CN110229236B - CAR for inducing tumor cells to upregulate the expression of antigen MUC1 and its application - Google Patents

Abstract

This application belongs to the technical field of tumor cell immunotherapy, and specifically relates to a CAR capable of inducing tumor cells to up-regulate the expression of antigen MUC1 and its application for a patent application. The chimeric antigen receptor is an amino acid sequence connected by several protein fragments, specifically: signal peptide of human CD8a molecule, humanized MUC1 single-chain antibody, transmembrane region of human CD8 molecule and intracellular region of 41BB molecule, human Intracellular region of CD3z molecule; In a preferred design, the intracellular region of human CD3z molecule is further connected with the sequence of IL22 CDS region through a linker sequence, so as to further promote the expression of MUC1 through the cytokine IL-22. Through the further structural optimization of CAR, this application has shown good technical effects on effectively inducing the expression of tumor cell surface antigen MUC1, reducing the occurrence of off-target effects, and improving the application effect of CAR‑T cells.

Description

CAR for inducing tumor cells to upregulate the expression of antigen MUC1 and its application

technical field

This application belongs to the technical field of tumor cell immunotherapy, and specifically relates to a CAR (Chimeric Antigen Receptor, Chimeric Antigen Receptor) that can induce tumor cells to up-regulate the expression of antigen MUC1 and its application.

Background technique

CAR-T (Chimeric Antigen Receptor T-Cell, Chimeric Antigen Receptor T Cell), as an effective treatment for tumor immunotherapy, was approved by the US Food and Drug Administration (FDA) on August 30, 2017 For the treatment of acute lymphoblastic leukemia (ALL), which targets CD19 and can effectively eliminate CD19 ⁺ tumor cells, it has shown encouraging results in preclinical and clinical trials, and has proved that they have refractory Potent antileukemic effects in children and adolescents with relapsed and relapsed b-precursor acute lymphoblastic leukemia were safe and feasible, with all toxicities reversible and without persistent B-cell aplasia. However, its application in solid tumors is still unsatisfactory, and there are still many problems to be solved. The main reason is that there is no effective tumor-specific antigen similar to CD19 in solid tumors and the inhibitory effect of the tumor microenvironment.

Lentiviruses are RNA viruses belonging to the Retroviridae family, the best known of which is the human immunodeficiency virus (HIV-1). HIV-1 has a diameter of about 120nm and contains two positive strands of RNA, which can effectively enter the nucleus and have a high infection efficiency for dividing cells and non-dividing cells. After the lentivirus infects the cells, the genes it carries can be integrated into the cell genome, and can be stably expressed for a long time, and can be stably inherited with cell division, so it becomes an effective tool for introducing foreign genes. The lentiviral vector is a gene therapy vector modified on the basis of HIV-1. By removing the disease-causing gene and reducing the homology between the helper plasmid and the vector plasmid, the modified lentivirus has a higher titer , better biological safety, and stronger ability to import foreign fragments. At present, the lentiviral system has been widely used in cell and animal experiments of gene overexpression, RNA interference, miRNA research and gene knockout and other functional research.

MUC1 (Mucin1, mucin 1) is a macromolecular transmembrane glycoprotein that is widely and highly expressed in malignant tumor cells such as breast cancer, ovarian cancer, lung cancer, prostate cancer, colon cancer, liver cancer and pancreatic cancer. Normal cells and MUC1 on the surface of tumor cells have different glycosylation structures. In theory, based on the expression characteristics of MUC1, it can be better used to distinguish tumor cells from non-tumor cells, and overexpression and abnormal glycosylation also make MUC1 an ideal target for various immunotherapies, especially CAR-T therapy. point, but in practical application, limited by MUC1 expression and other reasons, no good application effect has been seen so far.

Contents of the invention

The purpose of this application is to provide a CAR that can induce tumor cells to up-regulate the expression of the antigen MUC1, so as to lay a certain technical foundation for related CAR-T in the treatment of solid tumors.

The technical solution adopted by this application is described in detail as follows.

