), such as a replication-defective human recombinant adenovirus V5 (HadV5). This technical solution operates on the same principle across all viruses with envelope components, and therefore does not differ based on the type of virus. It is applicable to all viruses with envelope components. Here, we will take lentivirus as an example to provide a detailed breakdown of this technical solution.

[0135] The conventional viruses used in this application refer to second- or third-generation lentiviral vector systems including psPAX2, pMDlg / pRRE, and pRSV-Rev. The psPAX2 plasmid also contains coding sequences for gag, pol, rev, and tat; the pMDlg / pRRE plasmid contains coding sequences for gag and pol; and the pRSV-Rev plasmid contains a coding sequence for rev. The virus in this embodiment is a lentiviral vector particle, comprising at least the following components: (i) an envelope component (in this application, "enveloping membrane" and "enveloping membrane" are used interchangeably), which consists of a phospholipid bilayer bound to envelope proteins, wherein the envelope proteins at least include chimeric or modified glycoproteins as defined above; the envelope surrounds (ii) a core component composed of gag protein binding; the core itself surrounds (iii) a genomic component typically composed of ribonucleic acid (RNA) and (iv) an enzyme component (pol). The biological material may be present within the envelope, the core, and / or the genomic component. Lentiviral vectors can be readily prepared by those skilled in the art, for example, by following the general guidelines provided by Sandrin et al. (2002) Bioood 100:823 832. In short, lentiviral vector particles can be generated by co-expressing packaging elements (i.e., core and enzyme components), genomic components, and envelope components in production cells (e.g., 293T human embryonic kidney cells or their derivatives).

[0136] Engineered viral particles: These refer to particles with an envelope containing one or more proteins that facilitate the mediating of lentiviral infection of immune cells. In some embodiments, the engineered viral particles may package nucleic acid molecules or be empty viral shell particles. Empty viral shell particles are viral particles that do not contain nucleic acid molecules; they may contain a capsid and an envelope, or may contain only an envelope. In some embodiments, the nucleic acid molecules contained in the viral particles contain a nucleic acid sequence encoding a chimeric antigen receptor (CAR). In some embodiments, the viral particles are lentiviral particles.

[0137] Molecules that help mediate lentiviral infection of immune cells: These are molecules that, when they bind to molecules on the membrane of immune cells, can prompt immune cells to recognize viral particles, thereby promoting lentiviral adsorption, invasion, uncoating, replication, and / or release. In some embodiments, the immune cells may include T cells (including αβT cells, γδT cells, Treg, and other phenotypic classifications), NK cells, macrophages, DC cells, etc.; in some embodiments, the immune cells are preferably T lymphocytes, which may be iPS-derived cells, or T lymphocytes derived from frozen, refrigerated, or fresh peripheral blood, umbilical cord blood, or tumor tissue; in some embodiments, the molecules that help mediate lentiviral infection of immune cells include molecules that can stimulate T cell activation, such as proteins or other compounds that can activate T cell co-receptors or co-stimulatory molecules, such as antibodies; in some embodiments, the molecules that can stimulate T cell activation may be molecules that stimulate the CD3 / TCR complex; or they may be molecules specifically expressed on the surface of T cells; in some embodiments, the molecules that help mediate lentiviral infection of T cells may be indicator molecules of T cell activation, such as CD69; in some embodiments, the molecules that help mediate lentiviral infection of T cells may be molecules expressed by T cells at different differentiation stages, such as CD27; in some embodiments, the molecules that help mediate lentiviral infection of immune cells may be expressed on various immune cells, such as CD7.

[0138] The chimeric antigen receptor (CAR) used in this embodiment is a CD19-targeting chimeric antigen receptor, and the CAR-T cells are T lymphocytes expressing a CD19-targeting CAR. Specifically, the amino acid sequence of the CD19-targeting CAR is shown below:

[0139] DIQMTQSPSSSLSASVGDRVTITCRASQDISKYLNWYQQKPGKAPRLLIYHTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPYTFGGGTRLEIKGSTSGSGKPGS GEGSTKGQVQLQESGPGLVKPSQTLSLTCTVSGVSLPDYGVSWIRQPPGKALEWLGVIWGSETTYYSTSLKTRLTISKDNSKNQVVLTTMTNMDPVDTATYYCAKHYYYGGSYAMDYWG QGSSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC ELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:1).

