Single blood sampling preparation and preparation method thereof

Abstract

The invention belongs to the field of cell cryopreservation, and particularly relates to a single blood sampling cryopreservation preparation and a preparation method thereof.The preparation contains single blood sampling and cryopreservation liquid, after the single blood sampling is centrifugally concentrated, the cryopreservation liquid is used for replacing supernate, and the replacement volume of the cryopreservation liquid is larger than or equal to 50%. The single blood sampling preparation provided by the invention provides an initial raw material which can be directly connected with a preparation process in the existing cell therapy industry, and the quality stability of cell cryopreservation is improved.

Description

Technical Field

[0001] This invention belongs to the field of cell cryopreservation, specifically relating to a single-donor blood cryopreservation preparation and its preparation method. Background Technology

[0002] Apheresis refers to blood collected using a blood component separator. It primarily consists of mononuclear cells, with a small number of red blood cells, granulocytes, and plasma components. Unlike PBMCs, apheresis blood is a composite component and does not require cell separation, making it more suitable for large-scale culture of target cells in practical applications.

[0003] Because apheresis blood still contains a certain amount of red blood cells and plasma components, these non-target cell extracellular components have a significant impact on the cryopreservation quality of the target cells, making cryopreservation somewhat challenging. Furthermore, the initial cell density of apheresis blood varies considerably, generally ranging from 2 × 10⁻⁶ cells / day. 7 ~1×10 8 Within the cells / mL range. To facilitate the standardization of subsequent products and the stability of cell quality, it is necessary to minimize components other than non-target cells (PBMCs) and control the target cells within an appropriate range. Summary of the Invention

[0004] The inventors discovered through research that the density of cryopreserved cells and the proportion of cryopreservation solution in the formulation have a significant impact on the cryopreservation effect of apheresis blood. Therefore, a cryopreservation formulation for apheresis blood is now provided, which can effectively control parameter fluctuations in apheresis blood samples and ensure the product quality of the cryopreserved formulation.

[0005] The specific technical solution is as follows:

[0006] A single-donor blood cryopreservation preparation contains single-donor blood and cryopreservation solution. After centrifugation and concentration of the single-donor blood, the supernatant is replaced with cryopreservation solution, wherein the replacement volume of cryopreservation solution is ≥50%, preferably ≥75%.

[0007] In some embodiments, the cryopreservation solution contains DMSO, preferably, the content of DMSO is ≥10%.

[0008] In some embodiments, the content of DMSO in the apheresis cryopreservation formulation is ≥5%.

[0009] In some specific implementations, the cryopreservation solution is CS10 (Biolife, USA) or PBMC cryopreservation solution (Cycio Biotech, CS-PM-D1).

[0010] In some embodiments, the cell density of the apheresis cryopreservation formulation is <1×10⁻⁶. 8 cells / mL.

[0011] In some embodiments, the initial hematocrit of the apheresis cryopreservation formulation is ≤15%.

[0012] The present invention also provides a method for preparing a cryopreservation formulation of apheresis blood, wherein the method comprises centrifuging and concentrating apheresis blood, and replacing the supernatant with cryopreservation solution, wherein the replacement volume of cryopreservation solution is ≥50%, preferably ≥75%.

[0013] In some specific implementations, the concentration conditions for the cryopreserved preparation are centrifugation at 300-500g for 5-10 minutes.

[0014] In some embodiments, the cryopreservation preparation is prepared manually or by an instrument; preferably by an instrument; more preferably, the instrument is a fully automated cell processing system from Zhongbo Ruikang.

[0015] Another object of the present invention is to provide a T cell, which is obtained by cryopreservation and thawing of the above-mentioned single-donor cryopreservation preparation or the single-donor cryopreservation preparation prepared by the above-mentioned preparation method.

[0016] Another object of the present invention is to provide a method for preparing engineered T cells, comprising transfecting the above-mentioned T cells with nucleic acids.

