WO2023178845A1

WO2023178845A1 - Method for purifying t cells and use thereof

Info

Publication number: WO2023178845A1
Application number: PCT/CN2022/098862
Authority: WO
Inventors: 王海鹰; 曹士婷; 顾明园; 胡红明
Original assignee: 上海恒润达生生物科技股份有限公司
Priority date: 2022-03-21
Filing date: 2022-06-15
Publication date: 2023-09-28
Also published as: CN116814731A

Abstract

Provided are a method for purifying T cells and use thereof, and specifically provided is a method for purifying CD3+ T cells. The method is characterized by comprising the steps of: (1) incubating a labeled anti-CD3 antibody and PBMCs in DPBS, the ratio of CD3+ T cells in the PBMCs being less than 30%, and (2) isolating the CD3+ T cells from the PBMCs by means of the label.

Description

Methods for purifying T cells and their uses

Technical field

The invention belongs to the field of biotechnology, and specifically relates to methods for purifying T cells and their uses.

Background technique

Over the past two decades, immune cell therapy has developed dramatically in cancer treatment. Immune cell therapy uses the patient's own or donor's immune cells, which are cultured, amplified or genetically modified in vitro and then reinfused into the patient's body to eliminate or control cancer cells. CAR-T cells are a type of immune cell therapy. T cells from patients or donors have been activated in vitro and genetically modified to express cancerous cell-related antigen recognition molecule fragments on T cells, and are connected to T cells intracellularly. Receptor activating molecules and co-stimulatory signaling molecules, after binding to cancerous cell antigens, transmit downstream signals, allowing CAR-T cells to perform the function of killing cancerous cells.

Currently, seven CAR-T cell products around the world have passed regulatory review and been launched on the market. From the laboratory to the industrialization stage, the production process of CAR-T cells has undergone many improvements. The main operating steps include the following processes: blood collection from patients or donors, isolation of PBMCs from apheresis, isolation of PBMCs, and Activated T cells, CAR gene modified T cells, CAR-T cell expansion, CAR-T cell filling and cryopreservation. T cells are the basic material for CAR-T cell production, so T cells need to be isolated from PBMC and unnecessary cell populations, such as CD19 ⁺ B cells and CD14 ⁺ monocytes, need to be removed. Since CD3 is an important antigenic marker on the surface of T cells, and CD3 is not expressed on the cell membranes of B cells and monocytes, T cells are mainly isolated using magnetic beads covalently coupled with anti-CD3 and anti-CD28 antibodies and PBMC. After co-incubation, T cells bound to the magnetic beads are magnetically separated. Although the magnetic bead sorting method can remove most of the suspended CD3-negative cells, due to the characteristic of monocytes adhering to the surface of the culture container when cultured in vitro, some monocytes still remain after magnetic sorting. In the culture container, according to past literature publications and our company's research and development experience, these monocytes may also phagocytose magnetic beads and affect the activation of T cells. Therefore, how to eliminate the influence of monocytes during magnetic bead sorting is a topic that needs to be studied.

In addition, although the above-mentioned CAR-T production operation procedures can be completed in the open environment of a biological safety cabinet using culture plates and culture bottles as culture containers, and using pipettes and centrifuge tubes, however, according to the National Medical Products Administration in November 2019 Article 13 [Closed System] of the "GMP Appendix - Cell Therapy Products" (Draft for Comments) issued by the Food and Drug Review and Inspection Center of the Administration: "It is advisable to use closed equipment and pipelines for the production operations of cell therapy products; sealed The cleanliness level of the equipment and pipeline installation environment can be appropriately reduced." Therefore, the production of CAR-T cells in a closed system is more in line with the guidance of GMP principles, which is also the method currently adopted by most in the industry.

However, the T cells used to prepare CAR-T cells need to use different methods and reagents according to the CD3 ⁺ T cell positive ratio during processing, making it difficult to achieve high sealing during the entire preparation process.

Contents of the invention

By optimizing the purification process of T cells, the present invention achieves efficient purification of CD3 ⁺ T cells using a blocking process and reduces the adverse effects of monocytes on the preparation of CAR-T cells.

A first aspect of the present invention provides a method for purifying CD3 ⁺ T cells, including the steps:

(1) Incubate labeled anti-CD3 antibody and PBMC in DPBS, and

(2) Isolating CD3 ⁺ T cells in PBMCs by the label,

Wherein, the method does not include the steps of selecting an incubation buffer, adjusting cell density and incubation time according to the proportion of CD3 ⁺ T cells in PBMC.

In one or more embodiments, the proportion of CD3 ⁺ T cells in the PBMC is less than 30%.

In one or more embodiments, the label is a substance that facilitates separation of the complex of antibody and surface antigen-containing cells from other components in the system.

In one or more embodiments, the incubation mixture also contains labeled anti-CD28 antibodies. In some embodiments, the anti-CD3 antibody and/or anti-CD28 antibody is coupled to the label.

In one or more embodiments, the label includes, but is not limited to, biotin, solid phase carrier. In some preferred embodiments, the solid phase carrier is magnetic particles.

In one or more embodiments, the CD3 ⁺ T cell density in the DPBS is 4-10*10 ⁶ cells/mL, preferably 4-7*10 ⁶ cells/mL or 7-10*10 ⁶ cells cells/mL.

In one or more embodiments, the incubation is a shaking incubation, and the shaking is 20-500 rpm, preferably 50-100 rpm. In some embodiments, the incubation temperature is 10-40°C. In some embodiments, the incubation time is 20-60 minutes, preferably 30-45 minutes.

In one or more embodiments, the method specifically includes the steps of:

(a) Resuspend PBMC in DPBS, where the CD3 ⁺ T cell density is 4-10*10 ⁶ cells/mL, preferably 4-7*10 ⁶ cells/mL or 7-10*10 ⁶ cells/mL ;

(b) Add an equal volume of anti-CD3 antibody-coupled magnetic particles, shake and incubate for 20-60 minutes;

(c) Separating magnetic particles bound to CD3 ⁺ T cells by magnetic capture;

(d) Separate the magnetic particles from the cells to obtain CD3 ⁺ T cells,

Moreover, the method does not include the step of obtaining the proportion of CD3 ⁺ T cells in PBMC.

