CN117126891A

CN117126891A - Retrovirus expression vector and CAR-T cell comprising same

Info

Publication number: CN117126891A
Application number: CN202311101740.XA
Authority: CN
Inventors: 王建勋; 史渊源; 冯娅茹; 孙睿; 王炳彰; 卢泯村
Original assignee: Shenzhen Cell Valley Biopharmaceutical Co ltd; Guangdong Junhou Biopharmaceutical Co ltd
Current assignee: Shenzhen Cell Valley Biopharmaceutical Co ltd; Guangdong Junhou Biopharmaceutical Co ltd
Priority date: 2023-08-30
Filing date: 2023-08-30
Publication date: 2023-11-28

Abstract

The application provides a retrovirus expression vector and CAR-T cells comprising the retrovirus expression vector, and the expressed amino acid residue sequence is SEQ ID NO. 2. The CAR-T cells prepared by the method are provided with a safety switch, so that the CAR-T cells can be treated under artificial control, and the brake can be stepped on in time before adverse reaction occurs. After the CAR-T cell prepared by the application is provided with the safety switch, the CAR-T cell has better curative effect and fewer side effects than the common CAR-T cell and the CAR-T cell without the safety switch, and improves the safety of CAR-T therapy.

Description

Retrovirus expression vector and CAR-T cell comprising same

Technical Field

The application relates to the technical field of tumor treatment, in particular to a CAR-T cell with a suicide switch iCASP9 for treating multiple myeloma.

Background

Multiple myeloma is a malignant proliferative disease of plasma cells, characterized by unlimited proliferation of plasma cells in the bone marrow like tumor cells, and most cases are accompanied by monoclonal immunoglobulin secretion, ultimately leading to organ or tissue damage.

The current treatment regimen is mainly chemotherapy, local radiotherapy, hematopoietic stem cell transplantation. However, radiation therapy and chemotherapy treatments can repeatedly cause infection and fever in the course of the disease, such as skin infection, lung infection and other complications, and the application of chemotherapeutics and adrenocortical hormone also increases the chances of infection. Medical clinical CAR-T treatment regimens directed to targeting myeloma-related antigens have created new opportunities for such problematic diseases.

CAR-T therapy has achieved significant clinical efficacy in the treatment of multiple myeloma disease, but CAR-T therapy also brings about related toxicities and limitations, including extra-tumor toxicity, antigen escape, non-optimal activation, etc., which may cause Cytokine Release Syndrome (CRS) in addition to exerting anti-tumor effects, with increasing safety issues.

In managing adverse reactions, timely pharmacological intervention is effective, and a recently demonstrated molecular switch, capable of inducing caspase 9 (iCASP 9), has the potential to eliminate the threat posed by T cell therapy, if necessary, to deplete transplanted T cells.

Disclosure of Invention

In order to solve the technical problems, the application provides a retrovirus expression vector, the expressed amino acid residue sequence of which is SEQ ID NO. 2:

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSTSFGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSATNFSLLKQAGDVEENPGPMEWSWVFLFFLSVTTGVHSDIEQKLISEEDLDIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQIQLV QSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSSAAAGGGGSGGGGSGGGGSGGGGSSQVQLVQSGGGLVQPGRSLRLPCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIAYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGSGSYYNPFYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSQAVLTQPPSASGTPGQRVTISCSGSSSNIGGNTVAWYQQLPGTAPKLLIYNYSQRPSGVPDRFSGSKSGTSSSLAIGGLQSEDEADYYCAAWDDSLNGVVFGGGTKLTVLGAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

in one embodiment, the application provides a polynucleotide encoding an amino acid according to 1.

In one embodiment, the application provides a CAR-T cell obtained after transduction with a retroviral expression vector as described above.

In one embodiment, the application provides a pharmaceutical composition for treating a tumor, the pharmaceutical composition comprising a CAR-T cell as described above, and a pharmaceutically acceptable carrier, diluent or excipient, the tumor preferably being human multiple myeloma.

