WO2023240483A1

WO2023240483A1 - Method for rapidly preparing t cell

Info

Publication number: WO2023240483A1
Application number: PCT/CN2022/098878
Authority: WO
Inventors: 王海鹰; 曹士婷; 顾明园; 熊青卉; 胡红明
Original assignee: 上海恒润达生生物科技股份有限公司
Priority date: 2022-06-15
Filing date: 2022-06-15
Publication date: 2023-12-21

Abstract

Provided are a method for preparing a T cell expressing a functional molecule, comprising the steps of: 1) resuscitating and alleviating a PBMC for 1-4 hours; 2) sorting and activating a CD3+ T cell for 10-36 hours by means of the PBMC; and 3) using a retrovirus comprising a coding sequence of the functional molecule to transduce the CD3+ T cell to obtain the T cell expressing the functional molecule. Also provided is a CAR-T cell prepared by the method.

Description

A method for rapid preparation of T cells

Technical field

The invention belongs to the field of biology, and specifically relates to the field of immune cell therapy.

Background technique

Chimeric Antigen Receptor (CAR) T cells are a type of cell therapy product that uses genetic engineering to modify T cells so that the cell surface expresses CAR molecules that recognize specific antigens of malignant tumor cells, guiding CAR-T cells to fight malignant tumors. Cells bind and initiate T cell killing function. The sequence structure of the CAR molecule includes a single-chain antibody sequence that specifically recognizes the antigen, a hinge region that affects targeting efficiency, a transmembrane region that fixes the CAR molecule on the cell membrane, a T cell activation domain that transmits intracellular signals, and a cell that enhances signal transmission. Internal costimulatory domain. By designing single-chain antibody sequences that target different antigens, it is possible to create tumors that target malignant cells expressing different antigens. The CAR gene sequence is carried by a retroviral vector and transduced into activated T cells derived from the patient's autologous or donor allogeneic source. After in vitro culture and amplification, the T cells are infused back into the patient's body. In the body, the single-chain antibody sequence of CAR-T cells combines with the tumor malignant cell antigen it targets, causing downstream signal transmission in the intracellular domain, initiating the T cell poisoning function, and achieving the effect of eliminating malignant tumor cells.

As the first commercialized CAR-T cell therapy, CD19 CAR-T cells have gradually begun to be widely used in the treatment of B-ALL, LBCL and FL in the United States, and have extremely high market returns. However, behind the high revenue is the ultra-high pricing of CAR-T products. The two products launched in China are priced at about 1.2 million yuan, and the US products are priced at about 400,000 US dollars, which is comparable to unconventional drugs. The high pricing of CAR-T products comes from the fact that the cost in the research and development stage is much higher than that of ordinary traditional drugs, and the production technology is complex, requiring high accuracy and stability of production personnel and related equipment. Such high prices make it difficult for products to reach patients in need. Therefore, in order to reach more patients, we need to think about how to reduce pricing, that is, start by reducing the cost of CAR-T cell production without affecting the quality of CAR-T cells. Generally, the total duration of the CAR-T cell production process is between 10 and 25 days, and the costs caused by the mobilization of production manpower and the use of various consumable reagents cannot be underestimated. Therefore, the most intuitive way to reduce the cost of CAR-T cell production is to shorten the preparation time of CAR-T cells. Shortening the preparation time of CAR-T cells can not only reduce costs, but also prevent patients from disease progression while waiting for the preparation of CAR-T cells to be completed.

The preparation of CAR-T cells in the prior art usually includes the following steps: 1. Blood collection from the patient or donor: using a blood cell separator to enrich white blood cells from the blood of the patient or donor and transporting them to the cell preparation center at low temperature; 2. Isolation of PBMC from apheresis: Manually or automatically separate and enrich PBMC from white blood cells, which contain lymphocytes and monocytes, and remove most red blood cells, multinucleated cells, platelets and plasma. 3. Isolate and activate T cells from PBMC: Use magnetic beads or other solid phase interfaces covalently coupled to anti-CD3 and/or anti-CD28 antibodies to incubate with PBMC, and then magnetically separate the T cells bound to the magnetic beads; 4. CAR gene modified T cells: Use viral vectors or other gene transduction methods to transfer CAR gene sequences into T cells, allowing them to express CAR molecules on the cell surface; 5. In vitro expansion of CAR-T cells: CAR- T cells are expanded and cultured in cell culture plates, cell culture bottles or cell culture bags; 6. CAR-T cell filling and cryopreservation: Harvest CAR-T cells and store them in liquid nitrogen for later use.