In one or more embodiments, the method further includes the step of activating CD3 ⁺ T cells, such as incubating the CD3 ⁺ T cells with IL-2-containing medium for at least 24 hours, preferably 48 hours.

In one or more embodiments, the method further includes the step of obtaining PBMC before purifying CD3 ⁺ T cells.

The invention also provides a method for purifying CD3+T cells using the Sepax C-Pro cell processing system, which includes the steps of: (1) using the Sepax C-Pro cell processing system to separate and freeze PBMCs from blood samples, and remove residues in the samples. multinucleated cells in red blood cells and white blood cells, (II) use Sepax C-Pro cell processing system to wash and revive frozen PBMC, restore the viability and functionality of PBMC, and remove dead cells, (III) use Sepax C- The Pro cell processing system performs CD3+T sorting and activation of recovered PBMCs, including using the method described in any embodiment of the first aspect of the present invention.

Preferably, the method includes the steps:

(I) Use a blood cell separator to collect 20 mL to 200 mL of plasma, and store it in a refrigerator at 2 to 8°C.

(II) Use the Sepax C-Pro cell processing system and closed tube kit (such as CT-90.1), execute the NeatCell program, separate PBMC from a single blood sample, and resuspend the PBMC in PBMC freezing solution.

(III) Use the Sepax C-Pro cell processing system and closed tube kit (such as CT-90.1), use the CultureWash program, resuspend the thawed PBMC in the culture medium, and culture at 37±1°C and 5±0.5% CO2 Overnight, the proportion of CD3 ⁺ T cells in the PBMC is less than 30%,

(IV) Using the Sepax C-Pro cell processing system and closed tube kit (e.g. CT-90.1), wash the PBMC and the magnetic beads covalently coupled to anti-CD3 and anti-CD28 antibodies with DPBS and shake at room temperature at 60 rpm in DPBS Incubate the magnetic beads and PBMC for 30 minutes, wherein the CD3+T cell density is 4-10*10 ⁶ cells/mL (preferably 4-7*10 ⁶ cells/mL or 7-10*10 ⁶ cells/mL) mL) to obtain CD3+T cells enriched on magnetic beads; then resuspend the CD3+ cells in medium containing IL-2, culture at 37±1°C, 5±0.5% CO2, and obtain purified CD3 after separating the magnetic beads. +T cells.

The invention also provides a method for preparing CAR-T cells, which includes the following steps:

(1) Use a blood cell separator to collect 20 mL to 200 mL of plasma and store it in a refrigerator at 2 to 8°C.

(2) Use the Sepax C-Pro cell processing system and closed tube kit (such as CT-90.1), execute the NeatCell program, separate PBMC from a single blood sample, and resuspend the PBMC in PBMC cryopreservation solution.

(3) Use the Sepax C-Pro cell processing system and closed tube kit (such as CT-90.1), and use the CultureWash program to resuspend the thawed PBMC in the culture medium at 37±1°C and 5±0.5% CO ₂ Culture overnight,

(4) Use Sepax C-Pro cell processing system and closed tube kit (such as CT-90.1), wash PBMC and covalently coupled anti-CD3 and anti-CD28 antibody magnetic beads with DPBS, and shake in DPBS at 60 rpm at room temperature Incubate the magnetic beads and PBMC for 30 minutes, wherein the CD3 ⁺ T cell density is 4-10*10 ⁶ cells/mL (preferably 4-7*10 ⁶ cells/mL or 7-10*10 ⁶ cells/mL ) to obtain CD3+T cells enriched on magnetic beads; then resuspend the CD3 ⁺ cells in medium containing IL-2, culture at 37±1°C, 5±0.5% _CO2 , and obtain purified cells after separating the magnetic beads. CD3+T cells,

(5) Use the Sepax C-Pro cell processing system and closed tube kit (such as CT-90.1), in a RetroNectin-coated cell culture bag, mix the cell suspension and the virus containing the CAR coding sequence at 37±1°C and Culture overnight under 5±0.5% _CO2 conditions to obtain CAR-T cells.

(6) Use the Sepax C-Pro cell processing system and closed tube kit (such as CT-90.1), wash the CAR-T cells and resuspend them in IL-2-containing medium, at 37±1℃ and 5±0.5% CO Subculture to the required number of cells under the conditions of ₂ ,

(7) Use the Sepax C-Pro cell processing system and closed pipeline kit (such as CT-90.1), use sodium chloride injection to wash the CAR-T cells and resuspend the CAR-T cells in cryopreservation solution, and use a programmed cooling device Cryopreserved cells.

The present invention also provides the use of the method for purifying CD3 ⁺ T cells according to any embodiment of the first aspect of the present invention in preparing a reagent containing activated T cells.

In one or more embodiments, the activated T cells are CAR-T cells.

The present invention also provides a method for eliminating or weakening the non-specific adhesion of cells to a solid-phase carrier, which includes incubating the cells and the solid-phase carrier in the presence of DPBS.

In one or more embodiments, the cells are preferably mononuclear cells, such as PBMCs.

In one or more embodiments, the solid support is a container wall or magnetic particles.

The invention has the following beneficial effects:

The improved procedure can effectively prevent PBMC samples with low CD3 ratio and high CD14 ratio from monocytes phagocytosis of magnetic beads during magnetic bead sorting and affect cell activation, allowing cells to be activated and expanded normally. Secondly, the improved process can simplify the operation of CD3 ⁺ T cell sorting and activation. There is no need to perform different calculations based on the proportion of CD3 in the sample, achieving a truly fully closed process and reducing the possibility of contamination and errors.

Description of the drawings

Figure 1 is a schematic diagram of the CAR-T cell preparation process in the blocking process.

Figure 2 shows the test results of CAR-T cell characteristics prepared by the closed process and the improved open process.

Figure 3 shows the functional test results of CAR-T cells prepared by the closed process and the improved open process.

Figure 4 is a comparison of the operating steps of the CD3 ⁺ T cell sorting and activation process in the closed process and the steps and parameters of the open process before improvement.

Figure 5 shows the effect of incubation buffer on the intermediate properties of CAR-T cell preparation.