A CAR that can induce tumor cells to up-regulate the expression of antigen MUC1. The chimeric antigen receptor is an amino acid sequence connected by several protein fragments, named after MUC1-41BB-CD3z, specifically: human CD8a molecular signal peptide (CMV ), humanized MUC1 single-chain antibody (scFV.MUC1), human CD8 molecular transmembrane region and 41BB molecular intracellular region (41BB), human CD3z molecular intracellular region (CD3Zeta), namely: CMV-scFV.MUC1-41BB - CD3 Zeta (CD3ζ);

In a preferred design, the intracellular region of the human CD3z molecule (CD3Zeta) is further connected to the IL22 CDS region sequence (IL22) through a linker sequence (for example, using the TA2 linker sequence (T2A)), so as to further stimulate the IL-22 cytokine. Promote the expression of MUC1, that is, the overall structure is: CMV—scFV.MUC1—41BB—CD3Zeta (CD3ζ)—T2A—IL22, and the optimal design is named after MUC1-41BB-CD3z-IL22;

The human CD8a molecular signal peptide (CMV) includes 21 amino acids, and its sequence is shown in SEQ ID No.1, specifically:

MALPVTALLLPLALLLLHAARP;

The humanized MUC1 single-chain antibody (scFV.MUC1) includes 244 amino acids, and its sequence is shown in SEQ ID No.2, specifically:

EVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSSGGGGSGGGGSGGGSDIVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDH LFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSE;

The transmembrane region of human CD8 molecule and the intracellular region of 41BB molecule (41BB) include two parts: the transmembrane region of human CD8 molecule and the intracellular region of 41BB molecule, wherein:

The transmembrane region of human CD8 molecule includes 71 amino acids, and its sequence is shown in SEQ ID No.3, specifically:

LETTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLLSVITLYC;

The intracellular region of 41BB molecule includes 42 amino acids, and its sequence is shown in SEQ ID No.4, specifically:

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

The human CD3z molecular intracellular region (CD3Zeta) includes 112 amino acids, and its sequence is shown in SEQ ID No.5, specifically:

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

The TA2 connecting sequence (T2A) includes 18 amino acids, and its sequence is shown in SEQ ID No.6, specifically:

EGRGSLLTCGDVEENPGP;

The IL22 CDS region sequence (IL22) includes 179 amino acids, and the sequence is shown in SEQ ID No.7, specifically:

MAALQKSVSSFLMGTLATSCLLLLALLVQGGAAAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI;

Therefore, the preferred amino acid sequence of MUC1-41BB-CD3z-IL22 includes 730 amino acids, and the sequence is shown in SEQ ID No.8, specifically:

MALPVTALLLPLALLLHAARPGSMALPVTALLLPLALLLHAARPEVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVVTQ ESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSEMALPVTALLLPLALLLLHAARPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRK KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPMAALQ KSVSSFLMGTLATSCLLLLALLVQGGAAAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI.

The lentiviral expression plasmid constructed by using the CAR capable of inducing tumor cells to upregulate the expression of antigen MUC1 specifically includes the following steps:

(1) According to the central dogma, using existing technology to obtain the coding sequence DNA of the CAR;

(2) Using the Lenti-2G plasmid as the expression vector, perform double digestion with BsrGI and Not I, and then use T4 DNA ligase to integrate and recombine the coding sequence DNA in step (1) into the Lenti-2G plasmid;

(3) Transform the ligation product in step (2) into STABL3 competent cells, screen, expand and culture, and further extract the plasmid to obtain a recombinant lentivirus that can express MUC1-41BB-CD3z or MUC1-41BB-CD3z-IL22 Expression plasmids (respectively named: Lenti-2G-MUC1-41BB-CD3z, Lenti-2G-MUC1-41BB-CD3z-IL22).

The application of the lentiviral expression plasmid in the preparation of anti-tumor agents, the specific example of the tumor is HNSCC cancer (head and neck squamous cell carcinoma, mainly referring to nasal cavity, sinus, oral cavity, tonsil, pharynx and larynx and other tissue parts) cancerous cells) related tumors, further for example, human tongue squamous cell carcinoma cell line CAL33, human pharyngeal squamous cell carcinoma cell line HN4 and other related tumors; in application, CAR-T cells are prepared by transfection for application to increase tumor cell MUC1 expression level;

The specific application steps include the following steps:

(1) Lentivirus packaging; for details, please refer to the following operations:

293T cells were used as the target cells to be transfected, and first plated and incubated for 24 hours;