Most of the above steps can be completed within one day, but the in vitro expansion of CAR-T cells is a rate-determining step, and depending on the individual differences in cell raw materials and different required doses, the expansion time is generally not less than three days, and It may last more than ten days.

Contents of the invention

In order to shorten the preparation time of CAR-T cells, the present invention provides a method for preparing T cells expressing functional molecules. The method includes steps:

1) PBMC resuscitation and relief last for 1 to 4 hours, preferably 2 to 3 hours.

2) Sort and activate CD3+T cells from PBMC for 10 to 36 hours, preferably 15 to 24 hours,

3) Use a retrovirus containing a coding sequence of a functional molecule to transduce CD3+T cells to obtain T cells expressing the functional molecule. The transduction includes co-culturing the retrovirus and CD3+T cells for 12-36 hours, preferably 15-24 hours.

In one or more embodiments, the functional molecule is a CAR. Preferably, it is CD19-28z containing an anti-CD19 antibody or an antigen-binding fragment thereof, a hinge region, a transmembrane region, a CD28 intracellular costimulatory domain, and a CD3-zeta signal transduction domain. More preferably, the anti-CD19 antibody or antigen-binding fragment thereof is an anti-human CD19 antibody single chain variable region.

In one or more embodiments, the coding sequence is constructed in a retroviral plasmid.

In one or more embodiments, step 1) includes: culturing PBMC in culture medium under conditions suitable for PBMC growth for 1 to 4 hours, preferably 2 to 3 hours.

In one or more embodiments, the culture medium in step 1) includes AIM-V, X-VIVO, DMEM, RPMI1640, preferably X-VIVO15. The culture medium is also supplemented with one, more or all of acetylcysteine, GlutaMAX, HEPES and human plasma.

In one or more embodiments, suitable conditions for PBMC growth are about 37°C and about 5% _CO2 .

In one or more embodiments, step 2) includes:

2.1) Sorting CD3+ T cells from PBMC using an antibody, including an anti-CD3 antibody, in DPBS, and

2.2) Incubate CD3+T cells in culture medium under conditions suitable for CD3+T cell activation for 12-36 hours, preferably 15-24 hours.

In one or more embodiments, the antibody further includes an anti-CD28 antibody.

In one or more embodiments, the medium in step 2) contains IL-2. The culture medium includes AIM-V, X-VIVO, DMEM or RPMI1640, preferably X-VIVO15. The culture medium is also supplemented with one, more or all of acetylcysteine, GlutaMAX, HEPES and human plasma.

In one or more embodiments, conditions suitable for CD3+T activation are about 37°C and about 5% _CO2 .

In one or more embodiments, the antibody is a labeled antibody. Preferably, the antibody is coupled to a solid support. More preferably, the solid phase carrier is magnetic particles.

In one or more embodiments, step 3) includes: co-culturing the retrovirus and CD3+ T cells in culture medium under conditions suitable for retroviral transduction of CD3+ T cells for 12-36 hours, preferably 15-24 hours Hour.

In one or more embodiments, the medium in step 3) contains IL-2. The culture medium includes AIM-V, X-VIVO, DMEM or RPMI1640, preferably X-VIVO15. The culture medium is also supplemented with one, more or all of acetylcysteine, GlutaMAX, HEPES and human plasma.

In one or more embodiments, conditions suitable for retroviral transduction of CD3+ T cells are about 37°C and about 5% _CO2 .

In one or more embodiments, the transduction MOI of the transduction in step 3) is 0.1-2, preferably 0.5-1.5.

In one or more embodiments, in step 3), the density of CD3+ T cells is 1-5*10 ⁶ cells/mL, preferably 1*10 ⁶ cells/mL.

In one or more embodiments, the method further includes the step of removing the solid support. Preferably, the step of removing the solid phase carrier is located after step 3).

In one or more embodiments, the method further includes the step of: 4) cryopreserving the T cells expressing the functional molecules obtained in step 3); specifically including: washing the T cells with sodium chloride injection to cryopreserve The T cells were resuspended in liquid, programmed to cool down, and frozen in liquid nitrogen.

