CN113663061A

CN113663061A - Application of CD38 in preparation of CAR-T medicine

Info

Publication number: CN113663061A
Application number: CN202110895027.1A
Authority: CN
Inventors: 余宙; 叶晶; 徐南; 谭静雯; 康立清
Original assignee: Shanghai Unicar Therapy Bio Medicine Technology Co Ltd
Current assignee: Shanghai Unicar Therapy Bio Medicine Technology Co Ltd
Priority date: 2021-08-04
Filing date: 2021-08-04
Publication date: 2021-11-19
Also published as: WO2023010960A1

Abstract

The invention discloses application of CD38 in preparation of CAR-T drugs on the basis of no need of antibody blocking of CD38 antigen. The CAR-T medicine prepared by using the CD38 can be applied to preparing anti-tumor medicines, in particular malignant tumors, such as acute myelogenous leukemia. CAR genetically modified T cells (CART-38) negative for CD38 expression were obtained after treatment of CAR-T cells with CD38, CD38 negative CART cells have specific recognition and cytotoxicity against target cells carrying CD38 antigen. In the process of preparing CART-38, CART-38 cells can be expanded and cultured in vitro without the need to block CD38 antigen with antibodies. The invention can be applied to effectively treat the swellingTumor diseases, provides reference for the preparation mode of clinical-grade targeting CD38 CART cells, and is CD38⁺Hematological malignancy treatment offers more options.

Description

Application of CD38 in preparation of CAR-T medicine

Technical Field

The invention relates to the technical field of medicines, in particular to application of CD38, and especially relates to application of CD38 in preparation of CAR-T medicines.

Background

CD38 is proved to be widely expressed in various malignant blood tumors, so that CAR-T targeting CD38 is expected to become a drug for treating CD38⁺Effective measures for blood tumor. However, CD38, as a membrane-anti-biomarker expressed by most T cells, is theoretically difficult to successfully prepare CD 38-specific CART cells.

CD38 and hematologic tumors are potential therapeutic targets for a variety of hematologic malignancies. Statistically, 90% of Multiple Myeloma (MM) patients express high levels of CD38, and expression of CD38 is closely correlated with survival of the patients. In addition to MM, CD38 is also highly expressed in a variety of hematological malignancies, such as Acute Myeloid Leukemia (AML), acute B-lymphocytic leukemia (B-ALL), and the like. Is currently directed to CD38⁺The treatment of hematologic malignanes relies primarily on anti-CD38 monoclonal antibodies, and anti-CD38 antibodies target CD38 primarily through antibody-dependent cytotoxicity and antibody-dependent phagocytosis⁺Malignant cells. Currently, 2 monoclonal antibodies (Daratumumab, isatuximab) for treating CD38 are approved to be marketed, and have a certain treatment effect clinically. But for CD38⁺There is still a need to develop more effective treatment modalities for 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 chimeric variable single chain region scFv (antigen receptor) that can directly recognize tumor antigen in antibody and costimulatory signal to activation signal pathway CD3 zeta chain of T cell. The CART is not limited by MHC molecules for recognizing tumor cells, and can directly recognize tumor cell surface antigens to rapidly eliminate the tumor cells. One of the keys to the clinical success of CAR-T therapy is the selection of appropriate CAR-targeting antigens, the ectodomain of CAR molecules is usually derived from the antigen-binding region of monoclonal antibodies, and since CD38 is widely expressed in a variety of hematologic malignancies, CD38 can serve as a potential target for antigen-specific adoptive cell therapy. CD38Tightly regulated during T cell activation and differentiation, human CD38 is early in T cell precursors and CD4⁺veCD8⁺High expression on ve double positive thymocytes. During negative selection, CD38 expression decreased and mature single positive T cells expressed low levels of CD 38. However, mature T cells isolated from peripheral blood can regain CD38 expression in response to antigen activation or polyclonal stimulation. Studies have shown that CD38 is a Nicotinamide Adenine Dinucleotide (NAD)⁺) Glycohydrolases that deplete cellular NAD⁺Secondary messengers such as cADPR are generated to interfere with T intracellular signaling and metabolic processes. CD38 is generally reported to play an inhibitory role in the immune system and immune response, and overstimulation of CD38 inhibits anti-tumor immune communication and promotes tumor development.

Therefore, it is necessary to investigate the feasibility of preparing clinical grade CD38 negative CD38-targeted CART cells and the anti-tumor properties of these cells.

Disclosure of Invention

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

The invention adopts the following technical scheme:

the application of CD38 in preparing CAR-T medicine, in particular to preparing CAR-T medicine on the basis of CD38 antigen without antibody blocking.

Further, the CAR-T drug is applied to preparing antitumor drugs.

