CN111234031B

CN111234031B - Novel chimeric antigen receptor targeting MUC1 on surface of tumor cell and preparation method of MUC1 chimeric antigen receptor T cell

Info

Publication number: CN111234031B
Application number: CN202010062409.1A
Authority: CN
Inventors: 顾海华; 吴广; 李红智; 赵灵洁; 秦雅倩; 曹佳薇
Original assignee: Wenzhou Medical University
Current assignee: Wenzhou Medical University
Priority date: 2020-01-20
Filing date: 2020-01-20
Publication date: 2022-03-08
Anticipated expiration: 2040-01-20
Also published as: CN111234031A

Abstract

The invention provides a novel chimeric antigen receptor of targeted MUC1 on the surface of a tumor cell and a preparation method of a MUC1 chimeric antigen receptor T cell, and solves the technical defects that in all current clinical tests, a MUC1 antibody can identify the MUC-1N end in free blood which is separated from the cell, so that the tumor cell cannot be effectively targeted and the MUC1 positive tumor cannot be treated in a targeted manner. The humanized HzMUC1 antibody in the preparation technology only recognizes MUC1 on the surface of a tumor cell, does not recognize the N end of MUC-1 dissociated in blood, and can effectively treat MUC1 positive tumors in a targeted mode. After the novel MUC1.28.BB.z CAR-T cell and the MUC1 positive tumor cell are co-cultured, the target cell killing effect is obvious, a foundation is laid for enhancing the tumor immunotherapy effect of the anti-MUC1 CAR-T cell, and the kit has practical guiding significance on the immunotherapy of MUC1 positive solid tumors.

Description

Novel chimeric antigen receptor targeting MUC1 on surface of tumor cell and preparation method of MUC1 chimeric antigen receptor T cell

Technical Field

The invention specifically relates to the technical field of biology, and specifically relates to a novel chimeric antigen receptor targeting MUC1 on the surface of a tumor cell and a preparation method of a MUC1 chimeric antigen receptor T cell.

Background

Mucin 1(MUC1) is a high molecular weight cell surface transmembrane glycoprotein, and MUC1 is overexpressed in primary malignant tumors such as breast cancer, pancreatic cancer, ovarian cancer, endometrial cancer, prostate cancer, bladder cancer, lung cancer, kidney cancer and the like. The MUC1 protein has been proved to play a key role in promoting the growth of tumor cells, enhancing the characteristics of stem cells, resisting drugs and the like, and is one of the important targets of biological treatment of tumors.

The MUC1 protein is cleaved at the GSVVVV motif in the echinospermin, enterokinase and aggregatin (SEA) domains of the extracellular domain to form two subunits, MUC1-N (alpha subunit) and MUC1-C (beta subunit). The MUC1-N terminus consists of a signal peptide, variable tandem repeat region (VNTR) and SEA domain. The VNTR region comprises 20 amino acids in 20 to 125 tandem repeats, with serine and threonine being potential O-glycosylation sites. Also, MUC1-N and MUC1-C also contain small amounts of N-glycosylation sites. MUC1-C consists of an extracellular domain of 58 amino acids, a 28 amino acid transmembrane domain, and a 72 amino acid cytoplasmic domain. Under normal physiological conditions, MUC1-N and MUC1-C heterodimers formed by non-covalent bonds localize to the plasma membrane (plasma membrane). During tumorigenesis and progression, a large amount of MUC1-N is shed from the cell surface, and the molecular mechanism of how MUC1-N is shed from the cell surface is not clear.

Antibodies targeting MUC1 have been developed for 35 years of history for tumor therapy, and to date there are no FDA-approved therapeutic antibody drugs targeting MUC1. The MUC1 antibody in all clinical trials recognized only the epitope in MUC1-N, and the clinical trial was not satisfactory because, among other reasons, MUC1-N tended to shed from the tumor cell surface and circulate freely in the extracellular matrix and peripheral blood, and free MUC1-N neutralized most of the MUC1 antibody, which limited the amount of antibody that could be targeted to MUC1 protein on the tumor cell surface.

A Chimeric Antigen Receptor (CAR) is a fusion protein consisting of an extracellular, single-chain antibody scFv that binds to a tumor-associated Antigen, a transmembrane region, and an intracellular activation motif for T cells. The CAR-T cell therapy is to collect T cells in peripheral blood of a patient through a blood separation technology, perform gene modification on the T cells in vitro, enable chimeric antigen receptor CAR for recognizing specific tumor antigens to be expressed on T cell membranes, and then transfuse the CAR-T cells into the patient, wherein the CAR-T cells are activated while specifically targeting the tumor cell surface antigens (without presenting major histocompatibility complex), and can be expanded under the stimulation of the tumor cells in vivo, so that the cancer cells are killed efficiently.