[0017] In some embodiments, the nucleic acid contains a polypeptide coding sequence, preferably a CAR coding sequence.

[0018] In some specific embodiments, the CAR is an MSLN CAR, and more preferably, the amino acid sequence of the MSLN CAR is shown in SEQ ID NO:4.

[0019] The beneficial effects of the technical solution of this invention are as follows:

[0020] The single-donor blood cryopreservation formulation described in this invention provides a starting material that can be directly integrated with existing cell therapy industry preparation processes. The preparation method is highly operable, effectively controls the density range of target cells and the addition ratio range of cryopreservation solution, and improves the quality stability of cell cryopreservation. Attached Figure Description

[0021] Figure 1 Plasmid map of -P19V21 plasmid;

[0022] Figure 2 -CS10 cryopreservation solution ratio; T cell sorting yield after cryopreservation and thawing.

[0023] Figure 3 -Cell viability during CAR-T preparation after cryopreservation and thawing following the CS10 cryopreservation solution ratio;

[0024] Figure 4-Cell expansion of CAR-T cells after cryopreservation and thawing using CS10 cryopreservation solution; where 4-A is sample 1 and 4-B is sample 2.

[0025] Figure 5 - Cell expansion of CAR-T cells after cryopreservation and thawing at different cell densities. Detailed Implementation

[0026] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0027] One objective of this invention is to provide a single-donor blood cryopreservation formulation, which contains single-donor blood and cryopreservation solution. After the single-donor blood is centrifuged and concentrated, the supernatant is replaced with cryopreservation solution, and the replacement volume of cryopreservation solution is ≥50%, for example, 50%, 75%, or 85%.

[0028] In some embodiments, the replacement volume of the cryopreservation solution is preferably ≥75%, for example 75% or 85%.

[0029] The term "collected blood" refers to blood collected using a blood component separator, which mainly consists of mononuclear cells, along with a small number of red blood cells, granulocytes, and plasma components. Unlike PBMCs, collected blood is a composite component and does not require cell separation, making it more suitable for large-scale culture of target cells in practical applications.

[0030] In some embodiments, the target cells are immune cells, preferably any one or more of lymphocytes, dendritic cells, monocytes, macrophages, granulocytes, or mast cells.

[0031] In some specific implementations, the target cells are further preferably lymphocytes.

[0032] In some specific implementations, the target cell is more preferably a T lymphocyte, or simply a T cell.

[0033] In some embodiments, the cryopreservation solution contains DMSO, preferably, the content of DMSO is ≥10%.

[0034] In some embodiments, the DMSO content in the apheresis cryopreservation formulation is ≥5%, for example 7.5%, 8%, or 8.5%.

[0035] The cryopreservation solution can be any cryopreservation solution known in the art. In some specific embodiments, the cryopreservation solution is CS10 (Biolife, USA) or PBMC cryopreservation solution (Cycion Biotech, CS-PM-D1).

[0036] The CS10 was purchased from Biolife in the United States. It is a clinical-grade, ready-to-use cryopreservation solution, pre-formulated with 10% DMSO, a penetrating cryoprotectant that helps reduce damage caused by ice formation.

[0037] The PBMC cryopreservation solution (Saiqiao Biotechnology, CS-PM-D1) contains a self-developed ice control protectant + 10% USP grade DMSO, and is free of serum, protein, and animal-derived components.

[0038] In some embodiments, the cell density of the apheresis cryopreservation formulation is <1×10⁻⁶. 8 cells / mL, for example 8 × 10 7 cells / mL, 4×10 7 cells / mL, 2×10 7 cells / mL, 1×10 7 cells / mL.

[0039] In some embodiments, the initial hematocrit of the apheresis cryopreservation formulation is ≤15%.

[0040] Hematocrit (HCT), also known as erythrocyte volume density (PCV), refers to the volume ratio of red blood cells that settle after centrifugation of a certain amount of anticoagulated whole blood. It is a simple method to indirectly reflect the number and size of red blood cells.