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

Further, CAR-T cells treated with CD38 resulted in CAR genetically modified T cells negative for CD38 expression (CART-38). In the process of preparing CART-38, CD38⁺The cells are attacked by CART-38 and then die, and CD38 negative T cells are screened and reserved in the preparation process, so the finally obtained CART-38 cells are CD38 negative, and the CD38 negative CART cells have specific recognition and cytotoxicity on target cells carrying CD38 antigen.

Further, in the process of preparing CART-38, an in vitro amplification culture mode is used, and a method for blocking CD38 antigen by using an antibody is not used.

The present inventors have discovered that, heretofore, those of ordinary skill in the art have recognized that CART-38 requires a CD38 antibody to block CD38 on T cells⁺The conclusion that specificity for tumors is not entirely correct, i.e., it is a technical prejudice that antibodies are required to block the CD38 antigen in order to prepare a CART-38 drug. Experiments prove that the CD38-targeted CART cells with negative CD38 can be used for treating acute myelogenous leukemia AML. Therefore, the invention provides an application of CAR-T antitumor drug preparation on the basis of no need of antibody blocking, in particular to the effect on AML treatment drug, overcomes the technical bias, adopts the technical means abandoned by people due to the technical bias, and solves the technical problems, thereby having creativity.

The invention researches the feasibility of preparing CAR gene modified T cells capable of specifically recognizing CD38 by the conventional CAR-T production process and treats CD38 by CART-38 negative in CD38 expression⁺Feasibility of AML. The results showed that CART-38 is on CD38⁺Tumor cells have specific recognition function and effective in-vitro killing capacity, and CART-38 is found to be capable of remarkably relieving the disease condition of a part of patients in experiments on refractory/relapsed AML patients. Therefore, the feasibility of manufacturing clinical-grade CAR-T cells targeting CD38 is verified, and CAR-T medicaments prepared by applying CD38 can be applied to preparation of malignant tumor medicaments.

The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.

Drawings

Figure 1 is a schematic representation of CART-38 cell generation and expansion (n ═ 17), wherein figure 1A represents a schematic representation of CAR-CD38 transmembrane structure, figure 1B represents the percentage of CAR-38 in T cells, figure 1C represents the expansion (-folding) of CART-38 cells after transduction, figure 1D represents the change in CD38 expression on the surface of patient T cells (upper panel) and CAR-T cells (lower panel), figure 1E represents the expression of CD38 on the cell surface during preparation, figure 1F represents the expression of CD38 in CART-38, (data expressed as mean ± SEM,. P < 0.0001. vs. D0 (pre-transduction assay));

FIG. 2 is a schematic representation of CD38+ target cells (n ═ 17) specifically lysing CD38 CART, wherein FIG. 2A shows the proliferative response of mitomycin transfected K562 target tumor cells, CART-38 was labeled with CFSE 5 days before co-culture with less replicating target cells, and t-cell proliferation was detected using flow cytometry, FIG. 2B shows the cytokine release levels in supernatants by quantitative assessment of LDH (arrows represent CD38) in supernatants, observing lysis of K652-CD38 cells by CART-38 cells, and FIG. 2C shows the cytokine release levels in supernatants co-cultured with K562-CD38 or K562 cells (data expressed as mean. + -. SEM,. P < 0.001;. P < 0.0001);

FIG. 3 is a schematic representation of the CART-38 phenotype; wherein, fig. 3A-3C show the expression of CD38 negative anti-CD38 CAR-T surface CD4, CD8 using flow cytometry, fig. 3D shows cell surface staining to detect CD45RA and CCR7 expression to characterize the CD38 negative anti-CD38 CAR-T cell subtype distribution, fig. 3E shows the CD4/CD8 ratio of T cells and CD38 negative anti-CD38 CAR-T cells, fig. 3F shows the change of T cells and CD38 negative anti-CD38 CAR-T cell subtype, fig. 3G-3I show the cell subtype distribution between T cells and CD38 negative anti-CD38 CAR-T.

Detailed Description

In order to make the technical means, the characteristics, the purposes and the functions of the invention easy to understand, the invention is further described with reference to the specific drawings. However, the present invention is not limited to the following embodiments.

It should be understood that the structures, ratios, sizes, and the like shown in the drawings and described in the specification are only used for matching with the disclosure of the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions under which the present invention can be implemented, so that the present invention has no technical significance, and any structural modification, ratio relationship change, or size adjustment should still fall within the scope of the present invention without affecting the efficacy and the achievable purpose of the present invention.