In one embodiment, the application provides the use of a CAR-T cell as described above or a pharmaceutical composition as described above for the preparation of a medicament or formulation for the prevention and/or treatment of a tumor, preferably human multiple myeloma.

In one embodiment, the application provides a method of inhibiting tumor cells in vitro comprising: contacting a tumor cell, preferably a human multiple myeloma, with a CAR-T cell as described above or a pharmaceutical composition as described above, thereby inhibiting the tumor cell.

The present application constructs a CAR sequence with a switch that will contain a safety switching mechanism for managing potential toxicity; this combination can overcome the current limitations of CAR-T cell therapies and help improve the therapeutic outcome of patients. The CAR-T cells with the switch prepared by the application have obvious killing effect on RPMI tumor cells.

The CAR-T cell prepared by the application is provided with a safety switch, so that the CAR-T cell can be treated under artificial control, and the brake can be stepped on in time before adverse reaction occurs. After the CAR-T cell prepared by the application is provided with the safety switch, the CAR-T cell has better curative effect and fewer side effects than the common CAR-T cell and the CAR-T cell without the safety switch, and improves the safety of CAR-T therapy.

The application researches find that the feasibility of preparing the ICASP9 CAR T switch modified T cells and the feasibility of killing RPMI by the ICASP9 CAR T are obtained through a conventional CAR T production process. The results show that ICASP9 CAR T has an effective in vitro killing ability against RPMI tumor cells. ICASP9 CAR T can effectively stop the expansion and persistence of CASP9 CAR T cells in vitro through a small molecule chemical induction drug AP1903, thereby being beneficial to controlling the generation of side effects such as CRS and the like, relieving toxicity problems and increasing safety.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph showing the positive rate results of pMFG-MYC-CD38-BCMA-CAR-T (hereinafter referred to as JHSC 005) and pMFG-MYC-CD38-BCMA-ICASP9-CAR-T (hereinafter referred to as JHSC 008) cells after detection of transduced for 48h by flow cytometry using MYC antibody staining, wherein FIG. 1A is JHSC005, FIG. 1B is JHSC008 and the abscissa is PE signal intensity; the ordinate indicates the number of cells.

FIG. 2 is a graph of killing efficiency results using flow cytometry to detect killing of RPMI tumor cells by JHSC005 cells, JHSC008 cells and normal T cells at different target ratios.

FIG. 3 is a graph of apoptosis results using flow cytometry to detect the effects of addition of different concentrations of AP1903 on JHSC005 cells, where FIG. 3A is the lack of addition of AP1903, FIG. 3B is the addition of AP1903 ng/mL, and FIG. 3C is the addition of AP190310ng/mL.

FIG. 4 is a graph of apoptosis results using flow cytometry to detect the effects of addition of varying concentrations of AP1903 on JHSC008 cells, where FIG. 4A is the absence of addition of AP1903, FIG. 4B is the addition of AP1903 ng/mL, and FIG. 4C is the addition of AP190310ng/mL.

Detailed Description

In order that those skilled in the art will better understand the technical solutions of the present application, the present application will be further described with reference to examples, and it is apparent that the described examples are only some of the examples of the present application, but not all of the examples. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, shall fall within the scope of the application.

Unless otherwise indicated, the following examples were conducted under conventional experimental conditions, or under conditions recommended by the manufacturer's instructions.

Example 1

Materials: RPMI 1640 serum-free medium, AIM-V serum-free medium, fetal Bovine Serum (FBS), PBS buffer, and penicillin-streptomycin were all purchased from Gibco corporation, U.S.A. Lymphocyte isolates were purchased from Stemcell, canada. Apoptosis kits (Annexin-V FITC, binding buffer) were purchased from Beijing Jin Pulai company. Interleukin 2 (IL-2) and OKT-3 were purchased from Beijing Yiqiao Shenzhou technologies Co., ltd. retroNectin was purchased from TAKARA Bio Inc. of Japan. MYC antibodies were purchased from R & D Systems, usa. RPMI cells were purchased from ATCC. AP1903 is available from MedChemExpress.

