CN111956795B

CN111956795B - Application of chimeric antigen receptor combined anti-tumor drug taking CD99 as target

Info

Publication number: CN111956795B
Application number: CN202010876364.1A
Authority: CN
Inventors: 张同存; 顾潮江; 祝海川; 周经姣; 周勇; 史江舟
Original assignee: Wuhan Bio Raid Biotechnology Co ltd
Current assignee: Wuhan Bio Raid Biotechnology Co ltd
Priority date: 2020-08-27
Filing date: 2020-08-27
Publication date: 2023-02-03
Anticipated expiration: 2040-08-27
Also published as: CN111956795A

Abstract

The invention provides a combined medication method for treating tumors and application thereof, wherein the combined medication method combines chimeric antigen receptor immune cell therapy with chemotherapy and has stronger anti-tumor effect than single chemotherapy or single immune cell therapy. Meanwhile, the dosage of the chemotherapeutic drug can be reduced, and the toxic and side effects of the chemotherapeutic drug can be reduced. The problems that the CAR-T cell is out of target due to high heterogeneity of the tumor and the treatment effect is not ideal due to the singleness of the target point in the process of treating the tumor are solved.

Description

Application of chimeric antigen receptor combined anti-tumor drug taking CD99 as target

Technical Field

The invention relates to the field of medical biology, in particular to application of a Chimeric Antigen Receptor (CAR) combined anti-tumor medicament for treating a broad-spectrum tumor by taking CD99 as a target spot.

Background

The data published by the American cancer society in the early 2019 indicate that over 1810 new cancers exist in 2018 all over the world, 960 ten thousands of cancers die, and the cancer morbidity and mortality of Chinese people all occupy the first position of the world and are in a rapidly increasing state. Scientists around the world are also working on the study of various anticancer therapies, and immunotherapy has become a new generation of tumor treatment as the third revolution of anticancer therapy following chemotherapy and targeted therapy. In 2013, the American journal of science selects tumor immunotherapy as the largest scientific breakthrough in the current year, and in 2015, the tumor immunotherapy combination therapy is listed as one of the four most interesting scientific progresses.

The tumor immunotherapy refers to the application of immunological principles and methods to improve the immunogenicity of tumors and to utilize the immune system to attack tumor cells, thereby inhibiting or killing the tumor cells. The treatment strategies are divided into two main categories: immune checkpoint inhibitors (e.g., CTLA-4, PD-1, PD-L1, etc.) and cellular immunotherapy (e.g., CAR-T, etc.). Among them, CAR-T cells are all called Chimeric Antigen Receptor T-cells (Chimeric Antigen Receptor T-cells), and the principle is that an antibody single-chain variable region (Scfv) capable of recognizing a certain tumor Antigen is coupled to an intracellular region of CD 3-zeta chain in vitro by a genetic engineering method to form a Chimeric protein, and T cells of a patient cultured in vitro are transfected by a gene transduction method to express a Chimeric Antibody Receptor (CAR). After the T cells of the patient are "reprogrammed," a number of killer CAR-T cells are generated that can specifically target tumor cells. Compared with the traditional immunotherapy, the CAR-T has the remarkable advantages of more accurate treatment, more accurate target, wider tumor killing range, more lasting effect and the like. As a novel leading-edge treatment means, CAR-T treatment is mainly developed by the Chinese and American leaders, chinese performance is particularly prominent, and as long as 2019, 5 months, the global CAR-T treatment clinical trial registration item 507 is mainly distributed in China and the United states and accounts for 44.2 percent and 36.7 percent of the total number of trials respectively. CAR-T therapy has achieved great clinical success, especially with great success in the treatment of hematological tumors. In 2017, the FDA successively approved two CAR-T immunotherapeutic products, kymrian (CTL-019) and Yescara (axicabagene ciloleucel, KTE-C10), to be marketed by the US FDA for treatment of B-cell acute lymphoblastic leukemia in relapsed or refractory (r/r) children and young adults and in refractory, relapsed adult patients with large B-cell lymphoma, respectively.

The CD99 protein is a glycosylated protein located on a cell membrane and encoded by a gene MIC2 (MIC 2X, MIC 2Y) and has a molecular weight of 32kD. Mainly expressed in normal tissue cells such as thymic epidermal cells, islet cells, ovarian granulosa cells, testicular supporting cells and the like. Previous studies have shown that more than 95% of 20 tumors such as angiomatoid fibroblastic tumors are CD99 positive, more than 75% of 19 tumors such as T lymphoblastic leukemia/lymphoma are CD99 positive, and more than 55% of 14 tumors such as glioblastoma are CD99 positive. In particular, almost 100% of these tumors were found to be positive for CD99 in ewing's sarcoma and were clinically used as diagnostic markers for ewing's sarcoma. CD99 is not only highly expressed in tumors in the early stage, but in the case of T-cell acute lymphoblastic leukemia (T-ALL), the expression of CD99 is used for detecting immune minimal residual cells after chemotherapy and as a basis for diagnosing recurrence of T-ALL.