Example 1:

in the first step, the CAR structure is composed of a CD 8-derived linking and transmembrane domain, CD28 and 4-1BB costimulatory domain, and CD3-zeta signaling domain together, as shown in figure 1A. In the experiment, a total of 17T cells from healthy donors or patients were collected and CAR-modified T cells recognizing CD38 (CART-38) were generated by transduction with lentiviral vectors encoding the CAR. As shown in fig. 1B, the flow cytometry was used to verify CAR expression on the surface of transduced T cells, with a median CAR gene transduction efficiency of 59.63% ± 3.988%. As shown in fig. 1C, after 7 to 13 days of culture, the total number of cells reached 7 to 104-fold expansion, indicating that CART cells targeting CD38 can be expanded in vitro. During the culture, as shown in FIG. 1D, CD38 increased with the culture time⁺The cells gradually decrease. As shown in FIG. 1E, CD38 on the day following lentiviral transduction (D1)⁺The cells began to decrease (27.2% ± 6.766%), after which the proportion of CD38 positive cells continued to decrease in culture, with the CD38 positive cells essentially disappearing at day five (1.159% ± 0.348%). CD38 in finally obtained CART-38⁺The ratio was very low (5.143% ± 2.415%), as shown in fig. 1F. These results indicate CD38⁺Cells were attacked by CART-38 and then apoptotic, whereas CD38 negative T cells were screened and retained during the preparation process, so the final CART-38 cells were CD38 negative.

It was therefore necessary to verify whether CD 38-negative CART cells could be used as CD38⁺To validate this hypothesis, it was first determined whether T cells were also immune-competent in the absence of CD 38.

Second, it was examined whether the CART-38 cells are paired with CD38⁺Tumor cells have specific recognition. Constructing K562 target cells stably expressing CD38 antigen, co-culturing the target cells and CART cells after mitomycin treatment, CD38^－The K562 cell line of (a) was used as a control reference. As shown in fig. 2A, at CD38⁺The CART cells can be expanded in a short period of time under the stimulation of target cells, which shows that the CART-38 cells can recognize CD38⁺Tumor cells and expanded under stimulation of the CD38 antigen. To further assess the specific cytotoxicity of CART cells, CART was used-38 were incubated with K562-CD38 cells and K562 cells, respectively. As shown in FIG. 2B, CART-38 cells vs CD38^－K562 cells had no specific killing activity, whereas CD38⁺Tumor cells can be lysed by CART-38. Next, the secretion of cytokines such as IFN gamma, TNF alpha, IL-2, etc. of CART-38 after tumor stimulation activation was examined. As shown in fig. 2C, IFN γ cytokine levels were significantly elevated when CART-38 was co-cultured with K562-cd38 cells, compared to the K562 cell line. These results indicate that CD 38-negative CART cells have specific recognition and cytotoxicity against target cells carrying the CD38 antigen. Cytokine IL-4 was not detected at significant levels in our samples.

Thirdly, in order to observe the phenotype of the expanded CART-38 cells, the ATTUNE NxT flow cytometer is used for detecting CD4 in the CART-38 cells⁺And CD8⁺Proportion of subpopulations, as shown in FIG. 3A, the proportion of CD8+ subpopulations was significantly increased in CART-38 cells (64.9% + -4.0%, as shown in FIG. 3B), and the CD4/CD8 ratio<1(0.72 ± 0.26, as shown in fig. 3C). This indicates that the CART-38 obtained after amplification is mainly composed of CD38^-CD8⁺subset composition. Stem memory-like T cells (Tsccm) was defined as CD45RA⁺CCR7⁺The central memory T cells (Tcm) is defined as CD45RA^－CCR7⁺The effect memory T cells (Tem) is defined as CD45RA^－CCR7^－The effector T cells (Teff) is defined as CD45RA⁺CCR7^－. As shown in FIG. 3D, CD3 in CART-38⁺The proportion of the Tem subpopulation (41.89% + -5.14%) was significantly higher than the other subpopulations (Tsccm: 14.96% + -3.16%, Tcm: 22.72% + -2.79%, Teff: 20.41% + -3.83%), indicating that the level of differentiation of CART-38 is mainly in the Tem phase.

Next, phenotypic analysis was performed on CART-38 cells and T cells before preparation, respectively. CD38 has been described as activating a receptor expressed by T lymphocytes, and it was therefore hypothesized that CD38 was involved in the preparation process⁺Whether the loss of expression affects the phenotypic change of CART-38. The CD4/CD8 ratio of CART-38 cells decreased after transduction amplification, as shown in FIG. 3E. CD8 in CART-38 cells⁺Cell subsets were significantly elevated (═ P ═ 0.0012<0.01), and CD4⁺Cell subset reduction (═ P ═ 0.0272<0.1, as shown in FIG. 3F), which results in a CD4/CD8 ratioThe direct cause of the reduction. Unexpectedly, the CAR gene prior to transformation comprises CD38⁺The T cells of the subpopulation were also at the Tem stage, as shown in fig. 3G, indicating that the lack of positive expression of CD38 did not affect the level of differentiation of the T cells as a whole. However, further analysis of CD4 and CD8 subtypes revealed that CD4 is present in CART-38 cells⁺The Tcm subpopulation decreased (═ P ═ 0.0035<0.01, as shown in FIG. 3H), and CD4⁺The proportion of the Tem subgroup increases (. about.P ═ 0.0044)<0.01, as shown in fig. 3H). CD8 in CART-38 cells⁺The fraction of Tcm subgroup was significantly increased (P ═ 0.028)<0.1, as shown in FIG. 3I), although CD8⁺The proportion of the Tem and Teff subpopulations decreased in the subpopulations, but no statistical difference was found (as shown in fig. 3I). These results may indicate that CD4 in CART-38⁺The Tcm subtype further differentiates into a more mature Tem subtype, whereas CD8⁺The Tem and Teff subtypes may be excessive toward Tcm with a low degree of differentiation.