[0140] Pump: A machine for conveying or pressurizing fluids. The pump described in this application may be a positive displacement pump, a dynamic pump, or other types of pumps, such as piston pumps, plunger pumps, gear pumps, screw pumps, vane pumps, water ring pumps, centrifugal pumps, axial flow pumps, mixed flow pumps, vortex pumps, etc. In some instances, the pump may be a peristaltic pump.

[0141] Hollow fiber membrane module: Includes an inner interface, an outer interface, hollow fiber membrane bundles, and a sealing layer. The hollow fiber membrane bundles are located inside the hollow fiber membrane module, with pore sizes smaller than viruses and cells. Both ends are sealed and fixed within the hollow fiber membrane module using sealing rings. The inner interface of the hollow fiber membrane module is connected to the outer pipeline interface. The outer wall of the hollow fiber membrane module has two outer interfaces, which can serve as waste liquid discharge outlets. Specifically, the hollow fiber membrane module 28 includes end caps 281a and 281b at both ends, and an outer wall 283. The end caps 281a and 281b contain inner interfaces 30a and 30b, and the outer wall 283 contains outer interfaces 32a and 32b. Inside, there are numerous hollow fiber membrane bundles 284. The sealing layers 282a and 282b separate the internal space 285 of the hollow fiber membrane from the external space 286. The hollow fiber membrane bundles have numerous fine pores 287, allowing the matrix to pass through but preventing cells and viruses from passing through.

[0142] Substrate: This refers to a nutrient substrate composed of different nutrient components that supplies nutrients for cell growth and reproduction. Substrate can be clinical-grade reagents, such as PLA + 10% HSA, or research-grade reagents, such as 1640 + 10% FBS, or clinical basal culture media such as OpTmizer. TM CTS TM (LifeTech, Immunocult) TM XF (Stemcell Technologies), CellGro TM (CellGenix), TeXMacs TM Miltenyi (Miltenyi Corporation), Stemline TM (Sigma Corporation), Xvivo15 TM (Lonza Group, Switzerland), PrimeXV (Irvine Scientific), StemXVivo (RandD Group), basal media (MinimalEssential Media), α-MEM, RPMI 1640 medium, AIM-V, DMEM, F-12, X-vivo 15 (Lonza), X-Vivo 20, OpTmizer, and IMDM, etc.

[0143] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0144] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0145] It should be understood that the order of steps or the sequence of actions is not important as long as this application remains operational. Furthermore, two or more steps or actions may be performed simultaneously.

[0146] The use of any and all instances or exemplary language such as “e.g.” or “include” in this document is intended merely to better illustrate the application and does not constitute a limitation on the scope of the application. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of this application.

[0147] Furthermore, the numerical ranges and parameters used to define this application are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise explicitly stated, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately." Here, "approximately" generally means an actual value within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0148] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0149] To further illustrate the present invention, the following detailed description of an apparatus for bedside CAR-T preparation provided by the present invention is provided in conjunction with embodiments.

[0150] Example 1: Ultra-short-time and highly efficient gene modification of T cells using a cell gene modification device

[0151] The cell gene modification apparatus used includes two systems: a 40 mL system and a 1.5 mL system. The 40 mL system has a membrane area of ​​1.2 m². 2 Hollow fiber membrane module (polysulfone membrane hollow fiber dialyzer; manufacturer: Weigao; model: F12); 1.5 mL system with a membrane area of ​​75 cm² 2 Hollow fiber membrane module (trade name: hollow fiber column; manufacturer: Riplekin; model: D02-E100-10-N).