The research on the treatment of tumors by chemical anticancer drugs is early and the application is relatively wide. Cisplatin (DDP) is the first platinum compound approved by FDA in 1978 for cancer treatment, is commonly used for treating various cancers, including ovarian cancer, testicular cancer, head and neck cancer, colorectal cancer, bladder cancer, lung cancer and the like, and has the characteristics of wide anticancer spectrum, unique action mechanism, benefit of clinical combined medication and the like. The antitumor effect of cisplatin is associated with DNA damage and inhibition of DNA synthesis. Cisplatin interacts with DNA to form DNA adducts, leading to intra-or inter-strand cross-linking of DNA, activation of various signaling pathways, induction of oxidative stress, activation of apoptosis, and ultimately tumor cell death. Paclitaxel (PTX) was first approved by the FDA for the treatment of advanced breast cancer in 1992. 2018, was approved in the united states for the treatment of breast cancer, pancreatic cancer, ovarian cancer, kaposi's sarcoma and non-small cell lung cancer. Paclitaxel is natural alkaloid extracted from Taxus chinensis, has obvious stimulation effect on polymerization process of tubulin, and can inhibit depolymerization reaction of tubulin, thereby improving stability of tubulin, blocking mitosis process of tumor cell, and improving chemotherapy sensitivity.

Although CD99 as a target can be an effective method for treating tumors, the CAR-T cells still face the problems of off-target caused by high tumor heterogeneity and poor treatment effect caused by target singularity in the process of treating tumors. Where the high heterogeneity of tumor cells directly leads to limitations of CAR-T therapy during treatment, while the singleness of the target limits the therapeutic effect and tumor clearance. The CAR-T combined anti-tumor drug treatment can utilize the targeting property of CAR T cells and the universality of anti-tumor drugs, and the cure rate of monotherapy is improved. Therefore, CAR-T combined antitumor drug treatment can be used as a critical treatment means for tumor treatment and after healing.

The applicant prepares and obtains a Chimeric Antigen Receptor (CAR) taking CD99 as a target in earlier research, (patent publication No. CN 110590960A), the chimeric antigen receptor carries an ScFv sequence targeting CD99, CAR-T cells carrying the sequence can effectively kill any tumor cells expressing CD99 on the surface, and the broad spectrum of killing tumors is expanded. On the basis, in the process of further research on the clinical application of the chimeric antigen receptor, the applicant surprisingly finds that the CAR-T cell carrying the sequence is combined with a chemical anticancer drug to have a synergistic anti-tumor technical effect.

Disclosure of Invention

In view of the disadvantages of the prior art, the present invention aims to provide a method of combined administration for treating tumor, which combines chimeric antigen receptor immune cell therapy with chemotherapy and has stronger anti-tumor effect than chemotherapy or single immune cell therapy, and the use thereof. Meanwhile, the dosage of the chemotherapeutic drug can be reduced, and the toxic and side effects of the chemotherapeutic drug can be reduced. The problems that the CAR-T cell is out of target due to high heterogeneity of the tumor in the process of treating the tumor, the therapeutic effect is not ideal due to the singleness of target spots and the like are solved.

In order to achieve the purpose, the invention adopts the following technical scheme:

in one aspect, the invention provides a combination composition of a Chimeric Antigen Receptor (CAR) targeting CD99 and an anti-tumor drug for treating a broad spectrum of tumors.

The chimeric antigen receptor taking CD99 as a target spot sequentially splices a signal peptide, a single-chain antibody ScFv, strepII, CD8 hinge, a CD28 transmembrane region, a CD28 intracellular domain, an intracellular co-stimulatory domain 4-1BB and a CD3 zeta chain from the N end to the C end; preferably, the single-chain antibody ScFv is capable of recognizing CD99 antigen on the surface of tumor cells. The amino acid sequence of the signal peptide is shown as SEQ ID NO.5, the amino acid sequence of strepII is shown as SEQ ID NO.7, the amino acid sequence of CD8 hinge is shown as SEQ ID NO.9, the amino acid sequences of a CD28 transmembrane region (CD 28 TM) are shown as SEQ ID NO.11 and a CD28 intracellular domain (CD 28) are respectively shown as SEQ ID NO.17, the nucleotide sequence of an intracellular co-stimulatory domain 4-1BB is shown as SEQ ID NO.13, and the nucleotide sequence of CD3 zeta is shown as SEQ ID NO. 15.

In some embodiments of the invention, the amino acid sequence of the single chain antibody ScFv is shown in SEQ ID No.1, preferably, the nucleotide sequence of the single chain antibody ScFv is shown in SEQ ID No. 2.

In some embodiments of the invention, the amino acid sequence of the single chain antibody ScFv is shown in SEQ ID No.3, and correspondingly, the nucleotide sequence of the single chain antibody ScFv is shown in SEQ ID No. 4.