The study of this example was thought that CD 38-CART-38 cells could be enriched and processed, showing an expanded profile, with the ability to robustly and specifically kill target tumor cells. This example demonstrates that CART cells expressing a CD 38-specific transgene-encoding antibody can be prepared using conventional CAR-T cell production means. Obtaining CART-38 cells as CD38^-CD8⁺Mainly comprises subgroup, can be amplified and cultured in vitro, and is used for CD38⁺The tumor cells have specific recognition and cytotoxicity effects. Although CD38 is widely recognized as an early event in T cell activation, CD38 negative T cells have a complete cellular function and can enrich and process CART-38 cells. CD38 appeared in only one instance during the preparation of CART-38⁺Cell proliferation, but different from CD38^－CART-38, as shown in FIG. 2B, this example is CD38⁺CART-38 vs. CD38⁺The target cells do not have specific recognition or cytotoxicity.

In this example, CART-38 cells are expressed as CD38^-CD8⁺The subgroup is dominant. CD38 negative CDRT-38 cells predominate in CD 8. In addition to T cell subtypes, the differentiation state of CART cells also plays an important role in therapeutic success. In experiments, the deletion of CD38 expression does not change the differentiation state of the whole T cells, the differentiation level of CART-38 is mainly in the Tem stage, and the chronic diseaseThe differentiation state of T cells before toxin transduction is consistent.

Example 2:

first step, design and production of lentiviral vector constructs

The experimental apparatus is as follows in table 1:

TABLE 1

The experimental materials and reagents are as follows in table 2:

TABLE 2

The experimental method comprises the following steps:

in this example, CD8 leader-CD38-scFV-CD8 Hinge & transmembrane domain-stimulation domain-CD3 zeta CAR structures containing 41BB co-stimulatory domains were designed, and these CAR gene sequences were synthesized by Scopcodes, cloned into the lentiviral expression plasmid PSB1819 of Unicar therapy by enzymatic ligation, and then co-transfected with other 3 lentiviral backbone plasmids (Gag/pol, vsvg, Rev) into HEK293T cells to package the desired lentiviruses. This experiment was done with the help of the Unicar therapy lentiviral platform.

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

(1) Construction and extraction of recombinant plasmid

First, the p was cut with restriction enzymes EcoR I and BamH Isb1819 lentivirus expression plasmid backbone, and the resulting product was purified by gel electrophoresis to obtain linearized plasmids. Then designing and synthesizing primers, amplifying a large number of target fragments by using PCR, and purifying the PCR product by gel electrophoresis again. The linearized plasmid and the target fragment are mixed well in a suitable ratio, and a recombinase is added to perform homologous recombination. The plasmid after homologous recombination was heat-shocked by TOP10 competent cells, and the resulting bacterial suspension was spread on an agar plate containing AmpR and placed on a CO plate₂Culturing in an incubator. Selecting single colony, designing and synthesizing a primer pair to carry out PCR experiment, and identifying the positive and negative colonies of the product by gel electrophoresis. And selecting positive colonies, inoculating the positive colonies into a liquid culture medium containing AmpR, and transferring the positive colonies to a shaking table for culture. And finally, extracting plasmids in the bacterial colony by using a plasmid extraction kit, carrying out sequencing verification, comparing sequencing results by using Align software, and obtaining a target plasmid required by the experiment if the sequence is matched.

Collecting bacterial liquid of the positive bacterial colony, centrifuging at 8000rpm, removing supernatant, adding appropriate amount of PBS for resuspension, centrifuging, washing, adding RES-EF suspension, vortex oscillating, and mixing well. Adding LYS-EF lysis solution, slowly and uniformly mixing by inversion, and standing until the solution is clear. And finally adding NEU-EF neutralizing solution, slowly reversing and uniformly mixing until protein is separated out. Rinsing the filtration adsorption column with EQU-EF, adding the treated sample until complete adsorption, sequentially adding FIL-EF (eluent 1), ENDO-EF (eluent 2) and WASH-EF (eluent 3), collecting the product eluted by ELU-EF (eluent 4), adding isopropanol, performing vortex oscillation, standing at-20 deg.C for 20min, centrifuging at 12000rpm, discarding the supernatant, adding 70% ethanol, centrifuging at 12000rpm, washing twice, discarding the supernatant, and air-drying at 12000 rpm. And finally adding TE-EF into the plasmid precipitate, standing at 4 ℃ overnight, and storing after uniform vortex mixing.