The present invention also provides CAR-T cells prepared by the method of the present invention.

In one or more embodiments, the CAR is CD19-28z containing anti-CD19, CD28 intracellular costimulatory domain, and CD3-zeta intracellular costimulatory domain.

Advantages of the invention:

-Shortening the recovery and relief time of PBMC to 2 hours does not affect the characteristic of some dead cells clumping into flocculation after recovery, which can be easily removed by using a cell strainer;

-Shorten the sorting, activation and culture time of CD3+ T cells to 22-24 hours so that the cells can be properly activated without affecting subsequent viral transduction;

-The cell suspension does not remove magnetic particles, retains part of the activation function, and directly conducts viral transduction;

- Eliminate the in vitro expansion culture process of CAR-T cells. When viral transduction is completed, the magnetic beads are removed and the CAR-T cells are cryopreserved, which greatly shortens the total preparation time of CAR-T cells.

Description of the drawings

Figure 1 is a schematic diagram of the preparation process of Dash CAR-T cells.

Figure 2 is a comparison diagram of the preparation process of Dash CAR-T and general CAR-T.

Figure 3 is the test results of CAR-T cell characteristics prepared by Dash CAR-T process and general process.

Figure 4 is the functional test results of CAR-T cells prepared by Dash CAR-T process and general process.

Figure 5 shows the results of the in vivo pharmacodynamics study in mice of CAR-T cells prepared by Dash CAR-T technology and general technology.

Detailed ways

Based on the need to shorten the preparation time of CAR-T cells, the present invention provides a CAR-T cell process that can be prepared within 48 hours starting from PBMC (hereinafter referred to as Dash CAR-T).

the term

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.

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.

DPBS: The full name of DPBS is Dulbecco's Phosphate-Buffered Saline, which is Dulbecco's Phosphate-Buffered Saline. The main ingredients include NaCl, KCl, KH ₂ PO ₄ , Na ₂ HPO ₄ , etc., with a pH of 7.2-7.4. DPBS is a balanced salt solution for cells, usually used to resuspend cells and preserve cells in vitro for a short period of time. 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.

Medium: The medium described herein includes any medium suitable for immune cells, particularly CD3+ cells and other types of white blood cells. Including but not limited to AIM-V, X-VIVO, DMEM or RPMI1640.

X-VIVO: is the abbreviation of Lonza X-VIVO ^TM 15 medium. Suitable for the proliferation of T lymphocytes and can be used for the growth of CD3+ cells and other types of white blood cells.

CTS Dynabeads CD3/28: CTS ^TM Dynabeads ^TM CD3/CD28 is suitable for the isolation, activation and expansion of human T cells in vitro. Dynabeads are simultaneously coupled with anti-CD3 and anti-CD28 antibodies to provide primary and costimulatory signals required for T cell activation and expansion.

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 present invention first provides a method for preparing T cells expressing functional molecules, including the steps: 1) PBMC resuscitation and relief for 1 to 4 hours, preferably 2 to 3 hours, 2) sorting and activating CD3+ T cells from PBMC for 10 to 36 hours hours, preferably 15 to 24 hours, and 3) using a retrovirus containing a coding sequence of a functional molecule to transduce CD3+ T cells to obtain T cells expressing the functional molecule, the transduction includes co-culturing the retrovirus and CD3 + T cells 12-36 hours, preferably 15-24 hours.

The "functional molecule" described herein can be any protein or polypeptide that needs to be expressed in T cells, preferably CAR. The CAR described in the present invention can be various CARs well known in the art. The CAR can sequentially include a polypeptide (such as scFv) that binds tumor cell membrane antigens, a hinge region, a transmembrane region, and an intracellular signal region. The hinge region, transmembrane region and intracellular signal region well known in the art for constructing CAR can be used to construct the CAR of the present invention. Generally, polypeptides that bind to tumor cell membrane antigens can bind to membrane antigens widely expressed by tumor cells with medium affinity. The polypeptides are usually inserted with antigenic epitopes, and the inserted positions are selected from any 1, 2, or 3 of the following 3 positions. : The N-terminus of the polypeptide, between the polypeptide and the hinge region and within the polypeptide. The polypeptide that binds to tumor cell membrane antigens is a natural polypeptide or a synthetic polypeptide; preferably, the synthetic polypeptide is a single-chain antibody or Fab fragment.