Because of the encouraging progress CAR-T cells have made in the treatment of hematological malignancies, two CAR-T therapies have been approved by the U.S. Food and Drug Administration (FDA). The first approved CAR-T cell therapy was kymeriah from novain (targeting CD19 antigen known as CTL019), originally approved for treatment of B-cell acute lymphoblastic leukemia (B-ALL) patients under 25 years of age, refractory or with two or more relapses, with Complete Remission (CR) reaching 60 to 90% in patients with relapsed/refractory B-cell ALL as assessed by preclinical efficacy. Subsequently, another approved therapy is anti-CD 19 CAR-t (yescata) cell therapy from Kite Pharma for the treatment of large B cell lymphoma patients who had received at least two additional treatments, but had not experienced remission, or had relapsed disease. The rate of complete remission of yescata from refractory B cell lymphoma patients in a multi-central clinical trial reached 51%, with some patients having remission lasting more than three years.

Recently, the FDA has further approved kymeriah for use in adult patients with two or more treatments and disease progression, or unresponsive relapsed or refractory large B-cell lymphoma, including diffuse large B-cell lymphoma (DLBCL), high grade B-cell lymphoma, and DLBCL caused by follicular lymphoma. In addition to ALL and DLBCL, CAR-T cells also showed good results in early clinical trials of other hematological malignancies, including anti-BCMA CAR-T cells for the treatment of multiple myeloma.

These clinical trials have clearly demonstrated the efficacy of CAR-T cells in the treatment of malignancies, and CAR-T cell immunotherapy has achieved encouraging results to date in the treatment of hematological malignancies such as B cell malignancies, but with poor remission rates in patients with solid tumors.

Some of the major challenges facing CAR-T cell immunotherapy in solid tumors include: (1) lack of target antigens unique to tumors, (2) only a limited number of CAR-T cells infiltrate into the tumor stroma, (3) tumor heterogeneity and loss of associated antigens, (4) suppression of immune cells by the tumor microenvironment, etc.

Therefore, the search for an ideal tumor target antigen and the development of a CAR-T cell preparation technology which has more targeting property and low side effect and can efficiently kill tumor cells provides a new effective therapy for treating solid tumors.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a novel chimeric antigen receptor of targeted MUC1 on the surface of a tumor cell and a preparation method of a MUC1 chimeric antigen receptor T cell, which only recognizes MUC1 on the surface of the tumor cell and does not recognize the MUC-1N end dissociating in blood, thereby being capable of more effectively treating MUC1 positive tumors in a targeted manner.

The technical solution adopted by the invention is as follows: the novel chimeric antigen receptor targeting the tumor cell surface antigen MUC1 consists of a single-chain antibody scFv which is positioned outside the cell and is combined with the tumor cell surface MUC1, a transmembrane region and an activation motif of a T cell positioned inside the cell, and a novel fusion gene anti-MUC1-scFv-CD28-CD137-CD3 zeta is constructed.

A preparation method of a novel chimeric antigen receptor T cell targeting a tumor cell surface antigen MUC1 comprises the following steps:

(1) designing MUC1.28.BB.z CAR gene, and constructing and identifying recombinant lentivirus eukaryotic expression plasmid;

(2) preparation of a novel anti-MUC 1CAR recombinant lentivirus;

(3) preparation of novel MUC1.28.BB.z CAR-T cells.

The design of MUC1.28.BB.z CAR gene, the construction and the identification of the recombinant lentivirus eukaryotic expression plasmid in the step (1) comprise the following steps:

(A) design of muc1.28.bb.z CAR fusion gene: the fusion gene structure is EcoRI-Kozak sequence-Ig kappa signal peptide-VL-linker-VH-CD 8 hige-CD 28 transmembrane region-CD 28 intracellular region-CD 137/4-1BB-CD3 zeta-BamH I, wherein the Ig kappa signal peptide, the hige of CD8 gene, the transmembrane region and intracellular region of CD28 gene, the sequences of CD137 gene and CD3 zeta gene refer to genebank (NCBI), and the VL and VH are variable region light chain and variable region heavy chain sequence parts in humanized MUC1 scFv antibody.