(2) Packaging and concentration purification of lentivirus vector

Lentiviral (LV) vectors use a modified four plasmid expression system. HEK293T cells were observed under microscope for status and density, preferably for transfection. In the first step, the original culture medium is slowly discarded and fresh DMEM medium containing 4% FBS is added to avoid the cells being coveredAnd (4) blowing. Adding CaCl into a centrifuge tube₂The solution and a volume ratio of four plasmid, adding Bacterial Endotoxin Test Water (BETW) and adding HBS while vortex. Adding proper amount of prepared transfection reagent into transfected cells, slightly shaking and mixing uniformly, and then adding CO₂After the culture box waits for 6 hours, the culture solution is replaced, the original culture solution is discarded, and a fresh DMEM culture medium containing 4% FBS is supplemented. After the incubator is used for 24 hours, the liquid changing operation is repeated to continue the culture.

The cell state was examined under a microscope, the supernatant of LV vector after 48h addition of transfection reagent was collected and filtered by passing water for endotoxin examination of bacteria using a flow pump. The BETW, NaOH solution and ethanol were added again for filtration. Nuclease was added to the filtrate and left overnight at 4 ℃. The sample was concentrated using a tangential flow membrane, and the HBSS was stored after displacing the lentiviral vector in the membrane, and the biological titer was determined.

Second step, CAR-T cell preparation and expansion

The experimental apparatus is as follows in table 3:

TABLE 3

The experimental reagents are as follows in table 4:

TABLE 4

The experimental consumables are as follows 5:

TABLE 5

The experimental steps are as follows:

(1) isolation of Individual cell nuclei

Wiping and disinfecting the surfaces of consumable materials such as disposable sterile 50mL centrifuge tubes, 10mL pipettes and various specifications of gun heads with filter elements by using 75% alcohol, then placing the consumable materials into a biological safety cabinet, starting an ultraviolet lamp of the biological safety cabinet, carrying out ultraviolet sterilization on the consumable materials and an operation table top in the biological safety cabinet for 30min, and preparing before experiments.

Collecting 50mL of peripheral blood in a 5-tube 10mL EDTA-K2 anticoagulation tube, wiping the surface of the anticoagulation tube with alcohol, and sterilizing the anticoagulation tube and then operating in a biological safety cabinet. After the anticoagulation tube is gently inverted and uniformly mixed, peripheral blood in the anticoagulation tube is transferred to a 50mL centrifuge tube by using an electric pipettor for centrifugation at 2000rpm for 8 min. The upper plasma layer was carefully pipetted into a new 50mL centrifuge tube. The blood cell pellet was resuspended and diluted with D-PBS (-) at volume ratio blood: the mixing was performed at a ratio of D-PBS (-) to 1: 2. 25mL of diluted blood was slowly added to the top layer of 15mL of lymphocyte separation medium (Ficoll) along the inner wall of the centrifuge tube, taking care not to break the Ficoll interface layer. After the addition of blood was completed, the mixture was carefully placed in a centrifuge for horizontal centrifugation, and centrifuged at 800g at room temperature for 30 min.

After the centrifugation, the tube was removed and shaken. Carefully wiping the surface of the centrifuge tube, putting the centrifuge tube into a centrifuge tube rack in a biological safety cabinet, and observing that obvious layering exists in the centrifuge tube, wherein a macroscopic 'tunica albuginea' is the Peripheral Blood Mononuclear Cells (PBMC) obtained by the separation. Carefully aspirating the leukocyte membrane layer into a new centrifuge tube using an electric pipette, resuspending PBMC cells using 0.9% physiological saline, centrifuging at 1500rpm for 5min at room temperature, washing the PBMC cells for 2 times, resuspending PBMC using 0.9% physiological saline, performing cell counting using trypan blue counting method, and taking out 2X 10 cells according to the counting result⁸PBMC were then supplemented with 0.9% saline to 50mL at 1500rpm and centrifuged at room temperature for 5 min.