[0041] The second objective of this invention is to provide a method for preparing a cryopreservation formulation of apheresis blood. The method involves centrifuging and concentrating the apheresis blood, and then replacing the supernatant with a cryopreservation solution. The replacement volume of the cryopreservation solution is ≥50%, preferably ≥75%.

[0042] In some specific implementations, the concentration conditions for the cryopreserved preparation are centrifugation at 300-500g for 5-10 minutes.

[0043] In some implementations, the cryopreservation formulation is prepared manually or by instruments.

[0044] In some embodiments, the cryopreservation preparation is preferably configured with an instrument, and more preferably, the instrument is a fully automated cell processing system from Zhongbo Ruikang.

[0045] During preparation, blood bags, cryopreservation solution bags, and preparation bags are connected to sealed disposable consumables via sterile connectors. After gently mixing the blood sample, a small number of cells are taken for counting using the connector or the sampler provided with the consumables. After calculating the total cell count, the cells are centrifuged or concentrated using the corresponding function of the fully automated cell processing system (Zhongboruikang), and the corresponding volume of supernatant is removed. Then, the appropriate volume of cryopreservation solution is added, and the cell suspension is thoroughly mixed and dispensed using the instrument.

[0046] The third objective of this invention is to provide a T cell, which is obtained by cryopreservation and thawing of the above-mentioned single-donor blood cryopreservation preparation or the single-donor blood cryopreservation preparation prepared by the above-mentioned preparation method.

[0047] The cryopreservation process involves cooling the temperature to -80°C and then storing the sample in liquid nitrogen. For details on the temperature-controlled process, please refer to Chinese Patent CN202410960509.4, Example 3, the entire contents of which are incorporated herein by reference.

[0048] The fourth objective of this invention is to provide a method for preparing engineered T cells, comprising transfecting the aforementioned T cells with nucleic acids.

[0049] Physical methods for introducing nucleic acids into T cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation, etc. Biological methods for introducing nucleic acids into T cells include the use of DNA and RNA vectors. Chemical methods for introducing nucleic acids into T cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0050] In some embodiments, the nucleic acid contains a polypeptide coding sequence, preferably a CAR coding sequence.

[0051] In some specific embodiments, the CAR is an MSLN CAR, and more preferably, the amino acid sequence of the MSLN CAR is shown in SEQ ID NO:4.

[0052] The term "CAR" refers to a chimeric antigen receptor, which anchors specific molecules (such as antibodies) that recognize tumor cell surface antigens onto immune cells (such as T cells), enabling the immune cells to recognize tumor antigens or viral antigens and kill tumor cells or virus-infected cells. A CAR typically comprises, in sequence, an optional signal peptide, a polypeptide that binds to tumor cell membrane antigens, a hinge region, a transmembrane region, and an intracellular signaling region. Typically, the polypeptide that binds to tumor cell membrane antigens binds to membrane antigens widely expressed on tumor cells with moderate affinity. The polypeptide that binds to tumor cell membrane antigens can be a natural polypeptide or a synthetic polypeptide; preferably, the synthetic polypeptide is selected from single-chain antibodies, single-domain antibodies, Fab fragments, F(ab')2 fragments, and Fv fragments.

[0053] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Unless otherwise specified, the raw materials or processing techniques used in the following embodiments are conventional commercially available raw materials or conventional processing techniques in the art.

[0054] 1. Source of blood sample for apheresis:

[0055] Blood samples from volunteers were collected using a blood cell separator [SPECTRA OPTIA (Termall) or COM.TEC (Fresenius)], wherein the hematocrit (HCT) of the initial blood sample was not higher than 15%.