This example demonstrates the observation of the killing effect of ICASP9-CAR-T on human multiple myeloma (RPMI) tumor cells, and the specific procedure is as follows.

PBMC culture

Collecting peripheral venous blood of healthy people in a sterile way, performing density gradient centrifugation by using lymphocyte separation liquid, separating to obtain Peripheral Blood Mononuclear Cells (PBMC), adding a T cell culture medium for culturing, simultaneously adding OKT-3 with a final concentration of 100ng/mL and IL-2 with a final concentration of 100U/mL to stimulate T cell proliferation, and placing at 37 ℃ and 5% CO ₂ The cells were aseptically cultured in an incubator for 48 hours.

The T cell medium comprises: AIM-V serum-free medium (Gibco, USA) +10% fetal bovine serum FBS (Gibco, USA) +1% penicillin-streptomycin (Gibco, USA).

And collecting the cultured cells to obtain the primary human T cells.

Preparation of second-JHSC 005, JHSC008 cells

1. Retroviral expression vector construction

2 CARs containing CD38-BCMA tandem double targets are designed and constructed, wherein one CAR does not contain ICASP9 suicide gene, and is named pMFG-MYC-CD38-BCMA-CAR-T (hereinafter referred to as JHSC 005), and the expressed amino acid residue sequence is shown as SEQ ID NO. 1; another suicide gene containing ICASP9 is named pMFG-MYC-CD38-BCMA-ICASP9-CAR-T (hereinafter referred to as JHSC 008), and the expressed amino acid residue sequence is shown in SEQ ID NO. 2. The construction of retroviral expression plasmids is a routine experimental technique of molecular biology, and is carried out in accordance with techniques or conditions described in the literature in the art (for example, refer to the guidelines for molecular cloning experiments, fourth edition, scientific Press, et al, compiled by M.R. Green, J. Sam Broker et al, he Fuchu et al) and corresponding product specifications.

SEQ ID NO:1：

ATNFSLLKQAGDVEENPGPMEWSWVFLFFLSVTTGVHSDIEQKLISEEDLDIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSSAAAGGGGSGGGGSGGGGSGGGGSSQVQLVQSGGGLVQPGRSLRLPCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIAYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGSGSYYNPFYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSQAVLTQPPSASGTPGQRVTISCSGSSSNIGGNTVAWYQQLPGTAPKLLIYNYSQRPSGVPDRFSGSKSGTSSSLAIGGLQSEDEADYYCAAWDDSLNGVVFGGGTKLTVLGAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

SEQ ID NO:2

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSTSFGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSP EDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSATNFSLLKQAGDVEENPGPMEWSWVFLFFLSVTTGVHSDIEQKLISEEDLDIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSSAAAGGGGSGGGGSGGGGSGGGGSSQVQLVQSGGGLVQPGRSLRLPCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIAYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGSGSYYNPFYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSQAVLTQPPSASGTPGQRVTISCSGSSSNIGGNTVAWYQQLPGTAPKLLIYNYSQRPSGVPDRFSGSKSGTSSSLAIGGLQSEDEADYYCAAWDDSLNGVVFGGGTKLTVLGAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

2. Transfected phoenix-Eco cells

Retroviral expression plasmids carrying the gene of interest (JHSC 005, JHSC008 plasmids constructed in step 2.1) were transfected into phoenix-Eco cells with the transfection reagent Fugene. After 48h of transfection, the culture supernatant was collected, filtered through a 0.45 μm filter and the filtrate was kept ready for use, designated as acidophilic virus A.