Figure 6 shows the effect of magnetic bead incubation time on the intermediate properties of CAR-T cell preparation.

Figure 7 shows the impact of magnetic bead incubation on cell density on intermediate traits of CAR-T cell preparation.

Figure 8 is the experimental results of preparing CAR-T cells from three PBMC samples with low CD3 and high CD14.

Detailed ways

In the research experiment of process change, because the operation of CD3 ⁺ T cell sorting in the open process before improvement was more complicated, the CD3 ratio in the PBMC sample needs to be used to determine the type of buffer, incubation time and cell density during magnetic bead incubation. . Therefore, in the process of developing a closed process to prepare CAR-T, the inventor tried to fix the magnetic bead incubation buffer into X-VIVO medium (Lonza X-VIVO ^TM 15 medium) containing 5% human plasma to replace the original X-VIVO or DPBS. However, during the experiment, it was discovered that when X-VIVO medium containing 5% human plasma was used as the magnetic bead incubation buffer, monocytes would phagocytose the magnetic beads, affecting the CD3 ⁺ T cell sorting and activation effects. In addition, the results of the Phase I clinical trial of our company showed that the CAR-T cell expansion rate of two donors with a low CD3 ratio and a high CD14 ratio using plasma-free X-VIVO as the magnetic bead incubation buffer was significantly lower than that using DPBS serves as an additional donor of magnetic bead incubation buffer. The above two phenomena indicate that using X-VIVO as a magnetic bead incubation buffer will have adverse effects on CAR-T preparation regardless of whether human plasma is added, and the reason may be related to monocytes.

Based on this, the present invention provides an improved closed process method for purifying CD3 ⁺ T cells and its use based on the original open process, and can solve the problem of CD14 ⁺ monocytes affecting the magnetic bead sorting step. In the CD3 ⁺ T cell sorting and activation process of the present invention, when PBMC are incubated with magnetic beads covalently coupled to anti-CD3 and anti-CD28 antibodies, the operation of selecting different incubation buffers due to different proportions of CD3 cells in PBMC is deleted. Fix using DPBS as incubation buffer.

Herein, a sample is a sample of any blood source containing PBMCs, such as whole blood. Preferably, the sample is blood collected by machine apheresis unit that complies with the collection procedures and transportation methods.

The full name of DPBS is Dulbecco's Phosphate-Buffered Saline, which is Dulbecco's Phosphate-Buffered Saline. Its main ingredients include NaCl, KCl, KH ₂ PO ₄ , Na ₂ HPO ₄ , etc., with a pH of 7.2-7.4. DPBS is divided into two types according to whether it contains calcium and magnesium ions. Unlike conventional PBS, DPBS has a slightly lower phosphate content.

Therefore, a first aspect of the present invention provides a method for purifying CD3 ⁺ T cells, including the steps:

(1) Incubate labeled anti-CD3 antibody and PBMC in DPBS, and

(2) Isolating CD3 ⁺ T cells in PBMCs by the label,

Wherein, the method does not include the steps of selecting an incubation buffer, adjusting cell density and incubation time according to the proportion of CD3 ⁺ T cells in PBMC. The proportion of CD3 ⁺ T cells in the PBMC is greater than or equal to or less than 30%. The DPBS also contains labeled anti-CD28 antibodies.

As used herein, a "label" is a substance that facilitates the separation of complexes of antibodies (eg, anti-CD3 antibodies and anti-CD28 antibodies) and cells containing surface antigens (eg, CD3 and CD28) from other components in the system, such as biotin, Solid phase supports in which anti-CD3 antibodies and/or anti-CD28 antibodies are coupled to these labels. The preferred solid phase carrier is magnetic particles. Of course, other solid phase carriers commonly used in the art for coupling antibodies can also be used in the present invention.

Anti-CD3 antibodies and anti-CD28 antibodies can be any antibodies known in the art that can bind CD3 and CD28. Preferred sequences are shown in SEQ ID NO: 1 and 2.

The method of the present invention does not include the steps of selecting an incubation buffer, adjusting cell density and incubation time according to the proportion of CD3 ⁺ T cells in PBMC. The inventor found that when the cell density of PBMC is 4-10*10 ⁶ cells/mL, regardless of the proportion of CD3, DPBS can be used for cell resuspension and antibody incubation. In the existing technology, different cell densities and incubation systems need to be used according to the proportion of CD3 ⁺ T cells in PBMC: when the proportion of CD3 ⁺ T cells is greater than or equal to 30%, the cells need to be resuspended in DPBS, and the CD3 The cell density of ⁺ T cells needs to be adjusted to 1*10 ⁷ cells/mL; when the proportion of CD3 ⁺ T cells is less than 30%, the cells need to be resuspended in cell culture medium, and the total cell density during incubation needs to be adjusted to 3*10 ⁷ cells/mL. The entire process involves using the CD3 ratio in the PBMC sample to determine the type of buffer, incubation time and cell density during magnetic bead incubation, which increases the difficulty of the blocking operation. The method of the present invention does not need to obtain the proportion of CD3 ⁺ T cells in PBMC, and the entire purification process can be carried out in a fully closed manner. Preferably, the proportion of CD3+T cells in the PBMC is less than 30%.

Surprisingly, DPBS can eliminate or weaken the non-specific adhesion of monocytes to solid-phase carriers. Therefore, incubating monocytes and solid-phase carriers with DPBS can weaken the effect of monocytes occupying and engulfing solid-phase carriers. , reducing its impact on T cell activation. This may be one of the reasons why the method of the present invention does not require the selection of incubation buffer, adjustment of cell density and incubation time according to the proportion of CD3 ⁺ T cells in PBMC.

The inventors also found that according to the method of the present invention, there is no need to adjust the antibody incubation time according to the ratio of CD3 in DPBS. In the existing technology, when the proportion of CD3 ⁺ T cells is greater than or equal to 30%, the incubation time cannot exceed 30 minutes; when the proportion of CD3 ⁺ T cells is less than 30%, the incubation time needs to reach 60 minutes. In the method of the present invention, the incubation time does not need to be adjusted and can be 20-60 minutes, preferably 30-45 minutes.