[0056] 2. Resuscitation of cryopreserved cells

[0057] Prepare a DPBS solution containing 0.5% HSA and 1 mM EDTA (referred to as "washing buffer"), preheated to 37°C. Remove the frozen apheresis blood preparations from the liquid nitrogen container and quickly place them in a water bath preheated to 37°C, shaking rapidly until the ice is just completely thawed. Transfer the thawed cell suspension to pre-prepared centrifuge tubes, count a small number of cells, add washing buffer at more than twice the volume of the cell suspension, centrifuge at 500 x g for 8 min at room temperature, and discard the supernatant.

[0058] 3. Post-resuscitation cell treatment

[0059] Cells were resuspended at 1×10⁶ cells using AIM-V medium containing 0.5% HSA (referred to as "sorting medium"). 8 Cells / mL. Add 300 U / mL nuclease (Merck). Add CD4 magnetic beads and CD8 magnetic beads [1×10⁻⁶] according to the cell volume. 7 [2 μL (Miltenyi) per cell], incubated at room temperature for 30 min. After incubation, repeat the process at 1 × 10⁻⁶ cells / cell. 7 Add 1 mL of sorting buffer to the cells, mix well, and centrifuge at 300 x g for 10 min at room temperature.

[0060] 4. T cell sorting

[0061] Discard the upper part of the solution, each 1×10 8 Add 500 μL of sorting buffer to the cells, resuspend them, and thoroughly disperse them. Select and install an MS / LS sorting column (Miltenyi) according to the cell volume. Rinse the column with 3 mL of sorting buffer. When the liquid in the column is almost completely drained, slowly add the cell suspension into the column. After the solution has completely passed through the column, add 3 mL of sorting buffer to wash the column, repeating once. Remove the sorting column from the separator and place it on a suitable collection tube. Add 5 mL of sorting buffer and quickly push the plunger into the column to rapidly wash the target cells. After counting a small number of cells, centrifuge at 300 x g for 10 min at room temperature.

[0062] 5. T cell activation and culture

[0063] Discard the supernatant and adjust the cell culture density to 2.86 × 10⁶ cells / year using AIM-V medium (referred to as "complete medium") containing IL-7 / IL-15 and 5% serum substitute. 6cells / mL. Activation culture was performed by adding the appropriate amount of activator [TransAct volume is 1 / 35 of the cell volume (Miltenyi)] according to the cell volume. The culture flasks were placed in an incubator and activated for 2 days at a temperature of 37℃±1.0℃ and a CO2 concentration of 5.0%±0.2%.

[0064] After taking a small number of cells for counting, adjust the cell culture density to 1×10⁶ using complete culture medium. 6 cells / mL. Place the culture flask in an incubator at a temperature of 37℃±1.0℃ and a CO2 concentration of 5.0%±0.2%, and repeat the above operation every 2 days for a total of 9 days.

[0065] 7. CAR-T cell preparation

[0066] Activated T cells, 1×10 7 Cells / groups were treated with 320 μg / mL piggybac enzyme mRNA and 90 μg / mL plasmid P19V21 expressing the MSLN CAR sequence. The mixture was transferred to an electroporation cuvette and placed in a Nucleofactor 4D (Lonza) or GxT (Maxcyte) electroporator. Electroporation was performed using either FI-115 or Resting T / Expand T4 program. The electroporated cell suspension was then transferred to a culture flask, incorporating complete culture medium. The mixture was incubated at 37°C. Culture medium was replenished every 2-3 days to ensure optimal cell growth conditions. On day 11, cells were washed with physiological saline, added to cryopreservation solution, and stored in liquid nitrogen.

[0067] The amino acid sequence of the piggybac enzyme is shown in SEQ ID NO:1. The plasmid map of plasmid P19V21 is shown below. Figure 1 As shown, the sequence is as shown in SEQ ID NO:2. The structure of the MSLN CAR is as follows: from the N-terminus to the C-terminus, it contains the CD8α signal peptide, mesothelin VHH 1444, CD8α hinge region, CD28 transmembrane region and intracellular co-stimulatory signal region, and CD3ζ intracellular signal domain; the amino acid sequence of mesothelin VHH 1444 is shown in SEQ ID NO:3, and the amino acid sequence of the MSLN CAR is shown in SEQ ID NO:4.

