WO2023010960A1

WO2023010960A1 - Application of cd38 in preparation of car-t drug

Info

Publication number: WO2023010960A1
Application number: PCT/CN2022/095267
Authority: WO
Inventors: 余宙; 康立清; 叶晶
Original assignee: 上海优卡迪生物医药科技有限公司
Priority date: 2021-08-04
Filing date: 2022-05-26
Publication date: 2023-02-09
Also published as: CN113663061A

Abstract

Disclosed is an application of CD38 in preparation of a CAR-T drug on the basis of no need for blocking a CD38 antigen by an antibody. The CAR-T drug prepared from CD38 can be applied to preparation of antitumor drugs, especially malignant tumors such as acute myeloid leukemia. CAR-T cells are treated by using CD38 to obtain CAR gene modified T cells having negative CD38 expression (CART-38), and the CART cells having negative CD38 expression have specific recognition and cytotoxicity on target cells carrying the CD38 antigen. In the process of preparing CART-38, the CART-38 cells can be subjected to in-vitro multiplication culture, and an antibody does not need to be used for blocking the CD38 antigen.

Description

Application of CD38 in the preparation of CAR-T drugs

technical field

The present invention relates to the field of medical technology, in particular to the use of CD38, especially to the application of CD38 in the preparation of CAR-T drugs.

Background technique

CD38 has been confirmed to be widely expressed in a variety of malignant hematological tumors, so CAR-T targeting CD38 is expected to become an effective measure for the treatment of CD38 ⁺ hematological tumors. However, CD38, as a membrane anti-biomarker expressed by most T cells, is theoretically difficult to successfully prepare CD38-specific CART cells.

CD38 and hematological malignancies are potential therapeutic targets for a variety of hematological malignancies. According to statistics, 90% of multiple myeloma (MM) patients express high levels of CD38, and the expression of CD38 is closely related to the survival period of patients. In addition to MM, CD38 is also highly expressed in various malignant blood cancers such as acute myeloid leukemia (AML) and acute B-lymphoblastic leukemia (B-ALL). Current treatments for CD38 ⁺ hematologic malignancies mainly rely on anti-CD38 monoclonal antibodies, which target CD38 ⁺ malignant cells mainly through antibody-dependent cytotoxicity and antibody-dependent phagocytosis. At present, two CD38 monoclonal therapeutic antibodies (Daratumumab, isatuximab) have been approved for marketing, and have shown certain therapeutic effects in clinical practice. However, there is still a need to develop more effective treatments for CD38 ⁺ hematological malignancies.

In recent years, chimeric antigen receptor T cell therapy has made a breakthrough in the treatment of hematological malignancies. The CAR structure is to chimerize the variable single-chain region scFv (antigen receptor) and co-stimulatory signal in the antibody that can directly recognize tumor antigens to the CD3ζ chain of the activation signaling pathway of T cells. CART is not limited by the MHC molecules that recognize tumor cells, and can directly recognize tumor cell surface antigens to quickly eliminate tumor cells. One of the keys to the clinical success of CAR-T therapy is to select the appropriate CAR targeting antigen. The extracellular domain of CAR molecules is usually derived from the antigen-binding region of monoclonal antibodies. Since CD38 is widely expressed in a variety of malignant hematomas, CD38 can be used as an antigen Potential targets for specific adoptive cell therapy. CD38 is tightly regulated during T cell activation and differentiation, and human CD38 is highly expressed on early T cell precursors and CD4 ⁺ veCD8 ⁺ ve double-positive thymocytes. During negative selection, CD38 expression is reduced and mature single-positive T cells express low levels of CD38. However, mature T cells isolated from peripheral blood can regain CD38 expression upon antigen activation or in response to polyclonal stimulation. Studies have shown that CD38 is a nicotinamide adenine dinucleotide (NAD ⁺ ) glycohydrolase, which interferes with signaling and metabolic processes in T cells by consuming cellular NAD ⁺ to produce secondary messengers such as cADPR. CD38 is generally reported to play a suppressive role in the immune system and immune response, and overstimulation of CD38 suppresses antitumor immune contacts and promotes tumor development.

Therefore, it is necessary to explore the possibility of preparing clinical-grade CD38-negative CD38-targeted CART cells and the feasibility of these cells against tumors.

Contents of the invention

In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to provide the application of CD38 in the preparation of CAR-T drugs.

The present invention adopts following technical scheme:

The application of CD38 in the preparation of CAR-T drugs, especially the preparation of CAR-T drugs without antibody blocking CD38 antigen.

Further, the application of the CAR-T drug in the preparation of anti-tumor drugs.

Further, the tumor is a malignant hematological tumor. Especially acute myeloid leukemia.