The chimeric antigen receptor of the present invention can target one or more of the following antigens: CD19, CD20, CEA, GD2, FR, PSMA, PMEL, CA9, CD171/L1-CAM, IL-13RL1, MART-1, ERBB2, NY-ESO-1 family protein, BAGE family protein, GAGE family protein, AFP, MUC1, CD22, CD23, CD30, CD33, CD44v7/8, CD70, VEGFR1, VEGFR2, IL-11R/, EGP-2, EGP -40, FBP, GD3, PSCA, FSA, PSA, HMGA2, fetal acetylcholine receptor, LeY, EpCAM, MSLN, IGFR1, EGFR, EGFRvIII, ERBB3, ERBB4, CA125, CA15-3, CA19-9, CA72-4 , CA242, CA50, CYFRA21-1, SCC, AFU, EBV-VCA, POA and PROGRP. Preferably, the CAR is CD19-28z containing the single-chain variable region, hinge region, transmembrane region, CD28 intracellular costimulatory domain, and CD3-zeta signal transduction domain of an anti-human CD19 antibody. The method of the present invention shortens the recovery and remission time of PBMC to 2 hours, shortens the sorting, activation and culture time of CD3+T cells to 22-24 hours, and eliminates the need to remove the CAR-T cell in vitro expansion culture process, greatly shortening the CAR-T cell recovery time. Total cell preparation time. In addition, the inventors found that the CAR-T cells prepared according to the method of the present invention have a high activation state and higher T cell stemness, which may lead to better expansion and tumor killing capabilities.

The above results well demonstrate the high amplification ability and tumor killing ability of Dash CAR-T cells due to their strong T cell stemness.

As used herein, a "labeled" antibody 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, e.g., biological elements, solid phase carriers, 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.

In one or more embodiments, step 1) includes: culturing PBMC in culture medium (X-VIVO15 supplemented with acetylcysteine, GlutaMAX, HEPES and human plasma) under conditions suitable for PBMC growth for 1 to 4 hours, preferably 2-3 hours. In one or more embodiments, step 2) includes: 2.1) sorting CD3+ T cells from PBMC using an antibody, including an anti-CD3 antibody, in DPBS, and 2.2) in culture medium (supplemented with acetyl CD3+T cells are incubated in X-VIVO15) of cysteine, GlutaMAX, HEPES and human plasma under conditions suitable for CD3+T cell activation for 12-36 hours, preferably 15-24 hours. In one or more embodiments, step 3) includes: transducing CD3 with a suitable retrovirus in culture medium (X-VIVO15 supplemented with acetylcysteine, GlutaMAX, HEPES, IL-2 and human plasma) Conditions for + T cells Co-culture retrovirus and CD3 + T cells for 12-36 hours, preferably 15-24 hours. In one or more embodiments, the method further includes the step of: 4) cryopreserving the T cells expressing the functional molecules obtained in step 3); specifically including: washing the T cells with sodium chloride injection to cryopreserve The T cells were resuspended in liquid, programmed to cool down, and frozen in liquid nitrogen.

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 media are often commercially available, such as X-VIVO 15.

"Retroviruses" used to transduce cells herein include alpharetroviruses, betaretroviruses, gammaretroviruses, deltaretroviruses, and epsilonretroviruses; gammaretroviruses are preferred. Viruses used to transduce cells herein do not include lentiviruses. As used herein, "retroviral plasmids" are derived from retroviral vectors.

The present invention also provides CAR-T cells prepared by the method of the present invention. Preferably, the CAR is CD19-28z.

Specifically, the method of the present invention for rapidly preparing T cells (such as CAR-T) expressing functional molecules is shown in Figure 1, including: PBMC isolation, PBMC recovery and relief, CD3 ⁺ T cell sorting and activation, and retroviral transduction T cells and CAR-T cells are filled and frozen. The improvement points that are different from the general CAR-T cell preparation process are mainly reflected in shortening the T cell activation time to 22-24 hours and deleting the CAR-T cell in vitro expansion step. The samples applicable to this preparation process are blood collected by mechanical apheresis unit that complies with the collection procedures and transportation methods.