The incubation is shaking incubation, and the shaking is 20-500 rpm, preferably 50-100 rpm, such as 60 rpm.

The method for purifying CD3 ⁺ T cells of the present invention specifically includes the steps:

(1) Resuspend PBMC in DPBS, the CD3 ⁺ T cell density is 4-10*10 ⁶ cells/mL, preferably 4-7*10 ⁶ cells/mL or 7-10*10 ⁶ cells/mL ,

(2) Add an equal volume of anti-CD3 antibody-coupled magnetic particles, shake and incubate for 20-60 minutes.

(3) Separate magnetic particles bound to CD3 ⁺ T cells by magnetic capture,

(4) Separate magnetic particles from cells to obtain CD3 ⁺ T cells,

The method also includes the step of activating CD3 ⁺ T cells. Methods for activating CD3 ⁺ T cells are well known in the art, such as incubating CD3 ⁺ T cells with IL-2-containing medium for at least 24 hours, preferably 48 hours.

Before purifying CD3 ⁺ T cells, the method usually also includes steps of obtaining PBMC, such as blood cell extraction, PBMC separation, and PBMC recovery, which are all common knowledge of those skilled in the art. Exemplarily, the present invention provides a method for purifying CD3 ⁺ T cells using the Sepax C-Pro cell processing system: (1) using the Sepax C-Pro cell processing system to separate and freeze PBMCs from blood samples, and remove residual red blood cells in the samples and multinucleated cells in leukocytes, (2) use the Sepax C-Pro cell processing system to wash and revive the frozen PBMC to restore the viability and functionality of the PBMC, and remove dead cells, (3) use the Sepax C- The Pro cell processing system performs the method of purifying CD3+T cells of the present invention on the recovered PBMC, and performs CD3 sorting and activation on the PBMC. Typically, the Sepax C-Pro Cell Processing System is used with its corresponding closed circuit kit CT-60.1. Specifically, the specific steps of the method of purifying CD3 ⁺ T cells using the Sepax C-Pro cell processing system include:

Sample Collection:

Use a blood cell separator (

Spectra, Spectra

Fenwal ^TM

or equivalent standard mechanical collection equipment) to collect and separate white blood cells and plasma in the central clinical ward. The volume of single blood collection is about 20 mL to 200 mL, and the volume of plasma is about 20 mL to 200 mL. Place the collected blood and plasma samples in refrigerated transport boxes at 2 to 8°C and transport them to the cell preparation center using cold chain logistics. It is well known to those skilled in the field that according to the "Good Manufacturing Practice for Pharmaceuticals (2010 Revision)", clean areas are classified into ABCD. Level A: high-risk operation areas, such as filling areas, rubber stopper barrels and direct contact with sterile preparations. For areas in contact with open packaging containers and areas for aseptic assembly or connection operations, a one-way flow operating table (hood) should be used to maintain the environmental status of this area. The one-way flow system must supply air evenly in its working area, with a wind speed of 0.36-0.54m/s (guideline value). There should be data to prove the status of the unidirectional flow and be verified. In a closed, isolated operator or glove box, lower air speeds can be used. Level B: refers to the background area where high-risk operations such as aseptic preparation and filling are located in the Class A clean area. Level C and D: refer to clean areas with less important steps in the production of sterile drugs. In the present invention, the following cell processing steps are started in the B+A (i.e., grade A environment under grade B background) or C+A (grade A environment under grade C background) environment of the cell preparation center.

PBMC isolation:

PBMC (Peripheral Blood Mononuclear Cell, PBMC) refers to mononuclear cells in peripheral blood, mainly including lymphocytes, monocytes and other small amounts of cells. It is an important raw material for current T cell-related cell therapy.

This process uses the Sepax C-Pro cell processing system and the corresponding disposable closed tube kit CT-90.1 to separate PBMC from a single blood sample. First, the CT-90.1 kit is installed on the Sepax C-Pro cell processing system, then the apheresis bag containing the patient's apheresis blood is connected to the CT-90.1 kit, and the NeatCell program developed by the instrument manufacturer is executed to isolate PBMC. The NeatCell program finally resuspends PBMC in PBMC cryopreservation solution and outputs it to the cell cryopreservation bag. The cell cryopreservation bag is cryopreserved in a programmable cooling device, and finally transferred to a liquid nitrogen tank for storage. After the blood bag containing the donor's plasma is inactivated in a 56°C water bath for 30 minutes, the denatured proteins and other impurities in the plasma are centrifuged to settle to the bottom of the bag. Use a blood separator to transfer the supernatant plasma to a freezing bag and freeze it at -80°C for later use. The purpose of this process is to isolate the PBMC required for the preparation of CAR-T cells and remove the remaining red blood cells and multinucleated cells in the white blood cells from the single blood sample. The treated plasma was used to prepare the culture medium used throughout the preparation process, which contained 5% human plasma.

PBMC Resuscitation:

In this process, the Sepax C-Pro cell processing system and the corresponding disposable closed tube kit CT-60.1 are used to wash the thawed PBMC suspension and replace the frozen solution with culture medium to start PBMC relief culture. First, select the CultureWash program in the Sepax C-Pro cell processing system and install the CT-60.1 kit. Execute the program until the instrument prompts to connect the cell cryopreservation bag. Take out the frozen PBMC stored in liquid nitrogen, place it in a 37°C water bath, and gently shake the bag to melt the PBMC suspension. Attach the thawed cell cryopreservation bag to the kit and proceed with the CultureWash procedure. The CultureWash program finally resuspends the PBMC in culture medium and exports it to a PL325 cell culture bag (Origen Biomedical). After sampling and counting, the Dilution program of the Sepax C-Pro cell processing system is used to supplement the PBMC suspension. Adjust cell density. Place the PL325 cell culture bag containing the PBMC suspension into a carbon dioxide incubator and culture overnight at 37±1°C and 5±0.5% _CO2 . The purpose of this process is to restore the viability and functionality of PBMC and remove apoptotic cells.