Further, CD38-negative CAR gene-modified T cells (CART-38) were obtained after CD38 was used to treat CAR-T cells. During the preparation of CART-38, CD38 ⁺ cells were attacked by CART-38 and then apoptotic, while CD38-negative T cells were screened and retained during the preparation process, so the final CART-38 cells obtained were CD38-negative and CD38-negative CART cells It has specific recognition and cytotoxicity to target cells carrying CD38 antigen.

Furthermore, in the process of preparing CART-38, the method of in vitro expansion and culture is used, and the method of blocking CD38 antigen with antibodies is not used.

The research of the present invention found that in the past, those of ordinary skill in the art believed that CART-38 needs CD38 antibody to block CD38 ⁺ on T cells to have specificity for tumors. The conclusion is not completely correct, that is, it needs to block CD38 antigen with antibody to make CART -38 Drugs are technically biased. It has been verified by experiments that CD38-negative CD38-targeted CART cells can be used for the treatment of acute myeloid leukemia (AML). Therefore, the present invention provides an application for preparing CAR-T anti-tumor drugs without antibody blocking, especially for the effect on AML therapeutic drugs. The abandoned technical means solves the technical problem, so the present invention possesses an inventive step.

The present invention studies the feasibility of preparing CAR gene-modified T cells that can specifically recognize CD38 through conventional CAR-T production processes, and the feasibility of treating CD38 ⁺ AML with CD38-negative CART-38. The results show that CART-38 has specific recognition function and effective in vitro killing ability for CD38 ⁺ tumor cells. In the experiment for refractory/relapsed AML patients, it was found that CART-38 can significantly alleviate the disease status of some patients. Therefore, the feasibility of manufacturing clinical-grade CAR-T cells targeting CD38 has been verified, and CAR-T drugs prepared using CD38 can be applied to the preparation of malignant tumor drugs.

The idea, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings, so as to fully understand the purpose, features and effects of the present invention.

Description of drawings

Figure 1 is a schematic diagram of the production and expansion of CART-38 cells (n=17), wherein Figure 1A shows a schematic diagram of the transmembrane structure of CAR-CD38, Figure 1B shows the percentage of CAR-38 in T cells, and Figure 1C shows the percentage of CART-38 The expansion (-fold) of cells after transduction, Figure 1D shows the change of CD38 expression on the surface of patient T cells (upper panel) and CAR-T cells (lower panel), Figure 1E shows the expression of CD38 on the cell surface during the preparation process , Fig. 1F shows the expression of CD38 in CART-38, (the data are represented by means±SEM, ***P<0.001; ****P<0.0001vs.D0 (detection before transduction));

Figure 2 is a schematic diagram of CD38+ target cells that specifically lyse CD38 CART (n=17), wherein Figure 2A shows the proliferation response of target tumor cells transfected with mitomycin k562, co-cultured with target cells with weak replication ability 5 days ago, CART-38 was labeled with CFSE, and the proliferation of T cells was detected by flow cytometry. Figure 2B shows the observation of the effect of CART-38 cells on K652-CD38 cells by quantitatively evaluating LDH in the supernatant (the arrow represents CD38). Figure 2C shows the level of cytokine release in the supernatant of CAR-38 co-cultured with K562-cd38 or K562 cells (data are expressed as mean ± SEM, ***P<0.001; ****P<0.0001) ;

Figure 3 is a schematic diagram of CART-38 phenotype; among them, Figure 3A-Figure 3C shows the expression of CD4 and CD8 on the surface of CD38-negative anti-CD38 CAR-T detected by flow cytometry, and Figure 3D shows the detection of CD45RA and CCR7 on the surface of cells To characterize the subtype distribution of CD38-negative anti-CD38 CAR-T cells, Figure 3E shows the CD4/CD8 ratio of T cells and CD38-negative anti-CD38 CAR-T cells, and Figure 3F shows the ratio of T cells and CD38-negative anti- Changes in CD38 CAR-T cell subtypes, Figure 3G-Figure 3I show the distribution of cell subtypes between T cells and CD38-negative anti-CD38 CAR-T.

Detailed ways

In order to make the technical means, creative features, objectives and effects of the invention easy to understand, the present invention will be further elaborated below in conjunction with specific illustrations. However, the present invention is not limited to the following examples of implementation.

It should be noted that the structures, proportions, sizes, etc. shown in the drawings attached to this specification are only used to match the content disclosed in the specification, for those who are familiar with this technology to understand and read, and are not used to limit the implementation of the present invention. Limiting conditions, so there is no technical substantive meaning, any modification of structure, change of proportional relationship or adjustment of size, without affecting the effect and purpose of the present invention, should still fall within the scope of the present invention. The disclosed technical content must be within the scope covered.