Then, the constructed lentiviral expression plasmid and packaging plasmid were mixed, and the target cell 293T cells to be transfected were transfected for 48 hours with liposome transfection reagent;

After the transfection, the supernatant was collected for use in preparation for T cell infection;

(2) Preparation of purified T cells

First, mononuclear cells are isolated from human peripheral blood (for example, by density gradient centrifugation);

Then, separate and obtain purified CD ³⁺ T cells (specifically, for example, using T cell separation magnetic beads for separation and purification),

Finally, add an appropriate amount of CD3/CD28 magnetic beads to activate for 2 days for later use;

(3) T cell infection

Add the virus supernatant collected in step (1) and polybrene (polybrene, also known as hexadimethonium bromide) to the cells activated for 2 days in step (2), and incubate overnight for infection;

(4) Expansion of T cells and detection

After incubation of infected T cells in step (3), centrifuge and wash (generally not less than 3 times), add RPMI1640 medium containing 1000U IL-2 and 5% fetal bovine serum to further expand T cells;

For the expanded T cells, flow cytometry can be used to detect and determine the expression of CAR on the surface of T cells or cell proliferation. According to the test results, after further reinfusion, it can be used to stimulate and increase the expression of MUC1 in tumor cells.

IL-22 is a cytokine mainly secreted by Th22 cells and belongs to the IL-10 superfamily. Existing studies believe that IL-22 can enhance the body's innate immunity, protect the body's cells from damage and promote the regeneration and repair of tissue cells, and Participate in the regulation of various diseases and verification; and some studies have reported that IL-22 can promote the upregulation of the expression of tumor cell surface antigen MUC1. However, this application uses the tumor antigen MUC1 as the target, and further effectively enhances the expression of the tumor antigen MUC1 on the surface of tumor cells by optimizing the CAR structure, especially through the tandem IL22 molecule, which is helpful for improving the recognition of tumor cells by CAR-T cells , Improve the ability to target tumor cells, and then improve the killing effect of CAR-T cells on MUC1-positive tumor cells, laying a good technical foundation.

In general, in view of the off-target problem of existing CAR-T cells in the treatment of solid tumors and the problem of tumor immune escape, this application further optimizes the structure of CAR to effectively induce the expression of tumor cell surface antigen MUC1 and reduce the occurrence of off-target effects , and improving the application effect of CAR-T cells have shown good technical effects, so they have good practical value and significance for popularization and application.

Description of drawings

Figure 1 is a schematic diagram of the CAR structure of MUC1-41BB-CD3z (upper figure) and MUC1-41BB-CD3z-T2A-IL22 (lower figure);

Figure 2 is a schematic diagram of the structure of Lenti-2G-MUC1-41BB-CD3z-T2A-IL22;

Figure 3 is a diagram of CAR-T transfection efficiency detected by flow cytometry;

Figure 4 is the statistical result of cell proliferation and culture after transfection;

Figures 5 to 7 are graphs showing the killing efficiency of CAR-T cells on different tumor cells; Figure 5 is for OME cells, Figure 6 is for Cal33 cells, and Figure 7 is for HN4 cells;

Figure 8 is a summary of the effects of different tumor cells under different effect-to-target ratios (E/T);

Figure 9 shows that IL-22 can induce the upregulation of tumor cell MUC1 by flow cytometry;

Figure 10 is the secretion and expression of IL 22;

Figure 11 shows the monitoring of tumor growth and fluorescence by in vivo imaging technology.

Detailed ways

The present application will be further explained below in conjunction with the embodiments. Before introducing specific embodiments, a brief description of some experimental materials, experimental instruments, etc. in the following embodiments is as follows.

Experimental Materials:

Non-obese diabetic/severe combined immunodeficiency (nod/scid) mice, 6-8 weeks old female mice, purchased from Beijing Weitong Lihua Co., Ltd. (Beijing, China), cultured in a sterile environment;

Human tongue squamous cell carcinoma cell line (CAL33) and human pharyngeal squamous cell carcinoma cell line (HN4) were purchased from Shanghai Ninth People's Hospital of Shanghai Jiao Tong University School of Medicine;

Related cell lines were cultured in Dulbecco's Modified Eagle medium (DMEM-F12) containing 10% fetal bovine serum (FBS, HyClone, Chicago, IL, USA) and 100 U/ml penicillin, 100 μg/ml streptavidin Invitrogen (Invitrogen, Carlsbad, CA, USA);

Before the experiment started, tumor cells were transfected with luciferase-GFP viral supernatant, and the GFP channel was sorted with a FACS AriaTM cell sorter (BD Biosciences, San Jose, CA, USA) to finally obtain stable expression Luciferase-green fluorescent protein (GFP) cell line HN4 (cultured in DMED-F12 medium);

The immortalized cell line OME is an oral mucosal epithelial cell line overexpressing hTERT. This cell line is MUC1-negative and cultured in DMED-F12 medium.