Collection and transportation of apheresis blood. 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 and plasma processing. This process can be completed manually or automatically. Manually, use Ficoll density gradient centrifugation to slowly add apheresis blood to Ficoll, centrifuge with a centrifuge, and manually remove the separated PBMC layer. For automation, the Sepax C-Pro cell processing system and the corresponding single-use closed tube kit CT-90.1 are used. Ficoll density gradient centrifugation is also used and the NeatCell program developed by the instrument manufacturer is used to separate PBMCs. Finally, the PBMC are resuspended in PBMC cryopreservation solution and exported or transferred to a 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 to remove the remaining red blood cells and multinucleated cells in the white blood cells from a 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 eases. Place the PBMC cryopreservation bag into a 37°C water bath and shake it back and forth to melt it quickly and evenly. The thawed PBMC were transferred to culture medium (X-VIVO15 supplemented with acetylcysteine, GlutaMAX, HEPES, and human plasma), and centrifuged and washed once. Resuspend the washed PBMC in culture medium, place them in a cell culture bottle or cell culture bag, place them in a carbon dioxide incubator, and culture them at 37±1°C and 5±0.5% _CO2 for 2 hours. The purpose of this process is to restore the viability and functionality of PBMC and remove apoptotic cells.

CD3+ T cell sorting and activation. Take out the PBMC that have been cultured from the carbon dioxide incubator, centrifuge the cell suspension once, and wash it once with DPBS. After washing, PBMC were resuspended in DPBS and sampled and counted. Then the number of CD3 ⁺ cells was calculated based on the PBMC flow cytometry phenotyping results. DPBS was added to the PBMC suspension to adjust the CD3 ⁺ cell density (1-50*10 ⁶ CD3+ cells/mL). Add magnetic beads covalently coupled with anti-CD3 and anti-CD28 antibodies washed with DPBS (number of cells: number of magnetic beads = 1:1 (CTS ^TM Dynabeads ^TM CD3/CD28), and use a rotating mixer to mix the cells with magnetic beads. Get thorough mixing during co-incubation (30-60 minutes, room temperature). After the incubation is completed, place the cells on the DynaMag ^TM -5 magnetic stand. The CD3 ⁺ cells bound to the magnetic beads will be magnetically captured and retained in the centrifuge tube, and discarded Remove the supernatant and CD3 ^- cells. Add IL-2-containing medium (X-VIVO15 with acetylcysteine, GlutaMAX, HEPES, human plasma, interleukin 2) to resuspend the CD3 ⁺ cells (1-5*10 ⁶ cells/mL) and transfer to a cell culture flask or cell culture bag. Place the cells in a carbon dioxide incubator and activate them for 22-24 hours in an environment of 37±1°C and 5±0.5% _CO2 . The purpose of this step is The CD3 ⁺ T cells used to transduce the virus are sorted, other cell populations are further removed, and the T cells are costimulated with CD3 and CD28 antibodies to facilitate retrovirus transduction.

Retroviral transduction of T cells. First, coat the cell culture bottle or cell culture bag with RetroNectin working solution at 37°C. After the coating is completed, discard the supernatant in the culture bottle or culture bag. After the activated and cultured CD3 ⁺ T cells were washed once, they were resuspended in IL-2-containing medium (X-VIVO15 supplemented with acetylcysteine, GlutaMAX, HEPES, human plasma, and interleukin 2). After washing, the cells are sampled and counted, and the transduction MOI is calculated. The MOI should be 0.1-2 and the final cell density in virus transduction is 1-5*10 ⁶ cells/mL. Put the cell suspension and virus solution (MOI=1, cell density 1*10 ⁶ cells/mL) into the RetroNectin-coated culture bottle or culture bag, and incubate at 37±1°C and 5±0.5% CO ₂ Incubate in a carbon dioxide incubator for 16-24 hours. The purpose of this process is to transfer the CAR gene into T cells to prepare CAR-T cells.