Example 1:

In the first step, in this example, the CAR structure is composed of CD8-derived linkage and transmembrane domains, CD28 and 4-1BB co-stimulatory domains, and CD3-zeta signaling domains, as shown in Figure 1A. In the experiment, a total of 17 T cells from healthy donors or patients were collected and transduced with a lentiviral vector encoding CAR to generate CAR-modified T cells (CART-38) that recognize CD38. As shown in Figure 1B, the CAR expression level on the surface of the transduced T cells was verified by flow cytometry, and the median of the CAR gene transduction efficiency was 59.63%±3.988%. As shown in Figure 1C, after 7 to 13 days of culture, the total number of cells reached a 7- to 104-fold expansion, which indicated that CD38-targeted CART cells could be expanded and cultured in vitro. During the culture process, as shown in Figure 1D, the CD38 ⁺ cells gradually decreased as the culture time increased. As shown in Figure 1E, CD38 ⁺ cells began to decrease (27.2% ± 6.766%) on the second day (D1) of lentiviral transduction, and the proportion of CD38-positive cells continued to decrease in the subsequent culture, and CD38-positive cells basically disappeared on the fifth day (1.159 %±0.348%). The proportion of CD38 ⁺ in the finally obtained CART-38 was extremely low (5.143%±2.415%), as shown in Figure 1F. These results indicate that CD38 ⁺ cells undergo apoptosis after being attacked by CART-38, while CD38-negative T cells are screened and retained during the preparation process, so the finally obtained CART-38 cells are CD38-negative.

Therefore, it is necessary to verify whether CD38-negative CART cells can be used as a treatment strategy for CD38 ⁺ blood tumors. To verify this conjecture, it is first necessary to determine whether T cells still have the ability to respond in the absence of CD38.

The second step is to test whether CART-38 cells have specific recognition for CD38 ⁺ tumor cells. K562 target cells stably expressing CD38 antigen were constructed, and the target cells were co-cultured with CART cells after treatment with mitomycin, and the CD38 ^- K562 cell line was used as a reference for the control group. As shown in Figure 2A, under the stimulation of CD38 ⁺ target cells, CART cells can expand in a short period of time, which indicates that CART-38 cells can recognize CD38 ⁺ tumor cells and expand under the stimulation of CD38 antigen. To further evaluate the specific cytotoxicity of CART cells, CART-38 was co-incubated with K562-CD38 cells and K562 cells, respectively. As shown in Figure 2B, CART-38 cells had no specific killing activity on CD38 ^- K562 cells, while CD38 ⁺ tumor cells could be lysed by CART-38. Next, the secretion of IFNγ, TNFα, IL-2 and other cytokines of CART-38 after tumor stimulation and activation was detected. As shown in Figure 2C, the IFNγ cytokine levels were significantly increased when CART-38 was co-cultured with K562-cd38 cells compared with the K562 cell line. These results indicate that CD38-negative CART cells have specific recognition and cytotoxicity to target cells bearing CD38 antigen. The cytokine IL-4 was not detected at significant levels in our samples.

In the third step, in order to observe the phenotype of CART-38 cells after expansion, use the Attune NxT flow cytometer to detect the ratio of CD4 ⁺ and CD8 ⁺ subpopulations in CART-38, as shown in Figure 3A, in CART-38 cells The proportion of CD8+ subpopulation was significantly increased (64.9%±4.0%, as shown in Figure 3B), and the ratio of CD4/CD8 was <1 (0.72±0.26, as shown in Figure 3C). This indicated that CART-38 obtained after amplification was mainly composed of CD38 ^- CD8 ⁺ subset. Stem memory-like T cells (Tscm) are defined as CD45RA ⁺ CCR7 ⁺ , central memory T cells (Tcm) are defined as CD45RA ^- CCR7 ⁺ , effector memory T cells (Tem) are defined as CD45RA ^- CCR7 ^- , effector T cells (Teff ) is defined as CD45RA ⁺ CCR7 ⁻ . As shown in Figure 3D, the proportion of CD3 ⁺ Tem subgroup (41.89%±5.14%) in CART-38 was significantly higher than other subgroups (Tscm: 14.96%±3.16%, Tcm: 22.72%±2.79%, Teff: 20.41 %±3.83%), which indicated that the differentiation level of CART-38 was mainly in the Tem stage.