[0152] PBMCs (mononuclear cells) were separated using Ficoll separation solution (Tianjin Haoyang, catalog number HY2015). The Ficoll separation solution was slowly added to normal human blood to maintain a clear separation interface between the Ficoll separation solution and the normal human blood. A 50mL centrifuge tube containing the blood and separation solution was centrifuged at approximately 15°C for 20 minutes. After centrifugation, the liquid surface separated into four layers: an upper layer of plasma mixture, a lower layer of erythrocytes and granulocytes, and a middle layer of Ficoll fluid. At the boundary between the upper and middle layers, there was a narrow band of white, cloudy layer dominated by PBMCs, which was the PBMC cell layer. The upper plasma mixture was carefully aspirated using a sterile Pasteur pipette, and then the PBMCs were aspirated using a new sterile Pasteur pipette to obtain the separated PBMCs. PBMCs or T cells generated from PBMCs through sorting (or cells revived after cryopreservation of the above cells, where the PBMC cells and T cells are a mixed cell population containing CD4 and CD8 positive cells) were also obtained.

[0153] The PBMCs extracted using the density gradient method were activated by incubation with CD3 / CD28 beads (Dynabeads CD3 / CD28 CTS; manufacturer: Thermo Fisher Scientific; catalog number: 40203D) for 48 hours. The activated PBMCs were then subjected to the following experiments:

[0154] Methods for constructing lentiviral vectors are known in the art and are specifically described in references such as Naldini et al. (2000) Adv. Virus. Res. 55:599-609 and Negre et al. (2002) Biochimie 84:1161-1171. For example, lentiviral vectors can be readily prepared by those skilled in the art by following the general guidelines provided by Sandrin et al. (2002) Blood 100:823-832.

[0155] Gene modification was performed in a large system using the method described in patent CN115735006A. The groups were as follows: (1) Cells and viruses were subjected to convective gene modification in a gene modification device for 10 min; (2) Cell-virus suspension was placed in a culture plate and incubated for 10 min for static infection; (3) Cells and viruses were subjected to convective gene modification in a gene modification device for 90 min; (4) Cell-virus suspension was placed in a culture plate and incubated for 90 min for static infection; (5) Unmodified PBMCs. The matrix used was compound electrolyte injection PLA (manufacturer: Hebei Tiancheng Pharmaceutical Co., Ltd.; National Drug Approval Number: H20123411).

[0156] In the method of patent CN115735006A, activated PBMCs were added to the hollow fiber membrane module from both ends at a cell concentration of 1E6 cells / mL and a conventional virus concentration of 1 MOI, respectively, for 10 min and 90 min of convection. The cell and virus injection rates were 7.7 μL / min / cm. 2 The rate of convection gene modification was 0.42 μL / min / cm. 2 .

[0157] Viral gene modification verification was performed in large and small systems using the viral gene modification system described in this application. The groups were as follows: (1) Cell virus suspension was convectively modified in a gene modification device for 10 min; (2) Cell virus suspension was statically infected in a culture plate in an incubator for 10 min; (3) Cell virus suspension was convectively modified in a gene modification device for 90 min; (4) Cell virus suspension was statically infected in a culture plate in an incubator for 90 min; (5) Unmodified PBMCs.

[0158] In the method described in this application, activated PBMCs were mixed with 1 E6 cells / mL of conventional virus to form a homogeneous cytovirus suspension. This suspension was then added to the gene modification device via cyclic injection for 10 min and 90 min of convection (i.e., convection gene modification). The cell and virus injection rates in the large system were 4.6 μL / min / cm². 2 The rate of convection gene modification was 15.4 μL / min / cm. 2 In the small system, the cell and virus injection rate was 66.7 μL / min / cm. 2 The rate of convection gene modification was 6.7 μL / min / cm. 2 The cell-virus suspension was pumped at 4.6 μL / min / cm. 2 Or 66.7 μL / min / cm 2 The flow rate is increased by adding the gene modification device. The cell-virus suspension is introduced through tubing 56a, tubing 56b, and the gene modification device 28, filling the tubing (filling the tubing in 1-2 minutes). Then, the multi-port valve 18 is closed, the cell-virus suspension inlet tubing is closed, and the sample valve and access passage of matrix 4b are opened (e.g., ...). Figure 9 As shown), under the drive of pumps 22a and 22b, the matrix 4b simultaneously passes through pipes 56a and 56b, respectively, to perfuse the cell-virus suspension present in pipes 56a and 56b and the gene modification device 28 into the hollow fiber bundle inside the gene modification device 28 in the convection direction for gene modification. The pump speed is set to 15.4 μL / min / cm. 2 Or 6.7 μL / min / cm 2 The convection was set for 10 minutes and 90 minutes respectively.