Example 1

On the basis of existing CAR research, in order to improve the therapeutic effect of CAR-T cells and reduce the off-target effect of current CAR-T cell therapy, the inventors further optimized and modified the CAR structure. Two kinds of CARs have been prepared by humans, and the specific structural diagrams are shown in Figure 1.

in particular:

MUC1-41BB-CD3z is: human CD8a molecule signal peptide (CMV), humanized MUC1 single-chain antibody (scFV.MUC1), human CD8a molecule flexible fragment (CD8aTM), human CD8 molecule transmembrane region and 41BB molecule linked in sequence Intracellular region (41BB), intracellular region of human CD3z molecule (CD3Zeta), namely: CMV-scFV.MUC1-41BB-CD3Zeta (CD3ζ);

MUC1-41BB-CD3z-IL22 is: human CD8a molecule signal peptide (CMV), humanized MUC1 single-chain antibody (scFV.MUC1), human CD8 molecule transmembrane region and 41BB molecule intracellular region (41BB), Intracellular region of human CD3z molecule (CD3Zeta), TA2 junction sequence (T2A), IL22 CDS region sequence (IL22); namely: CMV-scFV.MUC1-41BB-CD3Zeta (CD3ζ)-T2A-IL22.

The human CD8a molecular signal peptide (CMV) includes 21 amino acids, and its sequence is shown in SEQ ID No.1, specifically:

MALPVTALLLPLALLLLHAARP.

The humanized MUC1 single-chain antibody (scFV.MUC1) includes 244 amino acids, and its sequence is shown in SEQ ID No.2, specifically:

EVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSSGGGGSGGGGSGGGSDIVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDH LFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSE.

The transmembrane region of human CD8 molecule and the intracellular region of 41BB molecule (41BB) include two parts: the transmembrane region of human CD8 molecule and the intracellular region of 41BB molecule, wherein:

The transmembrane region of human CD8 molecule includes 71 amino acids, and its sequence is shown in SEQ ID No.3, specifically:

LETTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLLSVITLYC

The intracellular region of 41BB molecule includes 42 amino acids, and its sequence is shown in SEQ ID No.4, specifically:

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

The human CD3z molecular intracellular region (CD3Zeta) includes 112 amino acids, and its sequence is shown in SEQ ID No.5, specifically:

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

The TA2 connecting sequence (T2A) includes 18 amino acids, and its sequence is shown in SEQ ID No.6, specifically:

EGRGSLLTCGDVEENPGP.

The IL22 CDS region sequence (IL22) includes 179 amino acids, and the sequence is shown in SEQ ID No.7, specifically:

MAALQKSVSSFLMGTLATSCLLLLALLVQGGAAAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI.

MUC1-41BB-CD3z amino acid sequence, including 490 amino acids, the specific sequence is:

MALPVTALLLPLALLLHAARPEVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVVTQESALTTSPGETVTLTCRSSTGAV TTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSELETTTPAPRPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC ELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

The amino acid sequence of MUC1-41BB-CD3z-IL22, including 730 amino acids, is shown in SEQ ID No.8, specifically:

MALPVTALLLPLALLLHAARPGSMALPVTALLLPLALLLHAARPEVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVVTQ ESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSEMALPVTALLLPLALLLLHAARPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRK KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPMAALQ KSVSSFLMGTLATSCLLLLALLVQGGAAAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI.

Example 2

On the basis of Example 1, the inventor further constructed a lentiviral expression vector, and the specific construction process can refer to the following steps.