Filling and cryopreservation of CAR-T cells. After completing viral transduction, first use the DynaMag ^TM -5 magnetic stand to remove the remaining magnetic beads in the cell suspension, then wash the CAR-T cells three times with sodium chloride injection (0.9%) and freeze them with cryopreservation solution (containing DMSO, Sodium Chloride Injection, Compound Electrolyte Injection, Glucose Injection, Human Albumin) were resuspended. After sampling and counting, the CAR-T cells were divided into cryopreservation bags and then transported to a programmed cooling device. After cooling is completed, transfer to liquid nitrogen for long-term storage. The cooling procedure is common knowledge to those skilled in the art. An exemplary cooling procedure includes the following steps: 1) pre-cooling the instrument to 20 degrees Celsius; 2) cooling down to -6 degrees Celsius at 1 degree Celsius per minute; 3) cooling down to -6 degrees Celsius at 25 degrees Celsius per minute. Down to -50 degrees Celsius; 4) Back to -14 degrees Celsius at 10 degrees Celsius per minute; 5) Down to -45 degrees Celsius at 1 degrees Celsius per minute; 6) Down to -90 degrees Celsius at 10 degrees Celsius every minute.

The DashCAR-T process has the following advantages compared with existing technology processes. In terms of PBMC resuscitation and relief, the original process requires 22±2 hours, while DashCAR-T only requires 2 hours of relief culture. In terms of CD3 ⁺ T cell sorting and activation, the Dash CAR-T process shortens the activation and culture time to 22-24 hours, and other operating steps remain unchanged. In terms of viral transduction, in the DashCAR-T process, the magnetic beads in the cell suspension are not magnetically removed first, and viral transduction is performed directly; while in the original process, the magnetic beads are removed first and then viral transduction is performed. There is no CAR-T cell expansion and culture process in the DashCAR-T process. After the viral transduction is completed, the CAR-T cells are filled and frozen directly. In terms of filling and freezing of CAR-T cells, since the magnetic beads have not yet been removed, in the Dash CAR-T process, the magnetic beads are first removed with a magnetic stand before filling and freezing. The other steps are the same as the original process. as shown in picture 2.

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, Viability, diameter, amplification multiple, flow cytometry phenotype and CAR infection rate of CAR-T cells prepared by Dash CAR-T process and original process

Prepare DashCAR-T cells using the DashCAR-T process, and compare the differences between Dash CAR-T cells and CAR-T cells produced by the original process. DashCAR-T preparation: After resuscitation of frozen PBMC in a 37°C water bath, transfer to culture medium, centrifuge and wash once. After washing, the PBMC were resuspended in culture medium and seeded into culture bottles at a cell density of 5×10 ⁶ viable cells/ml. Place the culture flask with the cell suspension in a 37°C, 5% _CO2 incubator and incubate for 2 hours.

Remove the PBMC that have completed the remission culture from the incubator and transfer them to a centrifuge tube. After centrifugation and washing with DPBS, adjust the cell density to 1×10 ⁷ CD3 ⁺ cells/ml with DPBS. Magnetic beads covalently coupled to anti-CD3 and anti-CD28 antibodies were added to the cell suspension and incubated together on a rotating mixer at room temperature for 30 minutes. Transfer the incubated cell magnetic bead suspension to a magnetic stand and magnetically sort CD3 ⁺ cells. The sorted cells were adjusted to 1 × 10 ⁶ CD3 ⁺ cells/ml with IL-2-containing medium, placed in a culture flask, and activated for 22 hours in a 37°C, 5% CO ₂ incubator.

The activated cells were taken out from the incubator, centrifuged and washed once with IL-2-containing medium, and the cell density was adjusted to 1×10 ⁶ viable cells/ml. The cell suspension and virus liquid were mixed for CAR gene transduction, MOI=1. Transfer the cell suspension to a RetroNectin-coated cell culture bag and conduct viral transduction for 24 hours in a 37°C, 5% _CO2 incubator.

The transduced DashCAR-T cells are first removed from the suspension using a magnetic stand, then centrifuged and washed three times with sodium chloride injection, resuspended in cryopreservation solution, and transferred to a cryopreservation bag for programmed cooling. The instrument was cryopreserved and stored in liquid nitrogen for a long time.

The original production process of CAR-T cells has different preparation procedures. Among them, PBMC are relieved for 24 hours and CD3 ⁺ cells are activated and cultured for 48 hours. After viral transduction, the magnetic beads are removed and transferred to a culture bottle for expansion culture for 5 days. Perform cryopreservation.