The antitumor drugs comprise cisplatin and paclitaxel. Preferably, in the above technical scheme, the concentration of cisplatin is 32 μ g/mL, and the concentration of paclitaxel is 20nmol/L.

In another aspect, the invention provides an application of the above-mentioned pharmaceutical composition for combination in preparing an antitumor drug. Preferably, the tumor is preferably ewing's sarcoma, acute lymphoma/leukemia, acute myeloid leukemia, malignant glioma, breast cancer.

In a further aspect, the present invention provides a novel anti-tumor combination therapy of chemotherapy in combination with chimeric antigen receptor T cell immunotherapy.

The invention has the beneficial effects that:

1. the chimeric antigen receptor with CD99 as the target provided by the invention comprises a specific single-chain antibody ScFv which is used for modifying immune cells, and the modified immune cells can be used for treating surface CD99 positive tumors, and particularly have remarkable tumor killing effects on Ewing sarcoma, acute lymphoma/leukemia, acute myelogenous leukemia, malignant glioma and breast cancer.

2. The CAR T cell and the anti-tumor drug are used in combination, the CAR-T and the anti-tumor drug can generate a synergistic effect, and the tumor killing efficiency is remarkably improved. The combination regimen may better control tumor progression and reduce cancer recurrence, thereby improving patient survival.

3. The invention also provides a preparation method of the immune cell expressing the chimeric antigen receptor, which is to activate and activate the separated immune cell for 2-15 days and then infect the lentivirus expressing the chimeric antigen receptor, so that the original immune cell does not influence the tumor killing effect of the transfected immune cell expressing the chimeric antigen receptor, and further, when the in vitro function detection is carried out on the immune cell expressing the chimeric antigen receptor, the selected cell line is a cell line with high expression outside a cell membrane or a cell line with a CD99 target spot expressed or a cell line with a medium expression outside the cell membrane, thereby the tumor killing effect evaluation on the immune cell expressing the chimeric antigen receptor is more scientific.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 is a schematic representation of the DNA fragments of C1-CAR, C2-CAR in the examples;

FIG. 2 is a plasmid map of PTK881-EF1 alpha-C1 and PTK881-EF1 alpha-C2 in the examples;

FIG. 3 shows the results of the measurement of transduction efficiency of C1-CAR-T cells and C2-CAR-T cells in examples;

FIG. 4 killing efficiency on negative target cells Raji;

FIG. 5 killing efficiency on positive target cells TC71, 6647;

FIG. 6 killing efficiency on positive target cells jurkat, molt 4;

FIG. 7 killing efficiency on positive target cells U373-MG and U251-MG;

FIG. 8 shows the killing efficiency of the positive target cells H1299 and MCF-7.

Detailed Description

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are performed according to techniques or conditions described in literature in the art or according to the product specification. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1: construction of PTK881-EF 1. Alpha. -C1 and PTK881-EF 1. Alpha. -C2

1. Fragments shown in SEQ ID NO.2 and SEQ ID NO.4 are artificially synthesized to form SP-C1 and SP-C2 respectively.

2. A human cDNA library is taken as a template, primers are designed for PCR to respectively amplify fragments CD8 hinge, a CD28 transmembrane region, a CD28 intracellular domain, 4-1BB and CD3 zeta, and a Strep II fragment is obtained in a primer complementary mode. Respectively amplifying and connecting SP-C1 and SP-C2 with fragments Strep tag II, CD8 hinge, CD28 transmembrane region, CD28 intracellular domain, 4-1BB and CD3 zeta in sequence by using an Overlap PCR technology to form C1-CAR and C2-CAR with enzyme cutting sites EcoR I and BamH I, wherein the structural schematic diagrams of the C1-CAR and the C2-CAR are shown in FIG. 1;

wherein SP-C1 and SP-C2 are single-chain antibodies ScFv capable of recognizing CD99 on the surface of tumor cells. Single chain antibody ScFv: the amino acid sequences of SP-C1 and SP-C2 are respectively shown as SEQ ID NO.1 and SEQ ID NO.3, and the nucleotide sequences of SP-C1 and SP-C2 are respectively shown as SEQ ID NO.2 and SEQ ID NO. 4. The nucleotide sequence of the signal peptide SP is shown as SEQ ID NO.6, the nucleotide sequence of strepII is shown as SEQ ID NO.8, the nucleotide sequence of CD8 hinger is shown as SEQ ID NO.10, the nucleotide sequence of CD28TM is shown as SEQ ID NO.12, the nucleotide sequence of CD28ICD is shown as SEQ ID NO.18, the nucleotide sequence of 4-1BB is shown as SEQ ID NO.14, the nucleotide sequence of CD3 zeta is shown as SEQ ID NO.16, the amino acid sequence of the signal peptide SP is shown as SEQ ID NO.5, the amino acid sequence of strepII is shown as SEQ ID NO.7, the amino acid sequence of CD8 hinger is shown as SEQ ID NO.9, the amino acid sequence of CD28TM is shown as SEQ ID NO.11, the amino acid sequence of CD28ICD is shown as SEQ ID NO.17, the amino acid sequence of 4-1BB is shown as SEQ ID NO.13, and the amino acid sequence of CD3 zeta is shown as SEQ ID NO. 15.