[0159] After the gene modification period ended, cells and virus suspensions were harvested from the gene modification device, and the cell recovery rate was calculated [Cell recovery rate = Cells collected after entering the gene modification device / (Total number of cells to be gene-modified - Cells not entering the gene modification device) × 100%]. Residual viruses were then removed by centrifugation and washing, and the cells were seeded into culture plates. To more accurately detect the ability of cells to be gene-modified, the gene-modified cells were further amplified in vitro until day 7. Cells were then collected for flow cytometry analysis to determine the proportion of gene-modified cells. The cell recovery rate in the gene modification device and the gene modification efficiency of the above samples under different conditions are shown in Tables 1 and 2. Figure 12 , Figure 13 As shown. Figure 12 To determine the CAR gene modification efficiency (%) of PBMC cells at different durations in a large system using existing technologies; Figure 13Table 3 shows the gene modification efficiency (%) of CAR gene in PBMC cells at different durations in a large system using the technology of this application; Table 3 shows the gene modification efficiency (%) of the gene modification device described in this application in the small system scheme of this application.

[0160] from Figure 12 The results show that, according to the method of patent CN115735006A, the gene modification efficiency of activated PBMCs in a large system with infection times of 10 min and 90 min is less than 5% in both the short time (10 min) and the long time (90 min), and the gene modification efficiency is extremely low and cannot meet the product requirements.

[0161] from Figure 13 As can be seen, the gene modification device described in this application can achieve ultra-short (10 minutes) and highly efficient T cell gene modification, with a gene modification efficiency of 11.9%. Extending the gene modification time to 90 minutes can improve gene efficiency. In this embodiment, the gene modification device described in this application uses a 40mL large-system hollow fiber assembly. The gene modification device described in this application can be used for large-scale CAR-T cell preparation, meeting more clinical needs.

[0162] Table 1: Figure 12 Statistics on corresponding gene modification efficiency (%)

[0163]

[0164]

[0165] Table 2: Statistics on gene modification efficiency (%) corresponding to Table 13

[0166] Day 7 of culture after gene modification Large system 10min 11.9 Let the incubator stand for 10 minutes 0.82 Large system 90min 15.09 Let it stand in the incubator for 90 minutes 3.01 Unmodified 0.35

[0167] Table 3: Gene modification efficiency (%) of the small-scale system using the method of this application at 90 minutes

[0168] Day 7 of culture after gene modification This application's small system 7.00 Unmodified 0.35

[0169] In summary, the gene modification device described in this application can be used for the preparation of clinically modified cells, and the gene modification effect is superior to existing methods. This application uses a cell and virus injection rate of 66.7 μL / min / cm. 2 The rate of convection gene modification was 6.7 μL / min / cm. 2 The proposed method can also successfully complete viral transduction (gene modification) of cell-virus suspensions. The gene modification device described in this application operates at viral suspension circulation rates of 1.5–66.7 μL / min / cm. 2 The matrix convection velocity was 1.50–15.4 μL / min / cm. 2Under certain conditions, gene modification of cells in viral cell suspensions can be achieved.