CD3 ⁺ T cell purification:

This process uses the Sepax C-Pro cell processing system and the corresponding disposable closed tube kit CT-60.1 to wash the PBMC that have been cultured, and then use magnetic beads covalently coupled with anti-CD3 and anti-CD28 antibodies to PBMC undergo CD3 sorting and activation. First, install the CT-60.1 kit on the Sepax C-Pro cell processing system, take out the PBMC that have been cultured from the carbon dioxide incubator, connect the cell culture bag pipeline containing the PBMC to the 200 mesh filter, and then connect it to the kit. When the cell suspension enters the kit, it can intercept and relieve floc generated after culture. Attach the DPBS bag as washing solution and magnetic bead incubation buffer to the kit, and perform the CultureWash program to wash PBMC with DPBS. The CultureWash program finally resuspended PBMC in DPBS and exported it to PL240 cell culture bags (Origen Biomedical). After washing, PBMC were sampled and counted, and then the CD3 ⁺ cell number was calculated based on the PBMC flow cytometry phenotyping results. DPBS was added to the PBMC suspension using the Dilution procedure to adjust the CD3 ⁺ cell density. Add magnetic beads covalently coupled to anti-CD3 and anti-CD28 antibodies (CTS ^TM Dynabeads ^TM CD3/CD28) washed with DPBS into the above PL240 cell culture bag, and use a horizontal shaker to incubate the cells with magnetic beads. Mix thoroughly. After the incubation is completed, place the cell culture bag on the CTS DynaMag magnetic separator. The CD3 ⁺ cells bound to the magnetic beads will be magnetically captured and retained in the cell culture bag. The supernatant and CD3 ^- cells will be discarded. Reconnect the cell culture bag to the kit and add IL-2-containing medium using the Dilution program to resuspend the CD3 ⁺ cells. Place the PL240 cell culture bag containing the CD3 ⁺ cell suspension into a carbon dioxide incubator, and activate and culture it in an environment of 37±1°C and 5±0.5% _CO2 . The purpose of this process is to sort out CD3 ⁺ T cells used to transduce viruses, further remove other cell populations, and costimulate T cells with CD3 and CD28 antibodies to facilitate retrovirus transduction. A comparison of the steps and parameters of the CD3 ⁺ T cell purification operation steps of the closed process of the present invention and the open process before improvement is shown in Figure 4.

As used herein, "cryopreservation medium" refers to a medium used to cryopreserve cells therein. Those skilled in the art are aware of suitable cryopreservation solution compositions for cells, particularly immune cells (eg PBMC or CD3+ T cells). These cryopreservation solutions are usually commercially available.

As used herein, "culture medium" refers to the medium used to culture cells. Those skilled in the art are aware of suitable media components for cells, particularly immune cells (eg PBMC or CD3+ T cells). These cryopreservation solutions are usually commercially available, such as X-VIVO 15.

Purified CD3 ⁺ T cells can be used for subsequent research, such as identification of the expression of single-chain variable fragments on the cell surface, expression of CAR molecules on T cells, killing capacity of CAR-T cells, and cytokine secretion profiles of CAR-T cells. , CAR-T cell phenotype, in vivo pharmacodynamic studies and toxicity tests in animal models, etc. It can also be used to produce TCR-T cells and conduct non-clinical research on TCR-T cells. If T cells are not genetically modified, they can be used to perform T cell-related immunological research, such as T cell activation mechanism, T cell migration mechanism, T cell intracellular information transduction, etc.

Purified CD3 ⁺ T cells can also be used to prepare CAR-T cells. Therefore, another aspect of the present invention provides an improved method for preparing CAR-T cells, including the steps:

(1) Obtain CD3 ⁺ T cells using the method for purifying CD3 ⁺ T cells described herein;

(2) Introduce CAR into the CD3 ⁺ T cells to obtain CAR-T cells.

Correspondingly, the present invention also provides the use of the method for purifying CD3 ⁺ T cells in preparing reagents containing activated T cells. The activated T cells are CAR-T cells.

Step (2) can be performed using any method known in the art for introducing CAR into T cells. The CAR is preferably introduced into T cells via a retroviral vector.

After obtaining CAR-T, the method usually includes steps such as expansion and culture of CAR-T cells, filling and cryopreservation of CAR-T cells. Therefore, the specific steps of the preparation method of CAR-T cells of the present invention include: PBMC isolation, PBMC recovery and relief, CD3 ⁺ T cell sorting and activation, retroviral transduction of T cells, CAR-T cell expansion culture and CAR- T cells are filled and frozen, see Figure 1. Exemplarily, the present invention provides a method for preparing CAR-T cells using the Sepax C-Pro cell processing system: (1) using the Sepax C-Pro cell processing system to separate and freeze PBMCs from blood samples, and remove residual red blood cells in the samples and multinucleated cells in leukocytes, (2) use the Sepax C-Pro cell processing system to wash and revive the frozen PBMC to restore the viability and functionality of the PBMC, and remove dead cells, (3) use the Sepax C- The Pro cell processing system performs the method of purifying CD3+T cells of the present invention on the recovered PBMC, performs CD3 sorting and activation on the PBMC, (4) after separating the cells from the solid phase carrier (such as magnetic particles), use Sepax C- The Pro cell processing system washes the activated CD3+T cells, incubates the cell culture medium and virus liquid in the presence of RetroNectin, and transduces the CAR gene into the T cells. (5) Use the Sepax C-Pro cell processing system to treat the CAR- The T cells are washed and transferred to a larger volume cell culture bag to expand the cells. (6) Use the Sepax C-Pro cell processing system to wash and fill the CAR-T cells, and use a programmed cooling device to complete the CAR-T cells. Cell cryopreservation. Specifically, the specific steps of the above steps (1)-(3) are as described in the method for purifying CD3 ⁺ T cells; the specific steps of steps (4)-(6) include:

Retrovirally transduced T cells:

This process uses a magnetic separator and Sepax C-Pro cell processing system and the corresponding disposable closed pipeline kit CT-60.1 to wash the activated cells, and uses RetroNectin to mediate and increase the infection rate of retroviral liquid T cells. efficiency. First, coat the PL70-2G cell culture bag with 15 μg/mL RetroNectin working solution at 37°C. After the coating is completed, discard the supernatant in the culture bag.