(1) According to the central dogma, using existing technology to obtain the coding sequence DNA of the CAR;

(2) Using the Lenti-2G plasmid (Lenti-2G-MCS-conGFP) as the expression vector, perform double digestion with BsrGI and Not I, and then use T4 DNA ligase to integrate and recombine the coding sequence DNA in step (1) into the Lenti-2G plasmid;

(3) Transform the ligation product in step (2) into STABL3 competent cells, screen, expand and culture, and further extract the plasmid to obtain a recombinant lentivirus that can express MUC1-41BB-CD3z or MUC1-41BB-CD3z-IL22 Expression plasmids (respectively named: Lenti-2G-MUC1-41BB-CD3z, Lenti-2G-MUC1-41BB-CD3z-IL22). The schematic structure of the constructed Lenti-2G-MUC1-41BB-CD3z-IL22 plasmid is shown in FIG. 2 .

It should be noted that the plasmids used in this example were prepared and provided by PPL (Public Protein/PlasmidLibrary, China) company according to the above steps.

Example 3

On the basis of Example 2, the inventors further conducted a preliminary experiment by transfecting the constructed lentiviral expression plasmid into CAR-T cells. The specific process is briefly introduced as follows.

(1) Lentiviral packaging

293T cells were used as the target cells to be transfected, and first six-well plates were plated and incubated for 24 hours to cultivate 293T cells (DMEM complete medium at 37°C);

During transfection, the medium was replaced with OPTI-MEM (amount of 2ml), and then 1.5g of the constructed lentiviral expression plasmid, 1.5g and 1g of the packaging plasmid psPAX2 and Pmd2.G, respectively, 8ul of liposome transfection reagent, Mixing to transfect the target cell 293T cells to be transfected;

Replace with normal DMEM medium after 12 hours;

After continuing to culture for 48 hours, the virus supernatant was collected, centrifuged at 1500 rpm for 10 minutes, and the supernatant was taken and stored at -80°C for use in infecting T cells.

(2) Preparation of purified CD3+ T cells

First, mononuclear cells were isolated from human peripheral blood (specifically using density gradient centrifugation);

Then, isolate and obtain purified CD3 ⁺ T cells (specifically, use T cell separation magnetic beads for separation and purification);

Afterwards, the purified T cells were treated with IL-2 (100 IU/ml, PeproTech, Suzhou, Jiangsu, China) and L-glutamine (2 mM , Gibco/Life Technologies/ The RPMI-1640 of Thermo Fisher Scientific, Waltham, MA, USA) was cultivated;

Finally, T cells were stimulated and activated with CD3/CD28 activating antibody (1 ul / 10 ⁷ cells) for 2 days before use.

(3) T cell infection

Using a 24-well cell culture plate, add T cells activated for 2 days in step (2) to each well (10 ⁶ /well), and add 1 ml of the virus supernatant collected in step (1) and polybrene (8 μg/mL) to each well ), incubated overnight for infection;

After 24 hours, it was replaced with a normal protein containing IL-2 (100 IU/ml, PeproTech, Suzhou, Jiangsu, China) and L-glutamine (2 mM , Gibco/Life Technologies/Thermo Fisher Scientific, Waltham, MA, USA ) of RPMI-1640 for cultivation.

(4) Expansion of T cells and detection

For the post-infection T cells incubated in step (3), after centrifugation and washing (3 times), add RPMI1640 medium containing 1000U IL-2 and 5% fetal bovine serum (replace the medium every 2-3 days) to further T cells were expanded, and the prepared cells were designated as: MUC1-T (containing the lentiviral vector plasmid MUC1-41BB-CD3z), MUC1-IL22-T (containing the chronic disease that can stimulate the expression of MUC1.

It should be noted that, as a control, referring to the above operation, the inventor packaged the Lenti-2G plasmid containing GFP into a lentivirus and directly infected T cells, and recorded the prepared cells as: GFP-T (equivalent to the blank expressing only GFP control T cell group).

(5) Experimental detection

(1) Detection of virus packaging and evaluation of T cell proliferation

Five days after the infection of T cells, the expression of CAR on the surface of T cells was detected by flow cytometry. The results are shown in Figure 3. It can be seen that the positive rate of CAR expression reached 50% to 70%, which indicated that the constructed CAR expression plasmid was successfully packaged into lentiviral particles and transfected successfully.