3. Transduction of PG13 cells

The PG13 cells were transduced with the avirus a. Inoculating PG13 cells into a culture dish, adding the philic virus A, centrifuging at 30deg.C and 2500rpm for 1 hr, and adding 37 deg.C and 5% CO ₂ Incubate for 2h. The culture supernatant was removed and fresh PG13 cell culture medium (containing 90% DMEM complete medium, 10% FBS) was added at 37deg.C with 5% CO ₂ Culturing in an incubator. For improved transduction efficiency, transduction enhancing agents such as retroNectin or Polybrene may be used, and the amount of transduction enhancing agent used is referred to the product instructions. After 24h transduction, the culture supernatant was harvested, filtered through a 0.45 μm filter, and the filtrate was retained for use, which was the corresponding JHSC005 retroviral vector and JHSC008 retroviral vector.

4. Transduction of T cells

Retroviral vectors transduce human primary T cells. Resuspension of human primary T cells to 5-10x10 using AIM-V Medium ⁵ Per mL, centrifuge transduction with retroviral vector harvested in step 2.3 followed by centrifugation at 37℃in 5% CO ₂ Incubator incubation, adding IL-2 to a final concentration of 100U/mL,37℃and 5% CO ₂ Culturing in an incubator. The cells obtained after transduction of the retroviral vector are the corresponding JHSC005 cells and JHSC008 cells.

Detection of CAR-T cell transduction efficiency

JHSC005 cells and JHSC008 cells after 48h transduction were taken, MYC (R & D, USA) antigen expression was detected by flow cytometry using MYC-PE antibody after 48h transduction, transduction efficiency (i.e., MYC expression rate) was calculated=myc-PE positive cell number/total viable cell number×100%, transduction efficiency was shown in fig. 1, and the result showed that the transduction efficiency of JHSC005 was 80.4% and the transduction efficiency of JHSC008 was 29.5%.

In vitro culture of CAR-T cells

Culturing transduced JHSC005 cells, JHSC008 cells and common T cells in a T cell culture medium, adding 100U/mL IL-2, counting passages for 1 time every 48 hours, observing cell states, and when the survival rate of three groups of cells reaches more than 80%, indicating that the cell states are good, and carrying out killing experiments.

In vitro culture of RPMI tumor cells

Resuscitates RPMI cells at 5X 10 ⁵ cell/mL was inoculated in RPMI 1640 complete medium, 37℃at 5% CO ₂ The cells were cultured aseptically in a cell incubator. The cell state is observed after 1 passage every 48 hours, and when the survival rate of RPMI cells reaches more than 80%, the cell state is good, and a killing experiment can be carried out.

RPMI 1640 complete medium contains: RPMI 1640 serum free medium (Gibco, USA) +10% FBS (Gibco, USA) +1% Green-streptomycin solution (Gibco, USA).

In vitro killing ability verification of JHSC008 cells

The experimental group is a JHSC005 cell group and a JHSC008 cell group; the control group was not transduced, i.e., a normal T cell group. In killing experiments, JHSC005 cells were diluted with normal T cells to adjust their transduction efficiency to 29.5% and were aligned with JHSC008 cells.

And (3) carrying out a tumor cell killing experiment by grouping and incubating effector cells and target cells, wherein the experimental effect target ratio is respectively 1:1 group, 1:2 group, 1:4 group, 1:8 group and 1:16 group, and killing efficiency is detected by adopting flow cytometry after 8 hours.

The specific experimental steps are as follows:

target cell plating RPMI tumor cells were mixed well, sampled, stained with trypan blue and counted by a cytometer. Target cells were added 8X 10 per well of 96-well plate ⁴ 100. Mu.L wells, target cells were resuspended in T cell medium (AIM-V serum free medium +10% FBS +1% P/S) and plated in 96 well cell culture plates, depending on the desired cell amount.