Next, phenotype analysis was performed on CART-38 cells and T cells before preparation. CD38 has been described as a receptor that activates the expression of T lymphocytes, so it is speculated whether the disappearance of CD38 ⁺ expression during the preparation process affects the phenotypic changes of CART-38. The CD4/CD8 ratio of CART-38 cells after transduction and amplification decreased, as shown in Figure 3E. The CD8 ⁺ cell subsets in CART-38 cells were significantly increased (**P=0.0012<0.01), while the CD4 ⁺ cell subsets were decreased (*P=0.0272<0.1, as shown in Figure 3F), which is the cause of CD4/ Proximate cause of decreased CD8 ratio. Unexpectedly, T cells containing the CD38 ⁺ subset before CAR gene transduction were also in the Tem stage, as shown in Figure 3G, which indicated that the loss of positive expression of CD38 did not affect the overall differentiation level of T cells. However, further analysis of CD4 and CD8 subtypes found that the CD4 ⁺ Tcm subpopulation decreased in CART-38 cells (**P=0.0035<0.01, as shown in Figure 3H), while the proportion of CD4 ⁺ Tem subpopulation increased (**P =0.0044<0.01, as shown in Figure 3H). The proportion of CD8 ⁺ Tcm subpopulation was significantly increased in CART-38 cells (*P=0.028<0.1, as shown in Figure 3I), although the proportion of Tem subpopulation and Teff subpopulation in CD8 ⁺ subpopulation decreased, but no statistical The difference is as shown in Figure 3I). These results may indicate that the CD4 ⁺ Tcm subtype in CART-38 is further differentiated to the more mature Tem subtype, while the CD8 ⁺ Tem and Teff subtypes may be over-differentiated to the less differentiated Tcm.

The research idea of this example is that CD38-CART-38 cells can be enriched and processed, display an expansion profile, and have the ability to kill target tumor cells robustly and specifically. This example verifies that conventional CAR-T cell production methods can be used to prepare CART cells expressing CD38-specific transgene-encoded antibodies. The obtained CART-38 cells are mainly composed of CD38 ^- CD8 ⁺ subgroups, can be expanded and cultured in vitro, and have specific recognition and cytotoxic effects on CD38 ⁺ tumor cells. Although CD38 is widely recognized as an early event of T cell activation, CD38-negative T cells have complete cellular functions that can enrich and process CART-38 cells. During the preparation of CART-38, there was only one case of CD38 ⁺ cell proliferation, but it was different from CD38 ^- CART-38, as shown in Figure 2B, this case of CD38 ⁺ CART-38 did not have specific recognition of CD38 ⁺ target cells or cells toxicity.

In this example, CART-38 cells are dominated by CD38 ^- CD8 ⁺ subpopulation. CD38-negative CDRT-38 cells are mainly CD8. In addition to T cell subtypes, the differentiation status of CART cells also plays an important role in the treatment success. In the experiment, it was found that the loss of CD38 expression did not change the overall differentiation state of T cells, and the differentiation level of CART-38 was mainly in the Tem stage, which was consistent with the differentiation state of T cells before lentiviral transduction.

Example 2:

The first step, design and production of lentiviral vector constructs

The experimental equipment is as follows in Table 1:

Table 1

The experimental materials and reagents are listed in Table 2:

Table 2

experimental method:

In this example, CD8 leader-CD38-scFV-CD8 Hinge&transmembrane domain-Costimulation domain-CD3ζ CAR structures containing 41BB co-stimulatory domains were designed. These CAR gene sequences were synthesized by Qingke Biological Co., Ltd. and cloned into Unicar by enzyme digestion and enzyme ligation. therapy’s lentiviral expression plasmid PSB1819, and then co-transfect HEK293T cells with other three lentiviral backbone plasmids (Gag/pol, vsvg, Rev) to package the required lentivirus. This experiment was completed with the help of the Unicar therapy lentiviral platform.

The CAR sequence was cloned into psb1819, and the lentiviral vector contained promoter RSV, viral structural protein Gag, essential regulatory element Rev, target gene BCMA-CAR, resistance gene AmpR, etc.

(1) Construction and extraction of recombinant plasmids

First, the psb1819 lentiviral expression plasmid backbone was cut with restriction endonucleases EcoR I and BamH I, and the resulting product was purified by gel electrophoresis to obtain a linearized plasmid. Then design and synthesize primers to amplify a large number of target fragments by PCR, and purify the PCR products by gel electrophoresis again. The linearized plasmid and the target fragment are fully mixed in an appropriate ratio, and recombinase is added to make homologous recombination. Then, TOP10 competent cells were used to conduct heat-shock transformation of the homologously recombined plasmid, and the transformed bacterial solution was evenly spread on the agar plate containing AmpR, and cultured in a CO ₂ incubator. Select a single colony, design and synthesize primers to carry out PCR experiments on it, and use gel electrophoresis to identify positive and negative colonies. Positive colonies were selected and inoculated in liquid medium containing AmpR, and transferred to a shaker for culture. Finally, use the plasmid extraction kit to extract the plasmid in the colony, sequence and verify, and compare the sequencing results with the Align software. The sequence matching is the target plasmid required for the experiment.