The cell proliferation was counted, and the results are shown in Figure 4. It can be seen from the analysis that the cells in the GFP-T group proliferated the slowest, while the cells in the other two experimental groups proliferated faster. However, due to the influence of lentivirus transfection, the number of cells in each treatment group decreased with the prolongation of the proliferation time. All decreased gradually; in other words, from the perspective of cell proliferation efficiency, the amount of cell proliferation after proliferation culture for about 5 days was the highest.

(2) Detection of apoptosis in different tumor cells

After CAR-T cells were expanded for 15 days, tumor cells with different MUC1 expressions were used as experimental objects to detect and evaluate tumor cell apoptosis. The specific experimental settings were as follows:

Oral cancer tumor cells Cal33, HN4, and MUC1-negative immortalized oral mucosal epithelial cells OME were used as target cells, and T cells treated with different treatments were used as effector cells (GFP-T, MUC1-T, MUC1-IL22- T), according to different effect-to-target ratios (1:1, 10:1, 20:1), T cells and target cells were co-incubated for 6 hours in a 96-well plate, and three replicate wells were set up for each group.

Collect the supernatant after cell co-incubation, resuspend the cell pellet with Annexin V-binding buffer (BioLegend, San Diego, CA, USA), add 1ul CD326 antibody and 1ul Annexin Vantibody (BioLegend) to the cell suspension respectively , incubate at 4°C in a dark environment for 15 minutes; Propidium (propidium iodide, Sigma) was added to the cell suspension before going on the machine; all samples were analyzed using FACSCanto II or C6 flow cytometer (Becton Dickinson) , and the data were analyzed using FlowJo software (FlowJo, LLC, Ashland, Covington, KY, USA).

The results are shown in Figure 5, Figure 6, Figure 7, and Figure 8. It can be seen that even for different tumor cells, compared with GFP-T cells, MUC1-T cells can effectively and specifically kill MUC1-positive tumors cells, and the killing effect of MUC1-IL22-T cells is better than that of MUC1-T cells under the same ratio, which shows that MUC1-IL22-T cells have a better effect on the elimination of tumor cells. For example: when the effect-to-target ratio is 20:1, the difference is more obvious. MUC1-T cells can eliminate 50-60% of MUC1-positive tumor cells, while MUC1-IL22-T cells can eliminate MUC1-positive tumor cells. The removal effect of tumor cells can reach more than 90%.

(3) Detection of IL22 secretion and MUC1 expression

During the above (2) detection and evaluation of the apoptosis of different tumor cells, the inventors further detected and analyzed the secretion of IL22 and the expression of MUC1.

It should be noted that the specific reference for the MUC1 expression detection method is as follows:

First, use TRIzol reagent (Invitrogen) to extract mRNA from tumor samples;

Then, the RNA was reverse-transcribed into cDNA using the PrimeScript RT kit from TaKaRa;

Finally, the SYBR premix Ex Taq II (TaKaRa) was used to perform qRT-PCR analysis on the Agilent Mx3005P. When specifically analyzing MUC1, the primers were designed as follows:

Forward: 5'-TTTCCAGCCCGGGATACCTA-3',

Reverse: 5'-AGAGGCTGCTGCCACCATTA-3';

Glyceraldehyde 3-phosphate hydrogenase (GAPDH) was used as an internal reference, and the data were analyzed using 2- ^ΔΔCt .

During the experiment, in order to verify the stimulating effect of exogenous IL22 on the expression of MUC1 on the surface of tumor cells, the inventors added IL22 (20ng/ml) to different oral cancer tumor cells and immortalized oral mucosal epithelial cells in six-well cell culture plates. After 72 hours of incubation, MUC1 expression was detected by flow cytometry.

The specific results are shown in Figure 9. It can be seen that MUC1 on the surface of tumor cells is significantly up-regulated, while the expression of MUC1 on the surface of immortalized oral mucosal epithelial cells has no significant change. This result shows that IL22 can effectively and specifically induce the up-regulation of the expression of MUC1 on the surface of tumor cells, and has no effect on other cells. Obvious effect.

Further, the expression of IL22 and MUC1 after treatment of Cal33 cells by different effector T cell groups under different effect-to-target ratios was detected and counted, and the results are shown in FIG. 10 .