3. Plasmid PTK881-Kan was double digested with EcoR I and BamH I restriction enzymes, the product was electrophoresed through 0.8% agarose gel and recovered in a 1.5mL centrifuge tube by tapping, the corresponding digested fragments were recovered using the agarose gel recovery kit from Axygen, and the purity and concentration of the product were determined.

4. Adding the fragment into a 1.5mL centrifuge tube according to the 1:2 molar ratio, adding the Exnase II ligase and the homologous recombinase 5 XCE II buffer, and reacting for 0.5h at 37 ℃; taking out 10 μ L of the connecting liquid, adding 100 μ L of DH5 alpha competent cell, ice-cooling for 30min, heat-shocking at 42 ℃ for 90s, adding 500 μ L of soc culture medium at 37 ℃, culturing at 220rpm for 2h; after 2h, 400. Mu.L of excess liquid was removed by centrifuging 4000g of a 1.5mL centrifuge tube for 1 min. Coating the residual liquid on an LB flat plate and culturing at 37 ℃ for 12h; single colonies were picked on the plate and inoculated into 5mL of LB liquid medium at 37 ℃ and 220rpm for 12 hours.

5. Plasmids are extracted by an Axygen miniprep kit, plasmids PTK881-EF1 alpha-C1 and PTK881-EF1 alpha-C2 are obtained and sent to a first generation sequencing verification of science and technology company of Biotechnology engineering (Shanghai) GmbH, and then the DH5 alpha strain containing the plasmids PTK881-EF1 alpha-C1 and PTK881-EF1 alpha-C2 is preserved. The complete map schematic diagram of PTK881-EF1 alpha-C1 and PTK881-EF1 alpha-C2 is shown in FIG. 2.

Example 2 plasmid preparation and sequencing

1. Preparation of plasmids

The DH 5. Alpha. Strain containing plasmids PTK881-EF 1. Alpha. -C1 and PTK881-EF 1. Alpha. -C2 was inoculated into 250mL of LB medium containing 100. Mu.g/mL of ampicillin and cultured overnight at 37 ℃ and 220 rpm. The culture broth was centrifuged at 6000 g for 20 min at 4 ℃ and the supernatant was discarded.

Take out the buffer P1 in the Endo Free plasma mega kit (Qiagen), add 120 mL pre-cooled buffer P1 to the E.coli precipitate obtained by centrifugation, cover the centrifuge bottle cap, shake the centrifuge bottle vigorously to disperse the E.coli precipitate in the buffer P1 completely.

120 mL of Buffers P2 was added to the flask, the flask was covered with a cap and placed on a roller mixer, the speed was slowly increased to 50 rpm, and the mixture was thoroughly mixed and then left at room temperature for 5 min.

Adding 120 mL of Buffers P3 into a centrifuge bottle, covering the centrifuge bottle with a bottle cap, placing the centrifuge bottle on a roller mixing instrument, slowly increasing the speed to the maximum rotation speed of 70 rpm of the roller mixing instrument, and thoroughly mixing until the centrifuge bottle is white non-sticky and fluffy mixed liquid. Centrifuge at 9000 g for 15 min at 4 ℃.

50mL of Buffer FW was poured into the QIAfilter card, and the supernatant obtained by centrifugation was poured into the QIAfilter card, and gently stirred and mixed. And pumping and filtering the mixed solution into a corresponding marked glass bottle.

20 mL Buffer ER was added to each glass vial, mixed 6 times upside down and incubated at-20 ℃ for 30min.

The labeled mega columns were placed on corresponding racks, and 35 mL of Buffers QBT was added to each mega column to equilibrate and drain by gravity.

And (3) pouring all the liquid in the glass bottles into the corresponding marked mega columns in batches, and adding 200 mL of Buffer QC into each mega column in batches for washing after the liquid in the columns is drained. After the liquid in the column had run off, the waste liquid in the waste liquid collection tray was poured into a 50mL clean centrifuge tube.

Then, 40 mL Buffer QN was added to each mega column, the effluent was collected using a 50mL clean centrifuge tube, turned upside down for 6 times and mixed well, split-packed into 20 mL to another clean labeled 50mL centrifuge tube.

14 mL isopropanol (ambient temperature) was added to each 50mL centrifuge tube and mixed by inverting the tube 6 times. Centrifuge at 15000 g for 50 min at 4 ℃.

The supernatant was aspirated off the clean bench, and 3.5mL of endo-free water was added to each tube to rinse without dispersing the bottom precipitate. Centrifuge at 15000 g for 30min at 4 ℃. Buffer TE in an EndoFree plasma mega kit is put into an oven for preheating.