(2) T cell sorting

Resuspending the washed cell pellet with 0.5-1mL of sorting buffer per 2X 10⁷ Add 10 μ LCD3 magnetic beads to PBMC, blow and mix well, incubate 15min at 4 ℃ in the dark. After incubation was complete, 10mL of sort buffer was addedThe solution was washed once by centrifugation at 1500rpm for 5min at room temperature. During this period, a disposable sorting column was taken on the magnetic sorting rack and a new 50mL centrifuge tube, labeled "-", was placed under the sorting column for collection of CD3 negative cells; a new 50mL centrifuge tube, labeled "+", was prepared and used to collect CD3 positive cells for future use. 3mL of the sort solution was added to the disposable sort column for rinsing. The centrifuged cell-magnetic bead mixture pellet was resuspended in 3mL of sorting buffer and loaded onto the previously rinsed sorting column. And when the cell suspension does not drip any more, adding 3mL of sorting solution into the sorting column for washing the negative cells remained in the sorting column, and repeating the washing step for 2 times. When the liquid does not drip any more, adding 3mL of sorting solution into the sorting column again, taking down the sorting column, inserting the plug of the sorting column into the sorting column, pushing the liquid in the sorting column into a '+' centrifuge tube, and collecting to obtain CD3 positive T cells.

(3) T cell activation

The collected CD3 positive T cells are centrifuged at 1500rpm for 5min at room temperature, the supernatant is discarded, the cell pellets are resuspended by using a T cell culture medium, and CD3 and CD28 antibodies and IL-2 factor (100IU/mL) are added to the culture medium, wherein the CD3 and CD28 antibodies are used for activating the T cells, and the IL-2 cytokine is used for promoting proliferation. The culture medium used for the T cells was adjusted to 2X 10⁶Density in/mL, 5% CO at 37 ℃₂The carbon dioxide incubator of (1).

(4) Lentiviral transduction of cells

Taking out the T cell culture bottle from the carbon dioxide incubator, performing aseptic operation in a biological safety cabinet, gently blowing the T cells evenly by using a pipette gun, taking out 0.5-1mL of cells into a 1.5mL centrifugal tube, and counting by adopting a trypan blue staining method. The required amount of virus is calculated from the total amount of cells by adding the virus volume (cell amount) MOI/virus titer, and the MOI is usually used in an amount of 3-5. According to the calculation result, the lentiviral vector liquid is added into the cell suspension, and after being gently blown and uniformly mixed, the mixture is cultured in a carbon dioxide incubator with 5 percent CO2 at 37 ℃. Transduction efficiency can be measured 72h after lentiviral transduction.

(5) Expansion of CAR-T cells

Taking out cells from carbon dioxide incubator every day, observing cell morphology and culture medium color under inverted microscope, and counting cells according to 2-3 × 10⁵The density of/mL was supplemented with fresh medium and cultured in vitro for 7-14 days. During this time, cell expansion counts, detection of CD3+ CAR + cells and their CD4 and CD8 cell proportions, status assessment of T cell subtypes, detection of killing efficiency by LDH method, and detection of cytokines by CBA method can be performed.

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

Taking non-transduced T cells and transduced CART cells each about 1X 10⁶Each cell was individually placed in a 1.5mL EP tube, centrifuged at 1500rpm for 3min and the supernatant discarded, 1mL of 1xPBS was added to each tube to resuspend the cells, and centrifuged and washed using the same centrifugation conditions. After washing the cells repeatedly for 2 times, the cell pellet was resuspended in 0.1mL of 1 × PBS, 1 μ L of Protein L was added to each tube, pipetted and mixed well, and incubated at 4 ℃ for 45min in the dark. After the incubation was completed, each tube was washed twice with 1 × PBS, the cell pellet was resuspended with 0.1mL of 1 × PBS, 0.2 μ L of Streptavidin coupled to APC fluorescein was added to each tube, pipetted well and incubated at room temperature in the dark for 20 min. After incubation was complete, the cells were washed twice with 1mL of 1 × PBS, resuspended using 0.2mL of 1 × PBS, and the CAR + cell proportion was examined by flow cytometry.

(7) Trypan blue count

Taking the CART cells out of the carbon dioxide incubator every day, placing the CART cells in a biological safety cabinet, gently blowing and beating the cells in a mixing bottle by using a disposable pipette, sucking about 100 mu L of cell suspension into a 1.5mLEP tube, counting the cells by using a trypan blue staining method, calculating the number of the cells and the survival rate of the cells, and preparing a cell number expansion curve after seven continuous days.