[0068] 8. Culture of CAR-T cells

[0069] After electroporation, cells were replenished with complete culture medium every 2-3 days to ensure optimal growth conditions. On day 11, cells were washed with physiological saline, added to cryopreservation solution, and stored in liquid nitrogen. CAR-T cells were then incubated at 37°C in a 5% CO2 incubator for different times to characterize and assess their effects.

[0070] Example 1: Concentration of cryopreservation solution in apheresis blood cryopreservation preparation

[0071] The apheresis blood samples collected using Method 1 were divided into 5 groups, 20 ml per group, and transferred to 500 mL centrifuge tubes. After gentle mixing, the white blood cell count and viability were determined using a Biolife counter and Trypan Blue Stain staining. The cells were concentrated by centrifugation at 300–500 g for 5–10 min at room temperature. A certain volume of the supernatant was aspirated and discarded, and the same volume of CS10 cryopreservation buffer (purchased from Biolife, containing 10% DMSO) was added. The cell suspension was then thoroughly mixed using a pipette and aliquoted. The cells were then cooled to -80°C and stored in liquid nitrogen. The replacement volumes of CS10 cryopreservation buffer were 25%, 35%, 50%, 75%, and 85% of the original volume, with a cell density of 5 × 10⁶ cells / mL. 7 cells / mL.

[0072] The frozen apheresis samples were thawed according to methods 2-6, and T cells were sorted. Flow cytometry was used to detect the proportion of T cells before and after sorting. The T cell yield was calculated by combining the total cell count with the results. Figure 2 When the replacement volume of CS10 cryopreservation solution is less than 50% (i.e., DMSO is less than 5%), the cell state is poor and the cell sorting rate is significantly reduced when sorting T cells from apheresis after thawing.

[0073] Example 2: CAR-T cells obtained from apheresis blood cryopreservation formulation

[0074] The T cells obtained in Example 1 were prepared into CAR-T cells according to methods 7-8. The transfected CAR-T cells were collected, and the number and viability of CAR-T cells were detected by using a life counter and Trypan Blue Stain staining.

[0075] The results show that... Figure 3 Figure 4 shows that when the CS10 cryopreservation solution concentration is below 50% (i.e., DMSO concentration is below 5%), the cell viability during CAR-T cell preparation after thawing is significantly reduced, and in some cases, the cell quantity is too low to complete the preparation process. Figure 3 ).

[0076] When the CS10 cryopreservation solution concentration was below 50% (i.e., DMSO concentration below 5%) (Figure 4), the cell expansion capacity during CAR-T cell preparation after thawing was significantly reduced, and in one case, the donor cell quantity was too low to complete the preparation. Figure 4-B ).

[0077] Example 3: Cell density in apheresis cryopreservation formulations

[0078] The apheresis blood samples collected using Method 1 were transferred to 500 mL centrifuge tubes. After gently mixing the blood samples, a Life counter was used for Trypan Blue Stain staining to detect the number and viability of white blood cells. The cell density was approximately 1 × 10⁻⁶. 8 Cells / mL. Concentrate by centrifugation at 300–500g for 5–10 min at room temperature, adjusting cell density as follows, using PBMC cryopreservation solution (Cybridge Biotechnology, CS-PM-D1). Based on 20ml per batch, several batches of cryopreservation formulation can be prepared for each density.

[0079] Table 1. Preparation of each frozen formulation

[0080] Apheresis dosage The volume of the sample after centrifugation PBMC cryopreservation solution replenishment volume Cell density in cryopreserved formulations 2mL 1mL 19mL <![CDATA[1x10 7 cells / mL]]> 4mL 2mL 18mL <![CDATA[2x10 7 cells / mL]]> 8mL 4mL 16mL <![CDATA[4x10 7 cells / mL]]> 16mL 8mL 12mL <![CDATA[8x10 7 cells / mL]]> 20mL 10mL 10mL <![CDATA[1x10 8 cells / mL]]>

[0081] The cryopreservation preparation was cooled to -80°C and then stored in liquid nitrogen.