Effector cell suspensions were prepared by mixing JHSC005 cells, JHSC008 cells and normal T cells uniformly, sampling, staining with trypan blue and counting by a cell counter. According to the amount of effector cells required for killing, effector cells were taken, T cell medium (AIM-V serum-free medium+10% fbs+1% p/S) was used, and 100U/mL IL-2 was added to resuspend target cells.

Target cells have been added 8X 10 per well of 96-well plate ⁴ 100. Mu.L. Dilution of effector cells to 8X 10 ⁴ 100 μl was added to wells plated with RPMI cells, i.e. E: T (effector cells: target cells) =1:1 group (killing wells); dilution of effector cells to 4X 10 ⁴ 100 μl was added to wells plated with RPMI cells, i.e. E: T (effector cells: target cells) =1:2 group (killing wells); dilution of effector cells to 2X 10 ⁴ 100 μl was added to wells plated with RPMI cells, i.e. E: T (effector cells: target cells) =1:4 groups (killing wells); dilution of effector cells to 1X 10 ⁴ 100 μl was added to wells plated with RPMI cells, i.e. E: T (effector cells: target cells) =1:8 groups (killing wells); dilution of effector cells to 5X 10 ³ 100 μl was added to wells plated with RPMI cells, i.e. E: T (effector cells: target cells) =1:16 group (killing wells); three in parallel per group; and taking the hole with the RPMI cells as a non-killing hole; marking is carried out.

After the plate is finished, the 96-well plate is put into 5% CO at 37 DEG C ₂ The cells were co-cultured in a cell incubator for 8 hours.

Killing efficiency detection, centrifugation at 3000rpm for 5min after 8h, and collection of cells per well. Staining buffer (10% FBS in PBS) was washed 1 time, each group was stained with Annexin-V FITC antibody, RPMI cells without any treatment were antibody stained as negative control, incubated for 30min in the dark and then assayed with binding buffer suspension. The apoptosis rate of target cells was measured using a flow cytometer, wherein cells in the FITC positive area were apoptotic cells. Killing efficiency = killing Kong Ba apoptosis rate-no killing Kong Ba apoptosis rate.

As can be seen from FIG. 2, the result of killing effect of the three groups of cells on RPMI tumor cells at different target ratios is analyzed as follows, when the target ratio is 1:1, compared with the control group (common T cells), the experimental groups (JHSC 008 cells and JHSC005 cells) have obvious killing effect on the RPMI tumor cells, and the killing effect of the JHSC008 cells on the RPMI cells is highest; the killing efficiency is gradually reduced according to the target ratio of 1:1, 1:2, 1:4, 1:8 and 1:16, and the killing effect of the JHSC008 cells is obviously higher than that of the JHSC005 cells and the common T cells. The CAR-T cells transduced by the retrovirus vector, namely JHSC008 cells and JHSC005 cells, can effectively kill RPMI tumor cells compared with common T cells, and has remarkable effect of killing the RPMI tumor cells by the JHSC008 cells.

Example two

This example demonstrates that the small molecule drug AP1903 inhibits expression of JHSC008 cells, rimiducid (AP 1903) is a dimerizer that acts by cross-linking the FKBP domain, initiating Fas signaling, causing apoptosis. Rimiducid (AP 1903) dimerizes the Caspase 9 suicide switch and rapidly induces apoptosis (apoptosis) by sampling the JHSC005 and JHSC008 cells, respectively, being cultured in example one, staining with trypan blue and counting by a cytometer. JHSC005 cells and JHSC008 cells were diluted to 8X 10 respectively with T cell medium (AIM-V serum free medium+10% FBS+1% P/S) and 100U/mL IL-2 was added to resuspend the cells ⁵ /mL。

AP1903 (stock solution concentration: 1 mg/mL) was diluted stepwise 10-fold with PBS buffer (Gibco, USA) to 100. Mu.g/mL, 10. Mu.g/mL, 1. Mu.g/mL, 100ng/mL,10ng/mL.