The statistics and analysis of the results show that the smaller the E/T ratio, the weaker the cytotoxic function of T cells, the slower the proliferation of T cells, and the weaker the ability to secrete functional cytokines, while the CAR-T cells secreting IL22 Compared with other cells, it has a stronger cytotoxic effect. Therefore, it is a more feasible way to increase the cytotoxicity of T cells by enhancing the secretion and expression of IL22. Corresponding to this result, the expression of MUC1 in Cal33 cells was increased from 70% to about 95%, which further indicated that it is feasible to increase the expression of MUC1 by coupling IL-22 gene and increasing the expression of IL-22 .

(4) Detection of tumor growth in mice

On the basis of the above experiments, the inventors further carried out mouse animal experiments, and the specific process is briefly introduced as follows.

NOD/SCID immunodeficient mice were subcutaneously inoculated with 10 ⁶ HN4-fluc cells (100ul) resuspended in PBS to construct a mouse subcutaneous tumor-bearing model. After 12 days, 5×10 ⁶ different groups of T cells (PBS, GFP-T, MUC1-T, MUC1-IL22-T) were treated (5 rats in each group).

During the experiment, electronic scales and vernier calipers were used to detect mouse weight and tumor volume changes, and small animal in vivo imaging equipment was used to detect fluorescence changes expressed in tumors (bioluminescent photos were taken every 10 days, and mice were sacrificed on day 40 for analysis).

When using small animal in vivo imaging equipment to detect fluorescence changes expressed by tumors, first anesthetize mice with 3% isoflurane (RWD Life Sciences, Shenzhen, China) in the induction chamber; then each mouse was intraperitoneally injected with 100ul d- Fluorescein solution (0.15mg/ml, Yeasen Biotech Co., Ltd., Shanghai, China), after 10 minutes, use animal live imaging equipment IVIS Lumina, Series III spectrometer (Caliper Life Science) to detect fluorescence, and finally use live image 4.3. 1 software (PerkinElmer, Waltham, MA, USA) for analysis.

The experimental results are shown in Figure 11. Results statistics (based on the last measurement results on the 40th day) and analysis can be seen:

In terms of tumor fluorescence intensity: in the PBS and GFP-T treatment groups, the tumor fluorescence of the mice was at the level of 10 ⁹ , and there was no difference between the two, while the tumor fluorescence of the mice treated with CAR-MUC1-T cells dropped to the level of 10 ⁸ , the tumor fluorescence of mice treated with CAR-MUC1-IL22-T cells decreased to 10 ⁷ levels, compared among the groups, there was no difference between the first two groups, but there was a significant difference between the latter two groups and the former two groups, and the fluorescence was significantly reduced , while the fluorescence of the CAR-MUC1-IL22-T cell therapy group was further decreased by an order of magnitude compared with the CAR-MUC1-T cell therapy group, and the difference was obvious;

In terms of tumor volume: the tumor volume of the first two groups was around 1500mm ³ , the tumor volume of the mice in the CAR-MUC1-T cell treatment group dropped to 350mm ³ , while the tumor volume of the mice treated with CAR-MUC1-IL22-T cells Reduced to 150mm ³ , that is: the CAR-MUC1-IL22-T cell therapy group had a clear and inhibitory effect on the tumors in mice, and the effect was clearly stronger than that of the first three groups.

From the above experimental results, it can be seen that the effect of CAR-T cell therapy is related to the expression of tumor cell surface antigens. The present invention is optimizing the existing CAR structure targeting MUC1. The optimized CAR-MUC1-IL22 structure has undergone CAR-MUC1-IL22-T cells constructed by lentivirus packaging and T cell infection can effectively induce the upregulation of MUC1 expression on the surface of tumor cells, thereby playing a better role in killing and controlling tumors, and can effectively avoid the occurrence of off-target effects .