Collect the bacterial liquid of positive colonies, centrifuge at 8000rpm, discard the supernatant, add an appropriate amount of PBS to resuspend, wash by centrifugation, add RES-EF resuspension, vortex, and mix well. Then add LYS-EF lysate, mix evenly by inverting slowly, and let it stand until the solution is clear. Finally, add NEU-EF neutralizing solution, and mix evenly by inverting slowly until the protein precipitates. Rinse the filter adsorption column with EQU-EF, add the treated sample until complete adsorption, then add FIL-EF (eluent 1), ENDO-EF (eluent 2), WASH-EF (eluent 3) in sequence . Collect the product eluted with ELU-EF (eluent 4), add isopropanol to vortex, put it at -20°C for 20 minutes, centrifuge at 12000rpm, discard the supernatant, add 70% ethanol, 12000rpm Centrifuge and wash twice, discard the supernatant and air-dry at 12000rpm. Plasmid precipitation was added to TE-EF at the end, placed at 4°C overnight, vortexed to mix evenly, and stored.

(2) Packaging and concentration and purification of lentiviral vector

The lentiviral (LV) vector uses an improved four-plasmid expression system. Observe the state and density of HEK293T cells under a microscope, and transfect when the state is better. The first step is to slowly discard the original medium and add fresh DMEM medium containing 4% FBS to avoid blowing up the cells. Add CaCl ₂ solution and a certain volume ratio of four plasmids into the centrifuge tube, add bacterial endotoxin test water (BETW) and add HBS while vortexing. Add an appropriate amount of prepared transfection reagent to the transfected cells, shake gently to mix evenly, put it in a CO ₂ incubator and wait for 6 hours before changing the medium, that is, discard the original culture medium and add fresh 4% DMEM medium with FBS. After culturing in the incubator for 24 hours, repeat the operation of changing the medium above to continue culturing.

Check the state of the cells under a microscope, collect the supernatant of the LV carrier 48 hours after adding the transfection reagent, and use a flow pump to pass through water for bacterial endotoxin inspection for filtration. BETW, NaOH solution and ethanol were passed through again for filtration. Nuclease was added to the filtrate and placed at 4°C overnight. The sample was concentrated using a tangential flow envelope, and the lentiviral vector in the membrane envelope was replaced by HBSS and stored to determine its biological titer.

The second step, the preparation and expansion of CAR-T cells

The experimental equipment is as follows in Table 3:

table 3

The experimental reagents are listed in Table 4 below:

Table 4

The experimental consumables are listed in Table 5:

table 5

Experimental steps:

(1) Isolation of single nuclei

Use 75% alcohol to wipe and disinfect the surface of consumables such as disposable sterile 50mL centrifuge tubes, 10mL pipettes, and pipette tips with filter elements of various specifications, then put them into the biological safety cabinet, turn on the ultraviolet lamp of the biological safety cabinet, and put the biological The consumables in the safety cabinet and the operating table are sterilized by ultraviolet light for 30 minutes, and the preparations before the experiment are done.

Collect 50mL of peripheral blood into five 10mL EDTA-K2 anticoagulant tubes, wipe the surface of the anticoagulant tubes with alcohol, and operate in a biological safety cabinet after disinfection. After the anticoagulant tube was gently inverted and mixed, the peripheral blood in the anticoagulant tube was transferred to a 50mL centrifuge tube with an electric pipette for centrifugation at 2000rpm for 8min. Carefully pipette the upper layer of plasma and transfer to a new 50mL centrifuge tube. The blood cell pellet was resuspended and diluted with D-PBS(-), and the volume ratio was blood:D-PBS(-)=1:2 for mixing operation. Slowly add 25mL of diluted blood to the upper layer of 15mL lymphocyte separation medium (Ficoll) along the inner wall of the centrifuge tube, taking care not to damage the interface layer of Ficoll. After adding the blood, carefully take it to the centrifuge for horizontal centrifugation, and centrifuge at 800g room temperature for 30min.

After centrifugation, take out the centrifuge tube and remember to shake it carefully. Carefully wipe the surface of the centrifuge tube and put it into the centrifuge tube rack in the biological safety cabinet. Observe that there are obvious layers in the centrifuge tube. The "buffy coat" visible to the naked eye is the peripheral blood mononuclear cell (PBMC) obtained from this separation. . Use an electric pipette to carefully draw the buffy coat into a new centrifuge tube, resuspend the PBMC cells with 0.9% normal saline, centrifuge at 1500rpm for 5min at room temperature, wash the PBMC cells twice, resuspend the PBMC with 0.9% normal saline, and wash with trypan blue Cell counting was carried out by counting method, and 2×10 ⁸ PBMCs were taken out according to the counting results, added with 0.9% normal saline to 50 mL, centrifuged at 1500 rpm for 5 min at room temperature.