A96-well plate was used, 180. Mu.L of cell suspension and AP1903 at different concentrations were added to each well, and the following were set:

control, i.e., without the addition of AP1903, 180. Mu.L of JHSC005 cell suspension+20. Mu.LT cell culture medium, 180. Mu.L of JHSC008 cell suspension+20. Mu.LT cell culture medium; in the experimental group, 180. Mu.L of JHSC005 cell suspension+20. Mu.LAP 1903 (10 ng/mL) and 180. Mu.L of JHSC008 cell suspension+20. Mu.LAP 1903 (10 ng/mL) were used at an AP1903 concentration of 1 ng/mL; in the experimental group, 180. Mu.L of JHSC005 cell suspension+20. Mu.LAP 1903 (100 ng/mL), 180. Mu.L of JHSC008 cell suspension+20. Mu.LAP 1903 (100 ng/mL) at an AP1903 concentration of 10 ng/mL; marking is carried out. After the plate is finished, the 96-well plate is put into 5% CO at 37 DEG C ₂ The cells were co-cultured in a cell incubator for 6 hours.

After 6 hours, the cells were collected by centrifugation at 3000rpm for 5 min. Staining buffer (10% FBS in PBS) was washed 1 time, each group was stained with Annexin-V FITC antibody, no AP1903 added JHSC005 cells and JHSC008 cells were antibody stained as negative controls, incubated for 30min in the dark and then detected with binding buffer suspension. Apoptosis rates of JHSC005 cells and JHSC008 cells were examined by flow cytometry. As can be seen from FIGS. 3A and 4A, only 1% -2% of the cells of JHSC005 and JHSC008 without AP1903 are apoptotic, and as can be seen from FIGS. 3B and 3C, AP1903 with the concentration of 1ng/mL and 10ng/mL is added to act on the JHSC005 cells, and no obvious apoptosis phenomenon exists, but by comparing with FIGS. 4B and 4C, AP1903 with the concentration of 1ng/mL and 10ng/mL is added to act on the JHSC008 cells, obvious apoptosis phenomenon is generated, and the effect of a small molecular medicine AP1903 on the JHSC008 cells is proved, and activated cells expressing ICASP9 with a 'safety switch' is killed preferentially, so that the threat brought by CAR-T cell treatment can be eliminated, and the safety of treatment is improved.

It is to be understood that this application is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present application which will be limited only by the appended claims.

Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the application described herein. Such equivalents are also encompassed by the appended claims.

Claims

1. A retrovirus expression vector, characterized in that the amino acid residue sequence expressed by the retrovirus expression vector is SEQ ID NO. 2:MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGK QEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSTSFGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSATNFSLLKQAGDVEENPGPMEWSWVFLFFLSVTTGVHSDIEQKLISEEDLDIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSSAAAGGGGSGGGGSGGGGSGGGGSSQVQLVQSGGGLVQPGRSLRLPCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIAYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGSGSYYNPFYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSQAVLTQPPSASGTPGQRVTISCSGSSSNIGGNTVAWYQQLPGTAPKLLIYNYSQRPSGVPDRFSGSKSGTSSSLAIGGLQSEDEADYYCAAWDDSLNGVVFGGGTKLTVLGAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

2. A polynucleotide encoding the amino acid of claim 1.

3. A CAR-T cell obtained after transduction by the retroviral expression vector of claim 1.

4. A pharmaceutical composition for the treatment of a tumor, preferably human multiple myeloma, comprising the CAR-T cells of claim 3 and a pharmaceutically acceptable carrier, diluent or excipient.

5. Use of a CAR-T cell according to claim 3 or a pharmaceutical composition according to claim 4 for the preparation of a medicament or formulation for the prevention and/or treatment of a tumour, preferably human multiple myeloma.

6. A method of inhibiting a tumor cell in vitro comprising: contacting a tumor cell, preferably human multiple myeloma, with a CAR-T cell according to claim 3 or a pharmaceutical composition according to claim 4, thereby inhibiting the tumor cell.