(B) Construction of lentivirus expression plasmid: the MUC1.28.BB. zCAR fusion gene DNA fragment is inserted into a PLVX-EF1 alpha-IRES. ZsGreen lentivirus expression plasmid after double enzyme digestion by EcoRI and BamHI, and the generated PLVX-EF1 alpha-anti-MUC 1 CAR. IRES. ZsGreen plasmid is verified by sequencing after double enzyme digestion identification by EcoR I and BamH I.

The step (2) of preparing the novel anti-MUC 1CAR recombinant lentivirus comprises the following steps:

(a) packaging of muc1.28.bb.z CAR recombinant lentivirus: inoculation of 7X10 in a 10cm Petri dish⁶293T17 cells are transfected by DMEM medium containing 1% penicillin-streptomycin and 8% fetal calf serum at 37 ℃ and 5% CO2 overnight after 16-24 hours when the coverage rate of the 293T17 cells in a 10cm dish reaches 70-80%, and 6mL of the culture solution without penicillin-streptomycin is replaced 2 hours before transfection, and the plasmid is packaged according to the target plasmid: the mass ratio of the enveloped plasmid is 4: 3: 1, adding recombinant lentivirus expression plasmids PLVX-EF1 alpha-anti-MUC 1 CAR. IRES. ZsGreen1, PsPAX2 and PMD2G into a 1.5mL centrifuge tube, adding a transfection reagent Polyethyleneimine (PEI), and adding a plasmid: PEI 1: 3, fully mixing, standing for 20min, dropwise adding the mixture into a 10cm dish, and culturing overnight at 37 ℃ under 5% CO 2. 5mL of fresh culture solution is replaced 16 hours after transfection;

(b) concentration and purification of muc1.28.bb.z CAR recombinant lentivirus: after 24 hours of fluid exchange, the first viral supernatant was collected in a 50mL centrifuge tube and 5mL of fresh medium was added to the transfected 293T17 cells. The virus supernatant was centrifuged at 3000rpm for 10 minutes at low speed to remove cell debris, filtered through a 0.45 μm low protein adsorption filter to remove impurities, and stored at 4 ℃ for virus concentration. After 24 hours, a second virus supernatant was collected and processed as described above, and the virus solution after the primary treatment was added to an ultracentrifuge tube containing 20% sucrose solution: the virus solution was centrifuged at 4 ℃ for 2 hours at 1:4,82700g, the supernatant was discarded, and the virus pellet was resuspended in 1/100 virus stock volume of RPMI-1640 medium. The concentrated virus solution was transferred to a 1.5mL centrifuge tube and centrifuged at 14000rpm for 1 minute for further purification. The supernatant is then aspirated and used to transduce cells, or stored in aliquots at-80 ℃.

(c) Titer determination of muc1.28.bb.z CAR recombinant lentivirus: spreading culture plate with 24 wells at a speed of 1.5X10 per well⁵293T17 cells, 500uL culture solutionThe method comprises the following steps of setting 8 holes in total, wherein 1 hole is used for counting cells before transduction, 1 hole is used for a non-transduced control hole, the other 6 holes are respectively used for diluting holes for Vector and MUC1.28.BB.z CAR virus transduction, transduction is carried out when the cell coverage rate is 30-40%, liquid is changed 24 hours after transduction, expression of green fluorescent protein GFP is observed under a fluorescence microscope 72 hours after transduction, and dilution multiple holes with the green fluorescent protein GFP expression positive rate of 5-20% are selected for flow detection.

The step (3) of preparing the novel MUC1.28.BB.z CAR-T cell comprises the following steps:

isolation of human Peripheral Blood Mononuclear Cells (PBMCs): drawing 200mL of peripheral blood, subpackaging the peripheral blood into 50mL centrifuge tubes, centrifuging each tube for 50mL and 700g for 20 minutes to remove the plasma as much as possible, adding PBS to 50mL, adding 25mL of Ficoll lymphocyte separation solution into the centrifuge tube, adding 900g of blood diluted by PBS into the centrifuge tube, centrifuging for 15 minutes, slowly ascending and descending the centrifuge tube, sucking the leucocyte into a new centrifuge tube after centrifuging, adding PBS, centrifuging and washing the cell for 500g, and centrifuging for 10 minutes. One part of the cell pellet was resuspended in RPMI-1640 culture medium, and the other part was resuspended in a freezing medium, DMSO: FBS ═ 1: 9, resuspension, split charging and storing at-80 ℃, and transferring into liquid nitrogen after overnight.