DashCAR-T cells and CAR-T cells produced in the original process were tested before cryopreservation. The results are shown in Figure 3. The indicators of CAR-T cells produced by Dash CAR-T process that are highly related to product quality, including CAR infection rate, cell viability rate, CD3 ratio, CD19 ratio, etc. are all consistent with the quality of CAR-T cells produced by the original process. The difference is small. The expression ratios of CD14 and CD16+/CD56+ in the total cell population approached 0%. The results of cell activation-related indicators, such as CD25, CD69 and cell diameter, are quite different between the two processes. It can be seen from the figure that the activation status of Dash CAR-T cells is higher than that of CAR-T cells produced by the original process, especially the expression of CD25 and CD69 is significantly different. CD45RO-/CCR7+ symbol

There are also significant differences in the proportion of T cell populations, among which Dash CAR-T cells still account for nearly 80%

T cells and CAR-T cells in the original manufacturing process

The proportion of T cells has dropped below 20%. What CD45RO-/CCR7+ represents

T cells are T cells with a high degree of stemness. T cell stemness is related to cell expansion and tumor killing ability. According to the results of this in vitro experiment, it can be found that Dash CAR-T cells have a high activation state and higher T cell stemness, which may contribute to better expansion and tumor killing capabilities.

Example 2, CAR-T cell function test at the culture endpoint of Dash CAR-T process

The frozen DashCAR-T cells and the original manufactured CAR-T cells were revived in a 37°C water bath, centrifuged and washed once with IL-2-containing medium, resuspended, and inoculated into culture bottles. Incubate for 48 hours in a 37°C, 5% _CO2 incubator to allow the CAR-T cell function to be well restored before performing functional testing.

The functional test is to co-culture CAR-T cells with target cells expressing the corresponding target, take CAR-T cells to detect CD107a expression, and take target cells to detect the killing ratio. The CD107a molecule is a sensitive marker of degranulation of toxic T cells, which directly reflects the level of cell killing activity. The CD107a expression of Dash CAR-T cells is similar to that of the original process CAR-T cells. As shown in Figure 4, in the cell killing test, within the range of the effect-to-target ratio of 20:1 to 1.25:1, Dash CAR-T cells showed similar killing ability to the original process CAR-T cells.

Example 3, In vivo pharmacodynamic study of Dash CAR-T cells

Figure 5 shows the establishment of an in vivo pharmacodynamics study model of CD19Dash CAR-T cells using Nalm-6-luciferase-GFP human B lymphoid leukemia cells in NOG mice. Experimental groups and dosages include: vehicle group, 1×10 ⁶ T cells without CAR gene transduction, 1×10 ⁶ BCMACAR ⁺ cells, 1×10 ⁶ original process CAR ⁺ cells, 5×10 ⁶ Original process CAR ⁺ cells, 1×10 ⁶ DashCAR-T process CAR ⁺ cells. There were five mice in each group. In vivo fluorescence imaging and analysis of the proportion of T cells in the peripheral blood of mice were performed 1 day before reinfusion and every 7 days after reinfusion. It can be seen from the in vivo fluorescence images that the tumor fluorescence of the DashCAR-T cell group on the 7th day after reinfusion is almost invisible, while some tumor cells of the original process CAR-T cells at the same dose still remain until reinfusion. It cannot be cleared until 14 days after losing. Five times the dose of CAR-T cells from the original process also almost completely eliminated tumors on day 7, showing that Dash CAR-T cells only need one-fifth the dose of CAR-T cells from the original process to achieve significant and complete tumor killing. Effect. On the 14th day after reinfusion, 1×10 ⁶ CAR ⁺ cells of the original process completely eliminated the tumor, but there was obvious recurrence on the 21st day after the reinfusion. The tumor elimination effect of the 5×10 ⁶ original process CAR ⁺ cell group and the 1×10 ⁶ DashCAR-T process CAR ⁺ cell group lasted until the 21st day after reinfusion, but the original process CAR ⁺ cell group had tumors Signs of relapse. Comparing the total fluorescence flux, it can be found that the average total fluorescence flux of the DashCAR-T group has remained at the lowest value since the 14th day after reinfusion; in addition, the expansion times of peripheral blood T cells of mice in the DashCAR-T group are much higher In other groups, the cells had expanded more than 100 times on the 21st day after reinfusion. The above results well demonstrate the high amplification ability and tumor killing ability of Dash CAR-T cells due to their strong T cell stemness.