Third, cell CFSE proliferation assay

The experimental apparatus is as follows in table 6:

TABLE 6

The experimental materials and reagents are as follows in table 7:

TABLE 7

The experimental method comprises the following steps:

(1) effector cell staining

And (3) uniformly blowing the cells in the culture bottle, putting effector cells with proper volume in a 50mL centrifuge tube, lifting 9 and lowering 9 at 1500rpm, centrifuging for 5min, discarding supernatant, and adding a proper amount of PBS (phosphate buffer solution) to repeat the centrifugal washing operation. After being mixed well with a small amount of PBS, trypan blue was diluted to an appropriate multiple for counting. Based on the counting results, the cell density was adjusted to 1X 10 with PBS supplementation⁷Then Cell Trace dissolved in DMSO was added at a concentration of 0.5. mu.L/mL^TMCFSE reagent, 37 degrees C were incubated for 5min, adding equal volume staining stop solution (containing 5% FBS PBS), 1500rpm, rising 9 and falling 9, centrifugation for 5min, discarding the supernatant, adding appropriate amount of PBS and repeat the above centrifugation and washing operation. Removing supernatant, adding appropriate amount of CTS containing 4% FBS^TM AIM V^TMSerum-free medium was resuspended well mixed and trypan blue diluted to the appropriate fold for counting. According to the counting results, the cells were supplemented with the same medium to a final density of 4X 10⁶and/mL, mixing uniformly for later use.

(2) Target cell inactivation

And (3) taking a proper amount of target cells which are blown uniformly in a culture bottle, putting the target cells into a 50mL centrifuge tube, raising and lowering the target cells by 9 at 1500rpm, centrifuging for 5min, discarding supernatant, and adding a proper amount of PBS (phosphate buffer solution) to repeat the centrifugal cleaning operation. Based on the counting results, the cell density was adjusted to 1X 10 with PBS supplementation⁷Then adding Mitomycin C according to the concentration of 10 mu g/mL, placing the mixture in a water bath kettle at 37 ℃ for 1 hour, and reversing and mixing the mixture for one time in the middle of 10-15 min. Then adding a proper amount of PBS, rotating at 1500rpm, rising 9 and falling 9, centrifuging for 5min, discarding the supernatant, and adding a proper amount of PBS to repeat the above centrifugal cleaning operation. The supernatant was discarded and CTS containing 4% FBS was added^TMAIM V^TMThe serum-free culture medium is re-suspended and mixed evenly,trypan blue was diluted to the appropriate fold and counted. According to the counting results, the cells were supplemented with the same medium to a final density of 8X 10⁵and/mL, mixing uniformly for later use.

(3) Decking and testing

The experiment was plated at a ratio of 5:1 effector cells to target cells, and a blank was set for 500. mu.L total lines, i.e., 250. mu.L each of effector and target cells. Placing at 37 deg.C and 5% CO₂In the incubator, samples were taken on day 0 of co-incubation (D0), day D1, day D3 and day D5, respectively, and placed in a 1.5mL centrifuge tube, and centrifuged at 1500rpm, rising and falling to 9, and centrifuged for 5min, the supernatant was discarded, and the above centrifugation and washing operation was repeated with an appropriate amount of PBS. After mixing well with a small amount of PBS, flow cytometry was used for FACS detection and the data was analyzed using FlowJo V10 software.

Fourth step, LDH killing detection

The experimental apparatus is as follows in table 8:

TABLE 8

The experimental materials and reagents are as follows in table 9:

TABLE 9

The experimental method comprises the following steps:

the experimental principle of the LDH cytotoxicity detection kit is that the toxicity of cells is quantitatively determined by lactate dehydrogenase. The calculation formula is as follows: and = (experimental group LDH release amount-spontaneous group LDH release amount)/(maximum group LDH release amount-spontaneous group LDH release amount) × 100% for target cell lysis.

(1) Pretreatment of effector and target cells

Respectively taking appropriate amount of the raw materials in a culture flaskThe effector cells and target cells (K562 and 8226) which are blown uniformly in the medium are placed in a 50mL centrifuge tube, 1500rpm is carried out, 9 is lifted and lowered for 9 minutes, the centrifuge tube is centrifuged for 5 minutes, the supernatant is discarded, and the centrifugation and cleaning operation is repeated twice by adding a proper amount of PBS. The supernatant was discarded and a small amount of CTS containing 4% FBS was used^TM AIM V^TMSerum-free medium was resuspended well mixed and trypan blue diluted to the appropriate fold for counting. Based on the counting results, the target cells were supplemented with the same medium to a final density of 2X 10⁵and/mL, and mixing uniformly for later use. The effector cells were adjusted to a density of 2X 10⁶/mL，1×10⁶/mL，5×10⁵/mL，2×10⁵and/mL, and mixing uniformly for later use.