[0170] Example 2: Cyclic sample introduction extends the lifespan of gene modification devices

[0171] In existing technologies, to improve gene modification efficiency, convection injection is employed at both ends of the hollow fiber membrane bundle during sample introduction. During the gene modification stage, matrix convection promotes virus-cell contact. However, in practice, prolonged high-flow-rate convection can cause severe losses to the viral gene modification system or device due to convective impact. When the injection rate is set to 53 μL / min / cm... 2 At that time, cells were mixed with virus (2 MOI) at a cell density of 2E6 cells / mL and then entered the hollow fiber membrane bundle from both ends. During perfusion, a high-pressure alarm was triggered, and the external interface 30b of the gene modification device was suddenly disconnected from the connected tubing, causing the cell-virus suspension to be ejected from the hollow fiber membrane bundle. This application changes the convection injection method to a circulating injection method, retaining the convection protocol only during the gene modification stage. The circulating injection is gentler and extends the lifespan of the gene modification device.

[0172] The overall layout diagram of the cyclic injection in this application is shown below. Figure 9 As shown, the specific loop flow path is as follows: Figure 3 , Figure 4 As shown, in sample introduction mode, the cell-virus suspension, driven by pump 22b, flows from the inner interface 30b to the inner interface 30a of the gene modification device 28. Pump 22a is turned on, and the multi-way valve 18 is set, allowing the cell-virus suspension to form a circulating flow path between tubing 56a, tubing 56b, and the gene modification device, maintaining flow for 3–5 minutes to establish a stable flow path and complete cell introduction. The introduction rate is 1.5–66.7 μL / min / cm. 2 The flow rate.

[0173] Example 3: The gene modification device can perform gene modification on unsorted single-collection resting samples in a short time and with high efficiency.

[0174] Because the cells in the single-collected sample are in In the initial state (i.e., mature but not stimulated by antigens), the cell membrane and intracellular signaling pathways are inactive, making it difficult for exogenous genes to effectively enter the cell through viral vectors and integrate into the genome during gene modification. This results in low gene modification efficiency, which directly affects the success rate of CAR-T cell preparation. The specific example in patent CN115735006A uses isolated and activated PBMC cells for gene modification.

[0175] The gene modification device of the present invention was used to validate single-sample samples. Mononuclear cell samples obtained from the apheresis machine were not separated and were gene modified in the gene modification device (40 mL system). (1) Mononuclear cell samples and viral suspensions were convection-modified in the gene modification device for 10 min; (2) Mononuclear cell samples and viral suspensions were incubated statically in a culture plate at room temperature for 10 min; (3) Mononuclear cell samples were not gene modified. Single-sample samples obtained from the apheresis machine were not activated (resting cells) and were mixed at a cell concentration of 1E6 cells / mL with virus particles (engineered virus particles) containing the target CAR gene expressing anti-CD3 antibody and CD86 protein at a concentration of 3 MOI to form a cell-virus suspension. The cell-virus suspension was then mixed at a rate of 4.6 μL / min / cm². 2 The flow rate is increased by adding the gene modification device, and the cell virus suspension is introduced through tubing 56a, tubing 56b, and the gene modification device 28, filling the tubing; then the multi-port valve 18 is closed, the cell virus suspension inlet tubing is closed, and the sample valve and access passage of matrix 4b are opened (e.g., Figure 9 As shown), under the drive of pumps 22a and 22b, the matrix 4b simultaneously passes through pipes 56a and 56b, respectively, to perfuse the cell-virus suspension present in pipes 56a and 56b and the gene modification device 28 into the hollow fiber bundle inside the gene modification device 28 in the convection direction for gene modification. The pump speed is set to 15.4 μL / min / cm. 2 After 10 minutes of convection-based gene modification, the gene-modified cells in the gene modification device were collected, and residual viruses were removed by centrifugation. Finally, the cells were seeded into culture plates. Each group of cells was cultured for 5–7 days after gene modification, and then the cells were collected for flow cytometry analysis to determine the short-term gene modification efficiency of the gene modification device on a single sample.

[0176] The results are as follows Figure 14 As shown in Table 4, under ultra-short-time gene modification conditions, compared with the static gene modification group, the gene modification device can perform gene modification on single samples in a short time and efficiently, ****P<0.001.