[0082] Frozen apheresis samples were revived according to methods 2-6, with each group receiving 1×10⁻⁶ samples. 9 T cells were sorted; CAR-T cells were prepared according to method 7-8, transfected CAR-T cells were collected, and the number of CAR-T cells was detected using a Life counter and Trypan Blue Stain staining.

[0083] The results show that... Figure 5 When using PBMC cryopreservation solution for cryopreservation, the cell density is too high (1×10⁻⁶). 8 When the initial blood density is above a certain level (above a certain number of cells / mL), the effectiveness of CAR-T cells cultured after cryopreservation and thawing decreases. Therefore, if the initial blood density is high and not adjusted to an appropriate range, it will significantly affect the quality of cryopreservation.

[0084] Part of the sequence in this article

[0085] SEQ ID NO:1

[0086] MGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKPTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMGLTSSFMRKRLEAPTLKRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKASASCKKCKKVICREHNIDMCQSCF

[0087] SEQ ID NO:2

[0088]

[0089] SEQ ID NO:3

[0090] QVQVVESGGGFVQAGGSLRLSCAASTPIISIAYMGWYRQISEKERQLVATINSGGKTYYADSVKGRFTISRDNAKNTLYLQMNMLKPEDTGMYYCAASNKDYNDYDPDWGQGTQVTVSS

[0091] SEQ ID NO:4

[0092] MALPVTALLLPLALLLHAARPSQVQVVESGGGFVQAGGSLRLSCAASTPIISIAYMGWYRQISEKERQLVATINSGGKTYYADSVKGRFTISRDNAKNTLYLQMNMLKPEDTGMYYCAASNKDYNDYDPDWGQGTQVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

Claims

1. A single-donor blood cryopreservation preparation, characterized in that, The formulation contains apheresis blood and cryopreservation solution. After centrifugation and concentration of the apheresis blood, the supernatant is replaced with cryopreservation solution. The replacement volume of cryopreservation solution is ≥50%, preferably ≥75%.

2. The single-donor blood cryopreservation formulation as described in claim 1, characterized in that, The cryopreservation solution contains DMSO, preferably, the DMSO content in the cryopreservation solution is ≥10%; and / or the DMSO content in the apheresis blood cryopreservation preparation is ≥5%.

3. The apheresis cryopreservation preparation as described in claim 1 or 2, characterized in that, The cell density of the single-donor blood cryopreservation preparation is <1×10⁻⁶. 8 cells / mL.

4. The apheresis cryopreservation preparation as described in claim 1 or 2, characterized in that, The initial hematocrit of the apheresis blood cryopreservation preparation is ≤15%.

5. A method for preparing a cryopreservation formulation for apheresis, characterized in that, The method involves centrifuging and concentrating the apheresis blood, then replacing the supernatant with cryopreservation solution, wherein the replacement volume of the cryopreservation solution is ≥50%, preferably ≥75%.

6. The preparation method according to claim 6, characterized in that, The concentration conditions for the cryopreserved preparation are centrifugation at 300-500g for 5-10 minutes.

7. A T cell, characterized in that, The T cells are obtained by cryopreservation and thawing of the apheresis blood cryopreservation formulation according to any one of claims 1-4 or the preparation method according to claims 5-6.

8. A method for preparing engineered T cells, characterized in that, This includes transfecting nucleic acids into the T cells described in claim 7.

9. The method as described in claim 8, characterized in that, The nucleic acid contains a polypeptide coding sequence, preferably a CAR coding sequence.

10. The method as described in claim 9, characterized in that, The CAR is an MSLN CAR, and preferably, the amino acid sequence of the MSLN CAR is shown in SEQ ID NO:4.

Images