(2) T cell sorting

Resuspend the washed cell pellet with 0.5-1 mL of sorting buffer, and add 10 μl CD3 magnetic beads per 2×10 ⁷ PBMCs, mix well by pipetting, and incubate at 4°C in the dark for 15 minutes. After the incubation was completed, 10 mL of sorting buffer was added, centrifuged at 1500 rpm, and washed once at room temperature for 5 min. During this period, take a disposable separation column on the magnetic separation rack, and put a new 50mL centrifuge tube under the separation column, marked "-", for collecting CD3-negative cells; prepare a separate A new 50mL centrifuge tube marked "+" is used to collect CD3-positive cells and kept aside. Add 3mL of separation solution to the disposable separation column for rinsing. Resuspend the centrifuged cell-magnetic bead mixture pellet with 3 mL of sorting buffer, and add it to the sorting column that has been rinsed before. When the cell suspension is no longer dripping, add 3 mL of sorting solution to the sorting column to wash the negative cells remaining in the sorting column, and repeat the washing step 2 times. When the liquid is no longer dripping, add 3mL of separation liquid to the separation column again, remove the separation column, insert the plug of the separation column into the separation column, push the liquid in the separation column to the "+" centrifuge tube, collect CD3 positive T cells.

(3) T cell activation

The collected CD3 positive T cells were centrifuged at 1500rpm at room temperature for 5min, the supernatant was discarded, and the cell pellet was resuspended in T cell culture medium. CD3 and CD28 antibodies and IL-2 factors (100IU/mL) were added to the culture medium, in which CD3 and CD28 antibodies are used for the activation of T cells, while IL-2 cytokines are used to promote proliferation. The T cell use medium was adjusted to a density of 2×10 ⁶ /mL, and cultured in a carbon dioxide incubator at 37°C and 5% CO ₂ .

(4) Lentiviral transduction of cells

Take out the T cell culture bottle from the carbon dioxide incubator, operate aseptically in the biological safety cabinet, use a pipette gun to gently blow the T cells, and take out 0.5-1mL cells into a 1.5mL centrifuge tube, and use trypan blue staining method to count. Calculate the required amount of virus based on the total amount of cells, the calculation formula is volume of added virus = cell amount * MOI/virus titer, and the usual amount of MOI is 3-5. According to the calculation results, add the lentiviral vector solution into the cell suspension, mix it gently by pipetting, and culture it in a carbon dioxide incubator at 37°C and 5% CO2. 72h after lentiviral transduction, the transduction efficiency can be detected.

(5) Expansion of CAR-T cells

Take out the cells from the carbon dioxide incubator every day, observe the cell morphology and the color of the medium under an inverted microscope, and count the cells, supplement with fresh medium at a density of 2-3×10 ⁵ /mL, and culture in vitro for 7-14 days. During this period, cell expansion and counting, detection of CD3+CAR+ cells and their CD4 and CD8 cell ratios, status assessment of T cell subtypes, detection of killing efficiency by LDH method, and detection of cytokines by CBA method can be performed.

(6) Detection of CAR+ cell transduction efficiency and CD4/8 ratio

Take about 1× ¹⁰⁶ cells of non-transduced T cells and transduced CART cells, put them in 1.5mL EP tubes, centrifuge at 1500rpm for 3min, discard the supernatant, add 1mL 1xPBS to each tube to resuspend the cells , using the same centrifugation conditions for centrifugation. After washing the cells twice, use 0.1 mL of 1x PBS to resuspend the cell pellet, add 1 μL of Protein L to each tube, mix by pipetting and incubate at 4°C in the dark for 45 min. After incubation, wash each tube of cells twice with 1x PBS, resuspend the cell pellet with 0.1 mL of 1x PBS, add 0.2 μL of Streptavidin coupled with APC fluorescein to each tube, mix well by pipetting, and incubate at room temperature for 20 minutes in the dark. After incubation, add 1 mL of 1x PBS to wash twice, use 0.2 mL of 1x PBS to resuspend the cells, and use flow cytometry to detect the proportion of CAR+ cells.

(7) Trypan blue counting

Take the CART cells out of the carbon dioxide incubator and place them in a biological safety cabinet every day. Use a disposable pipette to gently blow and mix the cells in the flask, then draw about 100 μL of the cell suspension into a 1.5mL EP tube, and use a bench The cells were counted by Pan blue staining method, and the cell number and cell viability were calculated, and the cell number expansion curve was made after seven consecutive days.