The above results show that there is no significant difference between CAR-T cells made by Dash CAR-T process and CAR-T cells made by the original process in terms of cell viability, CD3+ cell proportion, non-T cell population and CAR infection rate; In terms of activation status and T cell stemness, Dash CAR-T cells are both higher than those of CAR-T cells derived from the original manufacturing process. Therefore, Dash CAR-T cells have better performance in cell expansion, cell killing capacity and mouse model drug efficacy tests. The performance is highly comparable to, and even higher than, the original process of CAR-T cells. In general, the key points of this patent are that in addition to shortening the CAR-T cell manufacturing process and reducing costs, it can also produce CAR-T with better killing effects, and have achieved results in both in vitro functionality and in vivo pharmacodynamics. verify.

Claims

A method for preparing T cells expressing functional molecules, including the steps:

1) PBMC resuscitation and relief last for 1 to 4 hours, preferably 2 to 3 hours.

2) Sort and activate CD3+T cells from PBMC for 10 to 36 hours, preferably 15 to 24 hours,

3) Use a retrovirus containing a coding sequence of a functional molecule to transduce CD3+T cells to obtain T cells expressing the functional molecule. The transduction includes co-culturing the retrovirus and CD3+T cells for 12-36 hours,

Preferably, the functional molecule is a CAR.
The method of claim 1, wherein step 1) includes: culturing PBMC in the culture medium under conditions suitable for the growth of PBMC for 1 to 4 hours,

Preferably,

The culture medium in step 1) includes AIM-V, X-VIVO, DMEM, RPMI1640, preferably X-VIVO15, and/or

The culture medium is also supplemented with one or more or all of acetylcysteine, GlutaMAX, HEPES and human plasma, and/or

Suitable conditions for PBMC growth are approximately 37°C and approximately 5% CO 2 .
The method according to claim 1 or 2, characterized in that step 2) includes:

2.1) Sorting CD3+ T cells from PBMC using an antibody, including an anti-CD3 antibody, in DPBS, and

2.2) Incubate CD3+T cells in culture medium under conditions suitable for CD3+T cell activation for 12-36 hours, preferably 15-24 hours.
The method of claim 3, characterized in that:

The antibodies also include anti-CD28 antibodies, and/or

The medium in step 2) contains IL-2, and/or

The culture medium includes AIM-V, X-VIVO, DMEM or RPMI1640, preferably X-VIVO15, and/or

The culture medium is also supplemented with one or more or all of acetylcysteine, GlutaMAX, HEPES and human plasma, and/or

The antibody is a labeled antibody; preferably, the antibody is coupled to a solid support.
The method according to claim 1 or 2, characterized in that step 3) includes: co-culturing retrovirus and CD3+T cells in the culture medium under conditions suitable for retroviral transduction of CD3+T cells 12-36 Hour,

Preferably,

The medium in step 3) contains IL-2, and/or

The culture medium includes AIM-V, X-VIVO, DMEM or RPMI1640, preferably X-VIVO15, and/or

The culture medium is also supplemented with one, more or all of acetylcysteine, GlutaMAX, HEPES and human plasma,

Suitable conditions for retroviral transduction of CD3+ T cells are about 37°C and about 5% CO 2 .
The method of claim 5, characterized in that:

Step 3) The MOI of the transduction is 0.1-2, and/or

In step 3), the density of CD3+T cells is 1-5*10 6 cells/mL.
The method according to claim 4, characterized in that the method further includes the step of removing the solid phase carrier, located after step 3).
The method according to claim 1 or 2, characterized in that the method further includes the step of: 4) cryopreserving the T cells expressing the functional molecules obtained in step 3),

Preferably, step 4) includes: washing the T cells with sodium chloride injection, resuspending the T cells with cryopreservation solution, programmed cooling, and freezing in liquid nitrogen.
CAR-T cells prepared by the method described in any one of claims 1-8.
The CAR-T cell according to claim 9, wherein the CAR contains an anti-CD19 antibody or an antigen-binding fragment thereof, a hinge region, a transmembrane region, a CD28 intracellular costimulatory domain, and a CD3-zeta Signal transduction domain of CD19-28z.