[0177] Table 4: Figure 14 Statistical data on gene modification efficiency (%)

[0178] sample Convection for 10 minutes Let stand at room temperature for 10 minutes Untransduced Sample 1 31.28 6.52 4.57 Sample 2 38.4 4.25 11.22 Sample 3 50.44 2.41 11.22 Sample 4 45.83 1.38 1.12 Sample 5 26.44 0.95 0.31

[0179] The results show that the gene modification device of this application can be used for unsorted single-collection resting samples, and can perform gene modification more efficiently in a short time when using viral particles (engineered viral particles) with surface expression of anti-CD3 antibody and CD86 protein.

[0180] Example 4: Gene modification efficiency of the gene modification device at convection velocities of 1.5–15.4 μL / min / cm²

[0181] In practice, excessively high convection velocities can lead to excessively high transmembrane pressure in the gene modification device, causing damage or even rupture. Conversely, excessively low convection velocities can affect short-term gene modification efficiency. Therefore, we validated the convection velocities. Cells from a single sample were mixed with a virus containing the target gene to form a viral-cell suspension.

[0182] (1) The viral cell suspension was prepared at a rate of 4.6 μL / min / cm 2 The matrix was loaded into the gene-modification device at a flow rate of 1.5 μL / min / cm from both ends of the device. 2 3.2 μL / min / cm 2 7.8 μL / min / cm 2 Or 15.4 μL / min / cm 2 (1) Genetically modify the convection velocity; (2) Incubate the virus cell suspension in a culture plate at room temperature for 10 min; (3) Cells without gene modification. In this example, a 40 mL large-system hollow fiber assembly was used, and the matrix used was compound electrolyte injection PLA (manufacturer: Hebei Tiancheng Pharmaceutical Co., Ltd.; National Drug Approval Number: H20123411). For specific convection methods, refer to Example 1.

[0183] In this embodiment, PBMCs were resuscitated and mixed with virus (3 MOI, the same engineered viral particles used in this embodiment as in Example 4) at a cell concentration of 1E6 cells / mL. In the gene modification device, the premixed cell-virus suspension was added to the device for convective gene modification for 10 min, at a rate of 1.5 μL / min / cm². 2 3.2 μL / min / cm 2 7.8 μL / min / cm 2 Or 15.4 μL / min / cm 2 The convection velocity was genetically modified. After convection gene modification was completed, cells and virus suspensions were harvested from the gene modification device, centrifuged and washed to remove residual virus, and the cells were seeded into culture plates. All cells were expanded for 5–7 days after gene modification, and then collected for flow cytometry analysis to detect the ultrashort-time gene modification efficiency. The results are shown in Figure 15. Figure 15A To determine the final cell recovery rate at different convection velocities; Figure 15B To assess the gene modification capability of the gene modification device under different convection velocities.

[0184] like Figure 15AIt can be seen that, within the same gene modification time, in a gene modification device filled with cell-virus suspension, the matrix at a flow rate of 1.5 μL / min / cm 2 3.2 μL / min / cm 2 7.8 μL / min / cm 2 and 15.4 μL / min / cm 2 The convection rate and cell recovery rate remained consistently above 70%; and as Figure 15B It is evident that both methods can achieve high gene modification efficiency, and the efficiency of gene modification gradually increases with the increase of convection velocity.

[0185] Example 5: Gene modification device can perform gene modification on bedside samples in a short time and with high efficiency.

[0186] When the gene modification device described in this application is used as a bedside device, the sample can be frozen PBMC extracted using a density gradient method, fresh PBMC extracted using a density gradient method, a single sample as described in Example 3, or a blood environment. For all of the above samples, the gene modification device described in this application can achieve short-time and efficient gene modification. The specific process in the gene modification device is described in Example 1.