SEQUENCE LISTING

<110> The First Affiliated Hospital of Zhengzhou University

<120> CAR and its application for inducing tumor cells to upregulate the expression of antigen MUC1

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<170> PatentIn version 3.5

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Claims (6)

1. A CAR that can induce tumor cells to up-regulate the expression of antigen MUC1, characterized in that the chimeric antigen receptor is an amino acid sequence connected by several protein fragments, named after MUC1-41BB-CD3z, specifically: sequentially connected human CD8a molecular signal peptide CMV, humanized MUC1 single-chain antibody scFV.MUC1, human CD8 molecular transmembrane region and 41BB molecular intracellular region 41BB, human CD3z molecular intracellular region CD3Zeta, namely: CMV-scFV.MUC1-41BB-CD3Zeta ; The human CD8a molecule signal peptide CMV has an amino acid sequence as shown in SEQ ID No.1, specifically: MALPVTALLLPLALLLHAARP; The humanized MUC1 single-chain antibody scFV.MUC1 has an amino acid sequence as shown in SEQ ID No.2, specifically: EVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSSGGGGSGGGGSGGGSDIVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDH LFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSE; The transmembrane region of the human CD8 molecule and the intracellular region 41BB of the 41BB molecule, wherein the amino acid sequence of the transmembrane region of the human CD8 molecule is shown in SEQ ID No.3, specifically: LETTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLLSVITLYC; The amino acid sequence of the intracellular region of the 41BB molecule is shown in SEQ ID No.4, specifically: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL; The amino acid sequence of the intracellular region CD3Zeta of the human CD3z molecule is shown in SEQ ID No.5, specifically: RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR. 2. The CAR capable of inducing tumor cells to upregulate the expression of antigen MUC1 as claimed in claim 1, wherein the intracellular region CD3Zeta of the human CD3z molecule is further connected to the IL22 CDS region sequence IL22 through a linking sequence; The IL22 CDS region sequence IL22, the amino acid sequence is shown in SEQ ID No.7, specifically: MAALQKSVSSFLMGTLATSCLLLLALLVQGGAAAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI. 3. The CAR capable of inducing tumor cells to upregulate the expression of antigen MUC1 according to claim 2, wherein the linking sequence is a TA2 linking sequence T2A; The amino acid sequence of the TA2 connecting sequence T2A is shown in SEQ ID No.6, specifically: EGRGSLLTCGDVEENPGP; Therefore, the overall structure of the formed CAR is: CMV-scFV.MUC1-41BB-CD3Zeta-T2A-IL22, named after MUC1-41BB-CD3z-IL22; The MUC1-41BB-CD3z-IL22 has an amino acid sequence as shown in SEQ ID No.8, specifically: MALPVTALLLPLALLLHAARPGSMALPVTALLLPLALLLHAARPEVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVVTQ ESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSEMALPVTALLLPLALLLLHAARPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRK KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPMAALQ KSVSSFLMGTLATSCLLLLALLVQGGAAAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI. 4. The lentiviral expression plasmid constructed by using the CAR capable of inducing tumor cells to upregulate the expression of antigen MUC1 according to any one of claims 1 to 3, characterized in that, it is specifically prepared through the following steps: (1) Obtain the coding sequence DNA of the CAR according to the central dogma; (2) Using the Lenti-2G plasmid as the expression vector, perform double digestion with BsrGI and Not I, and then use T4 DNA ligase to integrate and recombine the coding sequence DNA in step (1) into the Lenti-2G plasmid; (3) Transform the ligation product in step (2) into STABL3 competent cells, screen, expand and culture, and further extract the plasmid to obtain the recombined lentiviral expression plasmid. 5. The application of the lentiviral expression plasmid according to claim 4 in the preparation of anti-tumor agents, characterized in that, during application, CAR-T cells are prepared by transfection for application to increase the expression of MUC1 in tumor cells; The tumor is specifically HNSCC cancer, and the HNSCC cancer is specifically the corresponding cancer of human tongue squamous cell carcinoma cell line CAL33 or human pharyngeal squamous cell carcinoma cell line HN4; The specific application steps include the following steps: (1) Packaging and preparing lentivirus; (2) Obtain purified CD ³⁺ T cells; (3) T cell infection, in step (2) to activate CD ³⁺ T cells, add the virus and polybrene in step (1), and incubate overnight for infection; (4) Expand the T cells and test them for later use, incubate the infected T cells in step (3), recover and wash them, and then add the culture medium to further expand the T cells; test the expanded T cells for later use, or After further reinfusion, it can be used to stimulate and increase the expression of MUC1 in tumor cells. 6. The application of the lentiviral expression plasmid in the preparation of anti-tumor agents according to claim 5, characterized in that, in step (1), when packaging and preparing the lentivirus, 293T cells are used as the target cells to be transfected.

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