And (4) completely absorbing the centrifuged supernatant in the clean bench, and drying in the clean bench (volatilizing residual absolute ethyl alcohol for about 10 min).

Taking out the Buffer TE in the oven, adding 1mL of Buffer TE into each tube in a clean bench, blowing for 10 times by using a gun, and then putting the tube into the oven at 65 ℃, wherein the tube wall is uninterruptedly knocked to promote the precipitate to be completely dissolved. Centrifuging at 4 deg.C at 4000g for 1min to throw the liquid on the tube wall to the tube bottom, blowing, beating and mixing.

The whole liquid was transferred in a clean bench to endotoxin-free, pyrogen-free, nuclease-free EP tubes labeled accordingly. The plasmid concentration was measured by a micro-spectrophotometer at 2. Mu.L of the aspirated plasmid, and the plasmid was labeled on the corresponding EP tube to obtain plasmids PTK881-EF 1. Alpha. -C1 and PTK881-EF 1. Alpha. -C2.

2. Sequencing of target genes

20 mu L (about 500 ng) of plasmid DNA is respectively taken and sent out for sequencing, whether the target gene of a product obtained by plasmid production is changed or not is checked according to an original seed sequence, and the target gene cannot be changed in the process of fermentation culture and amplification of working seeds under a stable process, so that the method can be used for production and correct expression of protein in the next link.

Example 3 preparation and live-drop detection of Lenti3-C1-CAR, lenti3-C2-CAR Lenti-Lenti

1. Preparation of Lentiviral vectors

Connecting 130.0 to 140.0 × 10 cells into a multilayer cell culture bottle (Hyperflash) ⁶ A total of 560 mL DMEM complete medium (50 mL fetal bovine serum, 5mL of antimicrobial-antimicrobial (100X)) containing 5% CO at 37 ℃ was used in a number of 293T cells (Takara) ₂ Culturing in an incubator for 24h. Shuttle plasmid PTK881-EF1 alpha-C1/PTK 881-EF1 alpha-C2 was mixed with packaging plasmid in the following ratio, PTK881-EF1 alpha-C1/PTK 881-EF1 alpha-C2: BZ1 plasmid: BZ2 plasmid: BZ3 plasmid =12:10:5:6, obtain 320 μ g of mixed plasmid, add DMEM complete medium 15 mL. DMEM at 15 mL960 mug PEI is added into the complete culture medium, the DMEM complete culture medium mixed with the PEI is slowly added into the DMEM complete culture medium mixed with the plasmid, and the mixture is balanced for 10min at the room temperature. The 30mL of the mixture was mixed with 530mL of DMEM complete medium and the mixture was transferred to the multi-layer cell culture flask. Placing the multi-layer cell culture bottle at 37 deg.C with 5% CO ₂ After 3 days in the incubator, cell culture supernatant was collected.

After the supernatant was centrifuged at 4000 rpm (or 3000 g) for 30min, the supernatant after centrifugation was added with cryonase enzyme (Takara) and left at 4 ℃. After 6h, the lentivirus supernatant was suction filtered using a 0.22 μm filter and centrifuged at 30000 g for 2.5h at 4 ℃. The supernatant was removed and 1mL of T cell culture medium was added to resuspend the pellet. After resuspension, 20 mu L of the suspension is reserved for virus activity titer detection, and the remaining lentivirus concentrate is subpackaged and marked as Lenti3-C1-CAR and Lenti3-C2-CAR and is stored at-80 ℃ for later use.

2. Lentiviral vector activity titer detection

The principle is as follows: the anti-strepiI antibody is marked with fluorescein, and can be specifically combined with strepiI in the CAR, and the expression condition of the CAR in 293T cells is indirectly reflected by a fluorescence signal detected by a flow cytometer.

The method comprises the following steps: the 5.0 x 10 of the wells are connected into a 6-well plate ⁵ 293T cells are added into each well, 0.1. Mu.L, 0.5. Mu.L and 1. Mu.L of lentivirus concentrated solution are added into each well, and 1 negative control is arranged. Placing at 37 deg.C with 5% CO ₂ Culturing in an incubator. After three days, 293T cells are collected by Versene solution (Gibco) and sent to flow cytometry for detecting the proportion of the CAR-positive 293T cells, and the activity titer of Lenti3-C1-CAR and Lenti3-C2-CAR lentivirus concentrated solution is converted.

The results of flow-type detection of the activity titer of Lenti3-C1-CAR and Lenti3-C2-CAR lentivirus concentrate are shown in Table 1; the active titer of the current lentivirus concentrate is 1X 10 ⁸ ~10×10 ⁹ (TU/mL).