[0187] Sample 1: PBMCs extracted using the density gradient method and frozen in liquid nitrogen for thawing and resuscitation before use; Sample 2: Fresh PBMCs extracted using the density gradient method and used immediately for experiments; Sample 3: Diluted blood. The above samples were mixed with 3 MOI of virus particles (engineered virus particles) containing the target CAR gene and expressing anti-CD3 antibody and CD86 protein on the surface to form a cell-virus suspension, which was verified using a gene modification device (large system (40 mL)). The groups were: (1) Cell-virus suspension was convectively modified for gene modification in the gene modification device for 10 min; (2) Cell-virus suspension was statically infected in a culture plate for 10 min; (3) Unmodified PBMCs. The hollow fiber component used in this example was a 40 mL large system, and the matrix used was compound electrolyte injection PLA (manufacturer: Hebei Tiancheng Pharmaceutical Co., Ltd.; National Drug Approval Number: H20123411).

[0188] The gene modification efficiency of the above three different types of samples under different conditions is as follows: Figures 16A-16C As shown. Figure 16A The proportion of genetically modified cells obtained using the genetic modification device of this application for cryopreserved PBMCs (sample 1); Figure 16B The proportion of genetically modified cells obtained from fresh PBMCs (sample 2) using the genetic modification device of this application, ****P<0.001; Figure 16CThe proportion of genetically modified cells obtained from a blood sample (Sample 3) using the genetic modification device of this application.

[0189] In the gene-modification device, three different samples were premixed with a cell concentration of 1E6 cells / mL and a viral gene-modification system of 3 MOI, and then filled into the gene-modification device at a rate of 15.4 μL / min / cm³. 2 Gene modification was performed by convection at a flow rate of 10 min. After the gene modification time was completed, the cells were centrifuged and washed to remove residual virus, and then seeded into culture plates. All cells were amplified 5–7 days after gene modification, and then collected for flow cytometry analysis.

[0190] The results of this study can be found in Figures 16A-16C The gene modification device described in this application can be used to modify the genes of the three samples in a short time and efficiently.

[0191] Example 6: The gene modification device can be adapted to a variety of substrates

[0192] The matrix described in this application can be a cell culture medium, such as 1640 medium + 10% FBS, or a clinical-grade solution (e.g., PLA (compound electrolyte injection) + 10% HSA). The above two matrices were validated according to the protocol in Example 5, and the results are as follows: Figure 17 As shown, the results of this study demonstrate that various matrices, such as PLA+10% HSA and 1640+10% FBS, are suitable for the gene modification device of this application. This example uses a 40mL large-system hollow fiber assembly, and the matrices used are Compound Electrolyte Injection PLA (manufacturer: Hebei Tiancheng Pharmaceutical Co., Ltd.; National Drug Approval Number: H20123411) and RPMI Medium 1640 culture medium (manufacturer: Gibco; catalog number: C11875500BT).

[0193] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A gene modification system, characterized in that, It includes sample injection tubing, multi-way valve, pump, first tubing, second tubing, gene modification device, and collection tubing; The gene modification device includes a hollow fiber membrane assembly, comprising an outer wall and a hollow fiber membrane bundle, wherein the hollow fiber membrane bundle is disposed within the outer wall, the space within the hollow fiber membrane bundle is the hollow fiber membrane inner space, and the space between the outer wall and the hollow fiber membrane bundle is the hollow fiber membrane outer space. The outer wall is provided with a pair of inner interfaces and at least one outer interface. The two inner interfaces are respectively connected to both ends of the hollow fiber membrane bundle, so that the inner interfaces are connected to the inner space of the hollow fiber membrane and the outer interfaces are connected to the outer space of the hollow fiber membrane. The first pipeline and the second pipeline are respectively connected to the two internal interfaces; The sample inlet line, the first line, and the second line are connected by the multi-way valve; The first pipeline and / or the second pipeline are equipped with at least one of the pumps; The collection pipeline is connected to the multi-way valve, or connected to the first pipeline or the second pipeline through a second multi-way valve.

2. The gene modification system as described in claim 1, characterized in that, The pump includes a first pump and a second pump, the first pump being located in the first pipeline and the second pump being located in the second pipeline.

3. The gene modification system as described in claim 1 or 2, characterized in that, It also includes bubble sensors and / or pressure sensors.