Place the activated and cultured CD3 ⁺ T cells on the CTS DynaMag magnetic separator, magnetically capture the free magnetic beads that automatically fall off the cells, and transfer the CD3 ⁺ cell suspension without magnetic beads. Connect the cell suspension bag to the CT-60.1 kit installed on the Sepax C-Pro cell processing system, wash the cells with the CultureWash program, and finally resuspend the cells in IL-2-containing medium for export. After washing, the cells were sampled and counted, and the transduction MOI was calculated.

The production of virus liquid is from the retroviral vector stable transducer strain. The construction of the stable transducer strain is based on Loew R, Meyer Y, Kuehhlcke K, Gama-Norton L, Wirth D, Hauser H, Stein S, Grez M, Thornhill S, Thrasher. A, Baum C, Schambach AA new PG13-based packaging cell line for stable production of clinical-grade self-inactivating gamma-retroviral vectors using targeted integration. Gene Ther. 2010 Feb;17(2):272-80.doi:10.1038 /gt.2009.134.Epub 2009 Oct 29.PMID:19865181. The detailed steps are to thaw the frozen stably transduced cell lines and then expand the culture (the medium is DMEM+10% FBS, the culture conditions are 37°C, 5% CO ₂ ) , when the total viable cell number reaches between 1E8-1E9, the cells are seeded into multi-layer culture bottles. The culture medium is changed once after 24 hours of cell culture. The culture supernatant containing suspended virus particles is collected 24 hours and 48 hours after the medium change. The supernatant collected twice is mixed to become the virus liquid. Put the cell suspension and virus solution into the RetroNectin-coated cell culture bag and culture overnight in a carbon dioxide incubator at 37±1°C and 5±0.5% _CO2 . The purpose of this process is to remove the magnetic beads, one of the impurities in the finished product, and transduce the CAR gene into T cells to prepare CAR-T cells.

CAR-T cell expansion culture:

CAR-T, Chimeric Antigen Receptor-T cell (CAR-T) T cells refer to T cells that have been genetically modified to recognize specific target antigens in an MHC non-restrictive manner and continue to activate and expand. . The 2012 International Cell Therapy Association Annual Meeting pointed out that biological immune cell therapy has become the fourth method of treating tumors in addition to surgery, radiotherapy, and chemotherapy, and will become a necessary method of tumor treatment in the future. CAR-T cell reinfusion therapy is the most clear and effective form of immunotherapy in current cancer treatment. A large number of studies have shown that CAR-T cells can effectively recognize tumor antigens, induce specific anti-tumor immune responses, and significantly improve the survival status of patients.

This process uses the Sepax C-Pro cell processing system and the corresponding disposable closed tube kit CT-60.1 to wash the CAR-T cells and transfer them to a larger volume cell culture bag to expand the cells. First, install the CT-60.1 kit on the Sepax C-Pro cell processing system, then take out the CAR-T cell culture bag after viral transduction from the carbon dioxide incubator, connect it to the kit, and execute the CultureWash program to wash the CAR with culture medium. -T cells. The CultureWash program finally resuspends the CAR-T cells in IL-2-containing medium and exports them to PL325 cell culture bags. After washing, the CAR-T cells were sampled and counted, and then the IL-2-containing culture-based CAR-T suspension was added using the Dilution program of the Sepax C-Pro cell processing system to adjust the cell density. Place the cell culture bag back into a carbon dioxide incubator at 37±1°C and 5±0.5% _CO2 for culture, and then perform cell counting and fluid replenishment every 1-3 days. The purpose of this process is to clean and remove virus-related impurities and amplify CAR-T cells to the required number of cells.

CAR-T cell filling and cryopreservation:

In this process, the Sepax C-Pro cell processing system and the corresponding disposable closed pipeline kit CT-60.1 are used to wash and fill the CAR-T cells, and the cryopreservation of the CAR-T cells is completed with a programmed cooling device. First, install the CT-60.1 kit on the Sepax C-Pro cell processing system, then take out the CAR-T cell culture bag from the carbon dioxide incubator and connect it to the kit. Also connect the sodium chloride injection bag as the washing solution to the kit, and execute the CultureWash program to wash the CAR-T cells with sodium chloride injection three times. The CultureWash program finally resuspends the CAR-T cells in cryopreservation solution and exports them. After sampling and counting, the CAR-T cells are distributed into cryopreservation bags using the Dilution program of the Sepax C-Pro cell processing system, and then transported to programmed cooling. instrument. After cooling is completed, transfer to liquid nitrogen for long-term storage.

In addition, the present invention also provides a method for eliminating or weakening the non-specific adhesion of cells to a solid-phase carrier (such as a container wall or magnetic particles), including incubating the cells and the solid-phase carrier in the presence of DPBS. This prevents cells from occupying and engulfing the solid-phase carrier due to the adhesion effect and affecting the activation of T cells. The cells are preferably mononuclear cells, such as PBMC. In specific embodiments, the method includes the steps of a method for purifying CD3 ⁺ T cells described herein.

The present invention will be further described below in the form of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the invention. Unless otherwise stated, the methods and reagents used in the examples are conventional methods and reagents in the art.

Example

Example 1: Multi-factor study on magnetic bead sorting conditions

A multi-factor study on magnetic bead sorting conditions was conducted. Three repeated DOE experiments were conducted to study the magnetic bead incubation time (the research range was 30 to 60 minutes) and the magnetic bead incubation cell density (the research range was 4 to 10 × 10 ⁶ cells/ml). ), magnetic bead incubation buffer and other factors. In order to make the experimental results applicable to PBMC samples with different CD3 ratios, the CD3 ratio was also used as a research factor. However, the CD3 ratio is an uncontrollable variable during the production process because it completely depends on the properties of the PBMC sample. The specific experimental groups and cell concentration in the incubation solution are shown in Table 1 and Table 2 below.

DPBS (21-031-CVR, Corning) is 500mL per bottle, the concentration is 1X, and the components are: 0.20g/LKCl, 0.20g/LKH ₂ PO ₄ , 8.00g/LNaCl, 1.15g/LNa ₂ HPO ₄ .