(2) Decking and testing

Effector and target cells were plated in 96-well plates at a ratio of 10:1, 5:1, 2.5:1, 1:1, respectively, for a total of 100. mu.L, i.e., the effector and target cells were 50. mu.L in volume, respectively. After the cells are laid, the pore plate is sealed by using a sealing film, and the cells are centrifuged for 5min under the conditions of 250g, 3 rising and 1 falling, and then are put in CO with the temperature of 37 ℃ and the concentration of 5 percent₂Incubate overnight in an incubator. Then 10 mul of lysis buffer was added to each well of the target cells, and the well plates were incubated in the original incubator for 40 min. The well plate was removed, 250g was raised 3 and lowered 1, centrifuged for 5min, 50. mu.L of supernatant was pipetted into a clean 96 well plate and 50. mu.L of the assay substrate mixture dissolved in buffer was added to each well. Incubating for 10min in a dark environment at 20-25 ℃, observing the color development condition, and finally measuring the absorbance value by using an enzyme-labeling instrument under the condition of 490nm wavelength.

Fifth, cytokine detection

The experimental apparatus is as follows in table 10:

watch 10

The experimental materials and reagents are as follows in table 11:

TABLE 11

The experimental method is as follows:

(1) pretreatment of effector cells and target cells

And (3) uniformly blowing and beating the cells in the culture bottle, respectively taking a proper amount of effector cells and target cells (K562 and 8226) and placing the effector cells and the target cells in a 50mL centrifuge tube, lifting 9 and lowering 9 at 1500rpm, centrifuging for 5min, removing supernatant, and adding a proper amount of PBS to repeat the centrifugation and cleaning operation twice. The supernatant was discarded and a small amount of CTS containing 4% FBS was used^TM AIM V^TMSerum-free medium was resuspended well mixed and trypan blue diluted to the appropriate fold for counting. Based on the counting results, the target cells were supplemented with the same medium to a final density of 2X 10⁵and/mL, and mixing uniformly for later use. The same procedure was used to achieve a final effector cell density of 1X 10⁶and/mL, and mixing uniformly for later use.

(2) Decking and sampling

Effector cells and target cells were plated in 96-well plates at a ratio of 5:1, with a total of 100. mu.L, i.e., the effector and target cells were 50. mu.L in volume, respectively. After the cells were plated, the well plate was centrifuged at 250g, 3 g/1 g for 5min and then placed at 37 ℃ in 5% CO₂And taking out the pore plate after incubation for 18-24h in the incubator, taking 250g, lifting 3, lowering 1, centrifuging for 5min, and collecting 50 mu L of supernatant liquid as a sample to be detected.

(3) Standard Curve preparation and sample determination

Taking 2mL of diluent to fully dissolve the standard substance in the kit, and diluting 10 standard samples in total by a concentration gradient of 2 times. And (3) adding 50 mu L of capture microspheres into 50 mu L of sample, adding 50 mu PE detection reagent, mixing uniformly by vortex, and incubating for 3h at 20-25 ℃ in the dark. Then 1mL of buffer was added, 300g was centrifuged for 5min, and a portion of the supernatant was discarded and finally detected on an Attune NxT flow cytometer. Data were analyzed using legendedplex v8.0 and cytokine concentrations were calculated from a standard curve.

In the experiment, a third generation lentivirus CD 38-chimeric antigen receptor was constructed, transduced with CD38 from a healthy donor⁺T cells from hematological tumor patients prepare CART cells targeting CD38 (CART-38). And further evaluate CAThe immunophenotype of RT-38 and the ability to fight tumor cells. CART-38 cells can be expanded and cultured in vitro with CD8⁺CD38^－The cells are the main components. CART-38 vs. CD38⁺Tumor cells have specific recognition and cytotoxicity. The lack of positive expression of CD38 did not affect the overall differentiation level of T cells and killing ability of tumor cells. anti-CD38 CAR-T cells can be prepared by conventional methods, are characterized by CD 38-negative CD38-targeted CART cells, and can be used for clinically and effectively treating AML.

In summary, for CD38⁺For hematological tumors, the use of CART-38 is a safe and viable treatment. The invention verifies the feasibility, cell function and specific cytotoxicity of preparing the CD38 negative CART, and analyzes and characterizes the cell characteristics of the CD38 negative CART-38. The invention provides reference for the preparation mode of clinical-grade targeting CD38 CART cells and also provides CD38⁺The malignant hematological tumor treatment provides a new medicine.

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Claims

Use of CD38 for the manufacture of a CAR-T medicament, wherein the CAR-T medicament is manufactured without antibody blocking of the CD38 antigen.
2. The use of claim 1, wherein the CAR-T medicament is for the preparation of an anti-neoplastic medicament.
3. The use of claim 2, wherein the tumor is a hematological malignancy.
4. The use of claim 3, wherein the hematological malignancy is acute myelogenous leukemia.
5. The use of any of claims 1 to 4, wherein the CAR gene modified T cell CART-38, which is negative for CD38, obtained after treatment of CAR-T cells with CD38, specifically recognizes and is cytotoxic to target cells bearing the CD38 antigen.
6. The use of claim 5, wherein in vitro amplification culture is used in the preparation of CART-38.