4. The gene modification system as described in claim 3, characterized in that: (A1) The bubble sensor includes a first bubble sensor and a second bubble sensor, wherein the first bubble sensor is disposed in the first pipeline and the second bubble sensor is disposed in the second pipeline; and / or (A2) The pressure sensor includes a first pressure sensor and a second pressure sensor, wherein the first pressure sensor is disposed in the first pipeline and the second pressure sensor is disposed in the second pipeline.

5. The gene modification system according to any one of claims 1 to 4, characterized in that, The hollow fiber membrane module has a membrane area of ​​75 cm². 2 -1.2m 2 .

6. An apparatus for preparing genetically modified immune cells, characterized in that, Includes the gene modification system according to any one of claims 1 to 5.

7. The device as described in claim 6, characterized in that: (B1) The sample inlet tubing is connected to at least one of the cell container, virus container, and matrix container; and / or (B2) The sample injection line is connected to the mixing chamber, and the mixing chamber is connected to the cell container and the virus container through the pipeline respectively.

8. The device as described in claim 7, characterized in that, The mixing chamber is also connected to the matrix container via a pipeline.

9. The device according to any one of claims 6 to 8, characterized in that, The external interface is connected to the waste liquid collection device via a pipeline.

10. The device as claimed in claim 9, characterized in that, The waste liquid collection device has an exhaust port, and the exhaust port is equipped with an air filter.

11. The device according to any one of claims 6 to 10, characterized in that: (C1) The collection tubing is connected to the cell collection device; and / or (C2) The collection pipeline is connected to the virus removal device, and the virus removal device is connected to the cell collection device.

12. The device as claimed in claim 11, characterized in that, The virus removal device is connected to the waste liquid collection device or a second waste liquid collection device.

13. The device as claimed in claim 11 or 12, characterized in that, The virus removal device is connected to the matrix container or the second matrix container.

14. The device according to any one of claims 6 to 13, characterized in that, It also includes indicator lights and a computer control unit.

15. The device as claimed in any one of claims 10 to 14, characterized in that, The cell collection device is directly connected to the patient or connected to the patient through a reinfusion device.

16. A method for preparing CAR-T cells, characterized in that, Prepared based on the gene modification system according to any one of claims 1 to 5 or the device according to any one of claims 6 to 15.

17. The method as described in claim 16, characterized in that, include: Sample introduction, infection, and collection; The infection process includes: the first tubing, the second tubing, and the gene modification device are fully circulated with a cell-virus suspension; the matrix is ​​simultaneously introduced into the first and second tubings via the injection tubing, causing the cell-virus suspension to convect within the hollow fiber membrane. Cells and viruses in the cell-virus suspension are trapped and in contact within the hollow fiber membrane, while the matrix permeates from within the hollow fiber membrane to the outside. The convection velocity is 1.5–15.4 μL / min / cm. 2 .

18. The method as described in claim 17, characterized in that, The convection velocity is 1.5 μL / min / cm. 2 3.2 μL / min / cm 2 6.7 μL / min / cm 2 7.8 μL / min / cm 2 Or 15.4 μL / min / cm 2 .

19. The method as described in claim 17 or 18, characterized in that, The sample introduction includes: filling the first pipeline, the second pipeline, and the hollow fiber membrane bundle with the cell-virus suspension through the sample introduction pipeline, blocking the external interface, so that the multi-way valve connects only the first pipeline and the second pipeline to form a loop, and allowing the cell-virus suspension containing cells and viruses or engineered virus particles to circulate unidirectionally in the loop to complete the sample introduction.

20. The method as described in claim 19, characterized in that, The unidirectional circulating flow rate is 1.5–66.7 μL / min / cm. 2 .

21. The method according to any one of claims 17 to 20, characterized in that, The cells are those that have been refrigerated, those that have not been refrigerated, those that have been frozen, or those that have not been frozen; The cells are activated, unactivated, or partially activated cells; The cells include: (i) PBMC; or (ii) white blood cells; or (iii) Lymphocytes.

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