TABLE 1 lentivirus Activity titer assay results for Lenti3-C1-CAR, lenti3-C2-CAR

Example 4 preparation of C1-CAR-T, C2-CAR-T cells

1. Preparation of CAR-T cells:

100mL of peripheral blood of a healthy donor is collected, and mononuclear cells are separated by using a Ficoll lymphocyte separation medium. After counting, CD3 positive cells were sorted using appropriate amounts of CD3 MicroBeads, human (America, whirly) and sorted at 1.0 to 2.0X 10 ⁶ The Cell/mL density was cultured in a complete T-Cell culture Medium (OpTzer CTS-T-Cell Expansion basic Medium, opTzer CTS-T-Cell Expansion Supplement (Invitrogen), 500IU/mL IL-2 (double-Lut pharmaceutical industry)), at every 10 IU/mL ⁶ 25 μ L Dynabeads Human T-Activator CD3/CD28 (Invitrogen) was added to each cell to activate the T cells.

48h (Day 2), adding Lenti3-C1-CAR and Lenti3-C2-CAR lentiviral vectors according to the MOI of 3 for transduction, uniformly mixing, and placing in CO ₂ And (5) incubating in an incubator, and supplementing a proper amount of T cell complete culture medium for culturing after 4 hours.

After lentivirus transduction for 24h, the transduced C1-CAR-T, C2-CAR-T cells are replaced by fresh T cell complete culture solution, and the viable cell density is adjusted to 1.0-2.0 × 10 ⁶ Continuously culturing and expanding for 10 to 20 days per mL, observing and counting every day, performing liquid supplementing and expanding culture according to the counted cell number, and always keeping the cell culture density at 1.0-2.0 × 10 ⁶ /mL。

Collecting C1-CAR-T, C2-CAR-T cells according to the expected cell dosage, suspending in 100mL physiological saline containing 2% human serum albumin, transferring into a cell transfusion bag, and performing heat sealing to obtain a finished product of the C1-CAR-T, C-CAR-T cell preparation.

2. C1-CAR-T, C2-CAR-T cell transduction efficiency assay

Take 1.0X 10 ⁶ After each transduced T cell, incubated with 1. Mu.g/mL FITC-strepiI at room temperature for 30min, washed twice with physiological saline, FITC fluorescence signal was detected by flow cytometry, and the FITC positive cell ratio was measured, reflecting the ratio of CAR-T cells in total cells. The results of the C1-CAR-T, C2-CAR-T cell transduction efficiency assay are shown in FIG. 3 and Table 2, respectively. FIG. 3 and Table 2 show the successful preparation of C1-CAR-T, C2-CAR-T cells.

TABLE 2 C1-CAR-T, C-CAR-T cell transduction efficiency assay results

Example 5 in vitro functional assay of C1-CAR-T, C2-CAR-T cells in combination with antitumor drugs

And (3) respectively carrying out in-vitro tumor killing function detection on T, C1-CAR-T, C-CAR-T cells by adopting a calcein detection method.

The target cells are screened from six tumor cell lines, namely Ewing's sarcoma (EWS), acute lymphoblastic lymphoma/leukemia (T-ALL), glioblastoma (Malignant Gliomas), breast Cancer (Breast Cancer) and Lung Cancer (Lung Cancer), the screening standard is that the CD99 target can be highly expressed or expressed outside the membrane, the selected cell line is shown in Table 3, and the experimental group negative target cells are Raji (CD 99 negative cell line).

TABLE 3 selection of target cell lines for anti-CD99 CAR-T cells

The tumor killing experiment was performed as follows: taking 5X 10 ⁵ Each target cell was resuspended in 0.5mL of a tumor killing buffer (PBS containing 5% FBS), 5. Mu.L of Calcein-acetohydroxy-methyl ester (Calcein-AM) (final concentration: 25. Mu.M) was added, and gently mixed by pipetting. The cell suspension was incubated in an incubator for 30min. Centrifuging at room temperature for 5min, and discarding the supernatant. The operation was repeated twice. Adding 10mL of tumor-killing buffer, and adjusting the cell density to 0.5X 10 ⁵ One per mL. Adding 0.5X 10 of the active ingredient into each well of a 96-well plate respectively ⁵ Individual positive target cells (as shown in the table above) and negative target cells Raji. T, C1-CAR-T, C-CAR-T cell density was calculated, and T, C-CAR-T, C2-CAR-T cells were added to the control group at the target-to-effect ratio of 5:1, respectively. The experimental group is added with T, C-CAR-T, C-CAR-T cells according to the effective target ratio of 5:1, then cisplatin is added to the experimental group to achieve the final concentration of 32 mug/mL and paclitaxel is added to the experimental group to achieve the final concentration of 20nmoL/L. The control combined experimental group is incubated for 2 to 3 hours at 37 ℃. After the incubation, the supernatant was taken, the fluorescence intensity of calcein therein was measured, and the percentage of target cell lysis was calculated from the spontaneous release control and the maximum release control.