Table 1: DOE Experimental Design Grouping

Table 2. Cell concentration in incubation solution

Example 2: Preparation of experimental cells

PBMC were isolated from apheresis and frozen. After the thawed PBMC were placed in a T bottle and cultured overnight, magnetic separation was performed using CD3/28 magnetic beads (CTS Dynabeads CD3/CD28, Cat. No. 40203D, Thermo Fisher Scientific). The experimental conditions used during sorting are assigned according to the groups in the table above. The sorted CD3 ⁺ T cells were activated and cultured for 48±4 hours. The activated CD3 ⁺ T cells were transduced with retrovirus at the same MOI, and then CAR-T cells were expanded and cultured in T bottles.

During the experiment, samples were taken to measure cell number, cell viability, cell diameter, cell phenotype and other indicators that indicate cell growth, activation and CAR infection rate. Cell number, cell viability and cell diameter were detected by NC-200 cell counter. Cell phenotype was detected by flow cytometry. The sampling points were after PBMC recovery, after PBMC remission, after CD3 ⁺ T cell activation, after retroviral transduction, on the 6th day of CAR-T cell expansion culture, and on the 9th day of CAR-T cell expansion culture.

Figure 2 shows the comparability of the entire process of CAR-T cell viability, diameter, amplification fold, flow cytometry phenotype, and CAR infection rate before and after the improvement of the CAR-T cell preparation process. The quality of CAR-T cells produced by the improved process is different from that of CAR-T cells produced by the improved process in indicators that are highly related to product quality, including CAR infection rate, cell viability rate, CD3 ratio, CD19 ratio, etc. Small. The changes in cell activation-related indicators, such as CD25, CD69 and cell diameter, before and after the process improvement were consistent, and there was no abnormal phenomenon of delayed activation or non-activation. In terms of cell expansion, from the beginning of viral transduction to the end of culture, the cell expansion of the improved process is more than 50 times, which is enough to meet general reinfusion requirements. The expression ratios of CD19, CD14, and CD16/56 in the total cell population approached 0% after T cell sorting, and there was no abnormal increase during subsequent culture.

Figure 3 shows the CAR-T cell function test results at the culture endpoint before and after the improvement of the CAR-T cell preparation process. The functional test is to co-culture CAR-T cells with target cells expressing the corresponding target, take the culture supernatant to detect the release of cytokines, take CAR-T cells to detect CD107a expression, and take the target cells to detect the killing ratio. As can be seen from Figure 3, although individual differences due to different donors occurred in the six batches, resulting in different functional test results, in general, the CAR-T cells produced by the improved process expressed CD107a , target cell killing function, IFN-γ and IL-2 release capabilities are all much higher than those before the change.

The results show that the CAR-T cells produced after the improved process are comparable to those before the improvement, and have no impact on product quality. The entire process is carried out in a closed process, which reduces the risk of cross-contamination and external contamination, and is more in line with the requirements of gene therapy products. Regulatory requirements.

Example 3: Effect of incubation buffer on intermediate properties of CAR-T cell preparation

The results of the example are shown in Figure 5. First of all, it can be seen that the type of incubation buffer does not affect the cell viability. The average cell viability of the two treatments during the experimental cycle was higher than 90%. Observing the changes in cell diameter during the experimental period, it can be found that in the group using X-VIVO 15 basal medium as the incubation buffer, after activation for 48±4 hours, the average cell diameter was less than 10 microns, and the peak diameter was delayed until Day 7, and The average is below 11 microns. In the group using DPBS as the incubation buffer, the cell diameter reached its peak on Day 5, which was approximately 13 mm. The CD25 expression in the X-VIVO group also showed a delay and decrease in the peak value. Although the CD69 peak value was not delayed, the average maximum value was about 40%, which was significantly lower than that in the DPBS group (the average value was about 70%). The cells were not well activated, resulting in a low expansion fold. The expansion fold of the X-VIVO group was still less than 100 times on Day 13, while the expansion fold of the DPBS group was on average more than 400 times on Day 13. The type of incubation buffer does not affect the proportion of CD3 cells during the preparation process. The average CD3 proportion of both groups on Day 4 was greater than 80%, and it continued to exceed 90% from Day 10, indicating high purity of the final product. The CAR infection rate was also not affected by the type of incubation buffer. The CAR infection rate of the two groups at three time points remained consistent, about 60%-80%. In summary, using X-VIVO 15 basic medium as a magnetic bead incubation buffer has an adverse effect on cell activation, which in turn prevents cells from amplifying smoothly, but has no effect on other indicators.

Example 4: Magnetic bead incubation time and magnetic bead incubation cell density

As shown in Figure 6 and Figure 7, the magnetic bead incubation time (30 minutes, 45 minutes, 60 minutes) and the cell density of magnetic bead incubation (4×10 ⁶ cells/ml, 7×10 ⁶ cells/ml, 10 ×10 ⁶ cells/ml) does not affect the average cell viability (≥90%) and cell activation status during the experimental cycle (the average cell diameter peak appears on Day 5, greater than 11 microns; the average CD25 expression peak is close to 60%; the average CD69 The peak expression is about 40%-60%; the three incubation times have the same trend), the average cell expansion fold (more than 200 times, the three incubation times have the same trend), CD3 cell proportion (>80% after Day 4, after Day 5 >90%, the three incubation times have the same trend) and CAR infection rate (>60%, the three incubation times have the same trend).

Based on the above observed results, it can be concluded that in the CD3 + T cell sorting operation, the magnetic bead incubation time of 30 to 60 minutes and the magnetic bead incubation cell density of 4 to 10 × 10 ⁶ cells/ml do not affect CAR. - Various indicators during the preparation of T cells, so the process is improved and there is no need to change the incubation time and incubation cell density due to different proportions of CD3 cells in the sample. This experiment also confirmed that the magnetic bead incubation buffer has a significant impact on T cell activation and subsequent CAR-T cell expansion. Therefore, the improved process no longer uses different magnetic bead incubation buffers due to different proportions of CD3 cells in the sample. , fix the magnetic bead incubation buffer to DPBS.