The third step, cell CFSE proliferation experiment

The experimental equipment is shown in Table 6:

Table 6

The experimental materials and reagents are listed in Table 7:

Table 7

experimental method:

(1) effector cell staining

Pipette the cells in the culture flask evenly, take an appropriate volume of effector cells and put them in a 50mL centrifuge tube, centrifuge at 1500rpm for 5min, discard the supernatant, add an appropriate amount of PBS and repeat the above centrifugation and washing operation. After resuspending with a small amount of PBS and mixing evenly, dilute with trypan blue to an appropriate multiple for counting. According to the counting results, add PBS to adjust the cell density to 1×10 ⁷ /mL, then add Cell Trace ^TM CFSE reagent dissolved in DMSO at a concentration of 0.5 μL/mL, incubate at 37°C for 5 min, add an equal volume of staining stop solution (containing 5 %FBS in PBS), 1500rpm, up to 9 down to 9, centrifuged for 5min, discarded the supernatant, added an appropriate amount of PBS to repeat the above centrifugal cleaning operation once. Discard the supernatant, add an appropriate amount of CTS ^TM AIM V ^TM serum-free medium containing 4% FBS to resuspend and mix evenly, and dilute with trypan blue to an appropriate multiple for counting. According to the counting results, supplement the same medium to make the final density of cells 4×10 ⁶ /mL, mix well and set aside.

(2) target cell inactivation

Take an appropriate amount of target cells that have been pipetted evenly in the culture bottle and put them in a 50mL centrifuge tube, centrifuge at 1500rpm for 5min, discard the supernatant, add an appropriate amount of PBS, and repeat the above centrifugal cleaning operation. According to the counting results, add PBS to adjust the cell density to 1×10 ⁷ /mL, then add Mitomycin C at a concentration of 10 μg/mL, wait in a 37°C water bath for 1 hour, and mix evenly by inverting once in the middle of 10-15 minutes. Then add an appropriate amount of PBS, 1500rpm, increase by 9 and decrease by 9, centrifuge for 5 minutes, discard the supernatant, and then add an appropriate amount of PBS to repeat the above centrifugal cleaning operation. Discard the supernatant, add CTS ^TM AIM V ^TM serum-free medium containing 4% FBS to resuspend and mix evenly, and dilute with trypan blue to an appropriate multiple for counting. According to the counting results, supplement the same medium to make the final cell density 8×10 ⁵ /mL, mix well and set aside.

(3) Laying and testing

In the experiment, the ratio of effector cells to target cells was 5:1, and the total system was 500 μL, that is, 250 μL each of effector cells and target cells, and a blank control was set. Place in a 37°C, 5% CO ₂ incubator, take samples on day 0 (D0), D1, D3, and D5 of co-incubation, and put them in a 1.5mL centrifuge tube at 1500rpm, up to 9 down to 9, and centrifuge for 5min , discard the supernatant, add an appropriate amount of PBS and repeat the above centrifugal washing operation once. After adding a small amount of PBS and mixing evenly, the flow cytometer was used for FACS detection, and the data was analyzed using FlowJo V10 software.

The fourth step, LDH killing detection

The experimental equipment is shown in Table 8:

Table 8

The experimental materials and reagents are listed in Table 9 below:

Table 9

experimental method:

The experimental principle of the LDH Cytotoxicity Detection Kit is to quantitatively measure the toxicity of cells by lactate dehydrogenase. The calculation formula is: target cell lysis%=(LDH release amount in experimental group-LDH release amount in spontaneous group)/(LDH release amount in maximum group-LDH release amount in spontaneous group)×100%.

(1) Pretreatment of effector cells and target cells

Take an appropriate amount of effector cells and target cells (K562 and 8226) that have been evenly pipetted in the culture flask and put them in a 50mL centrifuge tube, centrifuge at 1500rpm for 5min, discard the supernatant, add an appropriate amount of PBS and repeat twice. Repeat the above centrifugal cleaning operation. Discard the supernatant, use a small amount of CTS ^TM AIM V ^TM serum-free medium containing 4% FBS to resuspend and mix evenly, and dilute to an appropriate multiple with trypan blue for counting. According to the counting results, supplement the same medium to make the final density of target cells 2×10 ⁵ /mL, mix well and set aside. Adjust the density of effector cells to 2×10 ⁶ /mL, 1×10 ⁶ /mL, 5×10 ⁵ /mL, 2×10 ⁵ /mL, mix well and set aside.