The results of the percentage lysis of T cells, DDP, PTX, C1-CAR-T cells, C2-CAR-T cells, C1-CAR-T cells plus DDP, C1-CAR-T cells plus PTX, C2-CAR-T cells plus DDP, C2-CAR-T cells plus PTX on the negative cells Raji are shown in FIG. 4.

The results of the percentage lysis of T cells, DDP, PTX, C1-CAR-T cells, C2-CAR-T cells, C1-CAR-T cells plus DDP, C1-CAR-T cells plus PTX, C2-CAR-T cells plus DDP, C2-CAR-T cells plus PTX against ewing sarcoma cell line TC71, 6647 target cells are shown in figure 5.

The results of the percentage lysis of T cells, C1-CAR-T cells, C2-CAR-T cells, C1-CAR-T cells plus DDP, C1-CAR-T cells plus PTX, C2-CAR-T cells plus DDP, C2-CAR-T cells plus PTX on acute lymphoblastic lymphoma positive cells Jurkat, MOLT-4 are shown in FIG. 6.

The results of the percentage lysis of T cells, C1-CAR-T cells, C2-CAR-T cells, C1-CAR-T cells plus DDP, C1-CAR-T cells plus PTX, C2-CAR-T cells plus DDP, C2-CAR-T cells plus PTX on malignant glioma-positive cells U251-MG, U373-MG are shown in FIG. 7.

The percentage results of lysis of T cells, C1-CAR-T cells, C2-CAR-T cells, C1-CAR-T cells plus DDP, C1-CAR-T cells plus PTX, C2-CAR-T cells plus DDP, C2-CAR-T cells plus PTX for breast cancer-positive cells MCF-7, and for lung cancer-positive cells H1299 are shown in FIG. 8.

From the results of fig. 4 to 8, it can be seen that the combination of DDP and PTX in the C1-CAR-T cells and C2-CAR-T cells has synergistic therapeutic effects on ewing sarcoma cells, acute lymphoblastic lymphoma positive cells, malignant glioma positive cells, breast cancer positive cells, and lung cancer positive cells. For example, as shown in fig. 5, the killing efficiency of T cells to TC71, 6647 is about 10%, the killing efficiency of DDP and PTX to TC71, 6647 is about 25%, and the killing efficiency of C1-CAR-T cells and C2-CAR-T cells to TC71 and 6647 is less than 45%, while the killing efficiency of C1-CAR-T cells plus PTX and DDP to target cells and the killing efficiency of C2-CAR-T cells plus PTX and DDP to target cells are both greatly improved, so that the combined use of C1-CAR-T cells and C2-CAR-T cells with PTX and DDP has an obvious synergistic effect on the lysis of target cells TC71, 6647, the tumor killing effect of CAR-T cells and antitumor drugs is obviously improved, and the effect of 3732 zxft 3425 is more than or equal to 2.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Sequence listing

<110> Wuhan Borui Rui Da Biotech Co., ltd

<120> application of chimeric antigen receptor combined antitumor drug taking CD99 as target

<130> CP20421

<141> 2020-08-27

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 241

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Lys Arg Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Gly Gly Val Asp Trp Gly Gln Gly Thr Leu Val Thr Val

100 105 110

Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile

130 135 140

Gly Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp

145 150 155 160

Gly Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln

165 170 175

Ser Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val

180 185 190

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

195 200 205

Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln

210 215 220

Gly Thr His Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

225 230 235 240

Lys

<210> 2

<211> 723

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gaggtgcagc tgcagcagag cggcgccgag ctggtgaagc ccggcgccag cgtgaagctg 60

agctgcaccg ccagcggctt caacatcaag gacacctaca tccactgggt gaagcgccgc 120

cccgagcagg gcctggagtg gatcggccgc atcgaccccg ccaacggcaa caccaagtac 180

gaccccaagt tccagggcaa ggccaccatc accgccgaca ccagcagcaa caccgcctac 240

ctgcagctga gcagcctgac cagcgaggac accgccgtgt actactgcgc ccgccgcggc 300

ggcgtggact ggggccaggg caccctggtg accgtgagcg ccggtggagg cggttcaggc 360

ggaggtggct ctggcggtgg cggatcggac gtggtgatga cccagacccc cctgaccctg 420

agcgtgacca tcggccagcc cgccagcatc agctgcaaga gcagccagag cctgctggac 480

ggcgacggca agacctacct gaactggctg ctgcagcgcc ccggccagag ccccaagcgc 540

ctgatctacc tggtgagcaa gctggacagc ggcgtgcccg accgcttcac cggcagcggc 600

agcggcaccg acttcaccct gaagatcagc cgcgtggagg ccgaggacct gggcgtgtac 660

tactgctggc agggcaccca cttcccccgc accttcggcg gcggcaccaa gctggagatc 720

aag 723

<210> 3

<211> 241

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Gly Gly Val Asp Trp Gly Gln Gly Thr Leu Val Thr Val