Example 5: Preparation of experimental cells

PBMC were isolated from apheresis and frozen. The thawed PBMC were placed in a cell culture bag and cultured overnight, and then magnetically sorted using CD3/28 magnetic beads. The sorted CD3 ⁺ T cells were activated and cultured in a cell culture bag for 48±4 hours. The activated CD3 ⁺ T cells were transduced with retrovirus in a cell culture bag at the same MOI, and then CAR-T cells were expanded and cultured in a larger cell culture bag.

During the experiment, samples were taken to measure cell number, cell viability, cell diameter, cell phenotype and other indicators that indicate cell growth, activation and CAR infection rate. Cell number, cell viability and cell diameter were detected by NC-200 cell counter. Cell phenotype was detected by flow cytometry. The sampling points were after PBMC recovery, PBMC remission, CD3 ⁺ T cell activation, retroviral transduction, day 3 of CAR-T cell expansion culture, day 6 of CAR-T cell expansion culture, and CAR-T cell expansion. Day 9 of culture.

Next, CAR-T cells from three donors were prepared using the above-mentioned improved CD3 ⁺ T cell sorting conditions. The PBMC samples from these three donors had a low CD3 ratio and a high CD14 ratio, thus proving that the process was improved. Use DPBS as a magnetic bead incubation buffer can successfully produce CAR-T cells and is not limited by the ratio of CD3 and CD14. The table below shows the CD3 and CD14 ratios of PBMC from three donors.

Table 3: Proportions of CD3 and CD14 in PBMC from three donors

供者编号Donor number	CD3+(％)CD3+(%)	CD14+(％)CD14+(%)
#1#1	12.912.9	56.756.7
#2#2	30.730.7	50.750.7
#3#3	7.977.97	7272

The experimental results of preparing CAR-T cells from three PBMCs with low CD3 and high CD14 are shown in Figure 8. In terms of cell viability, the viability of the three groups of cells was not less than 85% throughout the entire experimental process. After CD3 sorting, the cell viability remained above 90%, meeting the release standards. In terms of cell diameter, after 48±4 hours of cell activation, the diameter remained more than 10 microns, and the diameter peak appeared on Day 4 or Day 5, which was consistent with the general activation pattern. Two other cell activation-related indicators, CD25 and CD69, also showed the same trend. In terms of cell expansion, although the expansion times vary due to individual differences, the three groups of cells all expanded more than 100 times on Day 13, which can already meet production needs. In terms of the proportion of CD3 ⁺ cells, the proportion of sorted CD3 ⁺ cells continues to be higher than 89%, which also meets the CAR-T cell production standards. In terms of CAR expression rate, the CAR infection rate of the three groups of cells was greater than 45% from Day 7 and greater than 60% from Day 10, indicating that the virus was stably transduced and a corresponding proportion of CAR-T cells could be produced.

According to the above experimental results, it is proved that the improved process is feasible. The improved procedure can effectively prevent PBMC samples with low CD3 ratio and high CD14 ratio from monocytes phagocytosis of magnetic beads during magnetic bead sorting and affect cell activation, allowing cells to be activated and expanded normally. Secondly, the improved process can simplify the operation of CD3 ⁺ T cell sorting and activation, eliminating the need to perform different calculations based on the proportion of CD3 in the sample, reducing the possibility of human error.

Claims

A method for purifying CD3 + T cells from PBMC, characterized by comprising the steps:

(1) Incubate labeled anti-CD3 antibody and PBMC in DPBS, and

(2) Isolating CD3 + T cells in PBMCs by the label,

Wherein, the method does not include the steps of selecting an incubation buffer, adjusting cell density and incubation time according to the proportion of CD3 + T cells in PBMC,

Preferably, the method further includes the step of obtaining PBMC before purifying CD3+T cells.
The method of claim 1, wherein the label is a substance that facilitates the separation of the complex of cells with antibodies and surface antigens from other components in the system,

Preferably, the DPBS also contains labeled anti-CD28 antibodies,

Preferably, the anti-CD3 antibody and/or anti-CD28 antibody is coupled to the label.
The method of claim 1, wherein the label includes biotin or a solid phase carrier; preferably, the solid phase carrier is magnetic particles.
The method of claim 1, wherein the proportion of CD3 + T cells in the PBMC is less than 30%,

Preferably, the density of CD3 + T cells in the DPBS is 4-10*10 6 cells/mL.
The method of claim 1, wherein the incubation is shaking incubation, wherein

The shaking is 20-500rpm, preferably 50-100rpm, and/or

The incubation temperature is 10-40°C, and/or

The incubation time is 20-60 minutes, preferably 30-45 minutes.
The method according to any one of claims 1-5, characterized in that the method includes the steps:

(a) Resuspend PBMC in DPBS, where the CD3 + T cell density is 4-10*10 6 cells/mL;

(b) Add an equal volume of anti-CD3 antibody-coupled magnetic particles, shake and incubate for 20-60 minutes;

(c) Separating magnetic particles bound to CD3 + T cells by magnetic capture;

(d) Separate the magnetic particles from the cells to obtain CD3 + T cells,

Moreover, the method does not include the step of obtaining the proportion of CD3 + T cells in PBMC.
The method according to any one of claims 1 to 5, characterized in that the method further includes the step of activating CD3 + T cells, such as incubating CD3 + T cells with a medium containing IL-2 for at least 24 hours.
A method for purifying CD3 + T cells using the Sepax C-Pro cell processing system, including steps:

(I) Use the Sepax C-Pro cell processing system to separate and freeze PBMCs from blood samples to remove residual red blood cells and multinucleated cells in white blood cells.

(II) Use the Sepax C-Pro cell processing system to wash and revive the frozen PBMC, restore the viability and functionality of the PBMC, and remove dead cells.

(III) Using the Sepax C-Pro Cell Processing System to perform CD3 + T sorting and activation of recovered PBMCs, including using the method of any one of claims 1-7.
Use of the method according to any one of claims 1 to 8 in preparing a reagent containing activated T cells; preferably, the activated T cells are CAR-T cells.
A method for eliminating or weakening the non-specific adhesion of cells to a solid phase carrier, characterized in that the method includes incubating the cells and the solid phase carrier in the presence of DPBS,

Preferably,

The cells are monocytes, such as PBMC, and/or

The solid phase carrier is a container wall or magnetic particles.