(2) Laying and testing

Effector cells and target cells were plated in 96-well plates according to the ratio of 10:1, 5:1, 2.5:1, and 1:1, respectively. The total system was 100 μL, that is, the volume of effector cells and target cells was 50 μL, respectively. After laying the cells, the well plate was sealed with a parafilm, centrifuged at 250g, liter 3 down 1, for 5min, then placed in a 37°C, 5% CO ₂ incubator, and incubated overnight. Then 10 μL of lysate was added to each well of the target cells, and the plate was placed in the original incubator to incubate for 40 min. Take out the orifice plate, 250g, rise by 3 and drop by 1, centrifuge for 5min, pipette 50μL of supernatant into a clean 96-well plate, and then add 50μL of the detection substrate mixture dissolved in buffer. Incubate for 10 min at 20-25°C in a light-proof environment, observe the color development, and finally measure the absorbance value at a wavelength of 490 nm using a microplate reader.

The fifth step, cytokine detection

The experimental equipment is as shown in Table 10:

Table 10

The experimental materials and reagents are listed in Table 11 below:

Table 11

The experimental method is as follows:

(1) Pretreatment of effector cells and target cells

Pipette the cells in the culture flask evenly, take an appropriate amount of effector cells and target cells (K562 and 8226) and place them in a 50mL centrifuge tube, centrifuge at 1500rpm for 5min, discard the supernatant, add an appropriate amount of PBS and repeat twice. Repeat the above centrifugal cleaning operation. Discard the supernatant, use a small amount of CTS ^TM AIM V ^TM serum-free medium containing 4% FBS to resuspend and mix evenly, and dilute to an appropriate multiple with trypan blue for counting. According to the counting results, supplement the same medium to make the final density of target cells 2×10 ⁵ /mL, mix well and set aside. The same method was used to make the final density of effector cells 1×10 ⁶ /mL, mix well and set aside.

(2) Laying and sampling

Effector cells and target cells were plated in a 96-well plate at a ratio of 5:1, and the total system volume was 100 μL, that is, the volume of effector cells and target cells was 50 μL, respectively. After the cells are plated, the orifice plate is centrifuged at 250g, liters 3 to 1, for 5 minutes, then placed in a 37°C, 5% CO ₂ incubator, incubated for 18-24 hours, and then the orifice plate is taken out, 250g, liters 3 to 1, After centrifugation for 5 min, 50 μL of the supernatant was collected as the sample to be tested.

(3) Standard curve preparation and sample determination

Take 2mL diluent to fully dissolve the standard in the kit, and dilute a total of 10 standard samples with a 2-fold concentration gradient. Take 50 μL of capture microspheres, add 50 μL of sample, and then add 50 μL of PE detection reagent, vortex and mix well, and incubate at 20-25 ° C for 3 h in the dark. Then add 1mL buffer, centrifuge at 300g for 5min, discard part of the supernatant, and finally detect on the Attune NxT flow cytometer. Data were analyzed using LEGENDplex v8.0, and cytokine concentrations were calculated from standard curves.

In the experiment, a third-generation lentiviral CD38-chimeric antigen receptor was constructed, and T cells from healthy donors and CD38 ⁺ blood tumor patients were transduced to prepare CD38-targeted CART cells (CART-38). And further evaluate the immune phenotype and anti-tumor cell ability of CART-38. CART-38 cells can be expanded and cultured in vitro, with CD8 ⁺ CD38 ^- cells as the main component. CART-38 has specific recognition and cytotoxicity to CD38 ⁺ tumor cells. The loss of positive expression of CD38 does not affect the overall differentiation level of T cells and the ability to kill tumor cells. Anti-CD38 CAR-T cells can be prepared by conventional methods, which are characterized by CD38-negative CD38-targeted CART cells, and can be used for effective clinical treatment of AML.

In conclusion, the use of CART-38 is a safe and feasible treatment for CD38 ⁺ hematological tumors. The present invention verifies the feasibility, cell function and specific cytotoxicity of CD38-negative CART preparation, and analyzes and characterizes the characteristics of CD38-negative CART-38 cells. The present invention provides a reference for the preparation of clinical-level CD38-targeted CART cells, and also provides a new drug for the treatment of CD38 ⁺ malignant blood tumors.

The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make many modifications and changes according to the concept of the present invention without creative efforts. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art shall be within the scope of protection defined by the claims.

Claims

The application of CD38 in the preparation of CAR-T medicine is characterized in that the CAR-T medicine is prepared on the basis of not needing to block the CD38 antigen with an antibody.
The application according to claim 1, characterized in that the CAR-T drug is used in the preparation of anti-tumor drugs.
The use according to claim 2, characterized in that the tumor is a hematologic malignancy.
The use according to claim 3, characterized in that the malignant hematological tumor is acute myeloid leukemia.
The application according to any one of claims 1-4, wherein CD38 is used to treat CAR-T cells to obtain CD38-negative CAR gene-modified T cells CART-38, which are specific to target cells carrying CD38 antigens recognition and cytotoxicity.
The application according to claim 5, characterized in that, in the process of preparing CART-38, an in vitro expansion culture method is used.