100 105 110

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro

130 135 140

Gly Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp

145 150 155 160

Gly Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Lys Pro Gly Gln

165 170 175

Ser Pro Gln Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val

180 185 190

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

195 200 205

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln

210 215 220

Gly Thr His Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

225 230 235 240

Lys

<210> 4

<211> 723

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

caggtgcagc tggtgcagag cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg 60

agctgcaagg ccagcggctt caacatcaag gacacctaca tccactgggt gcgccaggcc 120

cccggccagg gcctggagtg gatgggccgc atcgaccccg ccaacggcaa caccaagtac 180

gaccccaagt tccagggccg cgtgaccatg acccgcgaca ccagcatcag caccgcctac 240

atggagctga gccgcctgcg cagcgacgac accgccgtgt actactgcgc ccgccgcggc 300

ggcgtggact ggggccaggg caccctggtg accgtgagca gcggtggagg cggttcaggc 360

ggaggtggct ctggcggtgg cggatcggac atcgtgatga cccagacccc cctgagcctg 420

agcgtgaccc ccggccagcc cgccagcatc agctgcaaga gcagccagag cctgctggac 480

ggcgacggca agacctacct gaactggctg ctgcagaagc ccggccagag cccccagcgc 540

ctgatctacc tggtgagcaa gctggacagc ggcgtgcccg accgcttcag cggcagcggc 600

agcggcaccg acttcaccct gaagatcagc cgcgtggagg ccgaggacgt gggcgtgtac 660

tactgctggc agggcaccca cttcccccgc accttcggcc agggcaccaa gctggagatc 720

aag 723

<210> 5

<211> 21

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

<210> 6

<211> 63

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atggccctgc ctgtgacagc tctgctcctc cctctggccc tgctgctcca tgccgccaga 60

ccc 63

<210> 7

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Asn Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Gly Ser

1 5 10

<210> 8

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

aactggagcc acccccagtt cgagaagggc ggtggcggaa gc 42

<210> 9

<211> 45

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 10

<211> 135

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210> 11

<211> 27

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 12

<211> 81

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60

gcctttatta ttttctgggt g 81

<210> 13

<211> 42

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 14

<211> 126

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 15

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 16

<211> 336

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 17

<211> 41

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 18

<211> 123

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120

tcc 123

Claims

1. A broad-spectrum antitumor drug composition, wherein the active ingredients of the antitumor drug comprise;

(1) An immune cell expressing a chimeric antigen receptor carries a single-chain antibody which specifically recognizes a CD99 antigen on the surface of a tumor cell, the single-chain antibody comprises SP-C1 or SP-C2, the amino acid sequences of the SP-C1 and SP-C2 are respectively shown as SEQ ID No.1 and SEQ ID No.3, and the nucleotide sequences of the SP-C1 and SP-C2 are respectively shown as SEQ ID No.2 and SEQ ID No. 4; the chimeric antigen receptor sequentially splices a signal peptide, a single-chain antibody, strepII, a CD8 change, a CD28 transmembrane region, a CD28 intracellular domain, an intracellular co-stimulatory domain 4-1BB and a CD3 zeta chain from the N end to the C end, the nucleotide sequence of the signal peptide is shown in SEQ ID NO.6, the nucleotide sequence of the strepII is shown in SEQ ID NO.8, the nucleotide sequence of the CD8 change is shown in SEQ ID NO.10, the nucleotide sequence of the CD28 transmembrane region is shown in SEQ ID NO.12, the nucleotide sequence of the CD28 intracellular domain is shown in SEQ ID NO.18, the nucleotide sequence of the intracellular co-stimulatory domain 4-1BB is shown in SEQ ID NO.14, the nucleotide sequence of the CD3 zeta is shown in SEQ ID NO.16, the amino acid sequence of the signal peptide is shown in SEQ ID NO.5, the amino acid sequence of the strepII is shown in SEQ ID NO.7, the amino acid sequence of the CD8 change is shown in SEQ ID NO.9, the amino acid sequence of the signal peptide is shown in SEQ ID NO. 28, the amino acid sequence of the CD3 is shown in SEQ ID NO.11, the amino acid sequence of the CD3 amino acid sequence of the SEQ ID NO.13 is shown in SEQ ID NO.13, the intracellular domain is shown in SEQ ID NO.13, the amino acid sequence of the co-1 is shown in SEQ ID NO. 13;

(2) A chemical antitumor drug selected from cisplatin or paclitaxel.

2. Use of the antitumor pharmaceutical composition of claim 1 in the preparation of a therapeutic antitumor pharmaceutical preparation.

3. An antitumor pharmaceutical preparation comprising the antitumor pharmaceutical composition according to claim 1 and a pharmaceutically acceptable pharmaceutical excipient.

4. The anti-neoplastic pharmaceutical formulation of claim 3, wherein the neoplasm includes Ewing's sarcoma, acute lymphoblastic lymphoma, glioblastoma, breast cancer and lung cancer.