Activation and expansion of human peripheral blood T lymphocytes: adding 100. mu.L of CD2-Biotin, 100. mu.L of CD3-Biotin and 100. mu.L of CD28-Biotin into a 2mL centrifuge tube, mixing well, swirling the magnetic beads, fully suspending, collecting 500. mu.L of magnetic beads, and multiplying the volume by the volume of 1X 10⁸Adding 200 μ L of prepared buffer solution, placing in a shaking table at 4 deg.C for 2 hr to fully coat antibody, taking out after 2 hr, storing at 4 deg.C for 4 months, and collecting 5 μ L of coated magnetic beads 5 × 10⁵One was mixed with basal RPMI-1640 prepared in advance, centrifuged horizontally for 5 minutes at 300g, washed twice and then resuspended in 100. mu.L of T cell culture. Will be 1 × 10⁶The PBMC cells and the magnetic beads 2:1 are fully and uniformly mixed, inoculated into a 24-hole culture plate and cultured in an incubator at 37 ℃ for 2 days.

③ the detection of positive rate of T cell of peripheral blood of human transduced by recombined slow virus and MUC1.28.BB. z CAR: after the recombinant lentivirus transduces human peripheral blood T cells by different MOI values for 5-8 days, detecting the GFP positive rate of the cells by flow; adding the freshly prepared or frozen concentrated virus into a T cell culture hole which is activated for two days according to the MOI value of 20, adding 1500g of polybrene 1200-one with the final concentration of 8ug/ml, centrifuging at 37 ℃ for 2 hours, placing the cell culture hole in an incubator, changing the liquid after 16 hours, carrying out secondary transduction on human peripheral blood T cells by the recombinant lentivirus 24 hours after the first transduction, and detecting the GFP positive rate of the green fluorescent protein of the T cell by flow cells 5-8 days after the lentivirus transduces the human peripheral blood T cells under the same conditions as the first transduction.

In the step (c) of measuring the titer of the MUC1.28.BB.z CAR recombinant lentivirus, a culture solution adopts a DMEM culture medium, 8% fetal bovine serum and 1% penicillin-streptomycin.

In the activation and amplification of the human peripheral blood T lymphocytes, the optimal concentration of the activated T cells is 10 mug/ml of final antibody concentration.

And step two, adding fetal calf serum and EDTA into PBS with pH 7.2 to ensure that the final concentration of the fetal calf serum is 0.5 percent and the final concentration of the EDTA is 2mM, fully mixing, and storing at 4 ℃ to obtain the final product.

The T cell culture solution in the activation and amplification of the human peripheral blood T lymphocytes is prepared from GTT551 culture solution, 10 percent fetal calf serum, 300 IU/mLIL-2.

The invention has the beneficial effects that: the invention provides a novel chimeric antigen receptor of MUC1 on the surface of a targeted tumor cell and a preparation method of a MUC1 chimeric antigen receptor T cell, which solve the technical defects that in all current clinical tests, a MUC1 antibody can not effectively target tumor cells and treat MUC1 positive tumors in a targeted manner because of recognizing MUC-1N end in free blood caused by cell shedding, and a humanized HzMUC1 antibody only recognizes MUC1 on the surface of the tumor cell and does not recognize MUC-1N end in free blood in the preparation technology of the patent, so that MUC1 positive tumors can be more effectively treated in a targeted manner, after the novel MUC1.28. BB.z-T cell and the MUC1 positive tumor cell are co-cultured, the target cell efficacy is obvious, the foundation is laid for enhancing the tumor immunotherapy effect of anti-MUC 1-T cells, and the method has a practical significance for the immunotherapy of MUC1 positive solid tumors.

Drawings

FIG. 1 shows that the HzMUC1 antibody does not recognize MUC1 protein in peripheral blood of pancreatic cancer patients.

Figure 2 is a schematic diagram of the novel muc1.28.bb.z CAR design structure.

Figure 3 is a map of the recombinant lentiviral expression plasmid PLVX-EF1 α -anti-MUC1 car.

Figure 4 shows the result of DNA sequencing of the muc1.28.bb.z CAR fusion gene in the recombinant lentiviral expression plasmid and the different functional regions in the CAR encoded by the different DNA fragments in the fusion gene.

FIG. 5 is a comparison of cells under visible light and fluorescent microscope fields (100X) after co-transfection of 293T17 cells 16, 40 and 64 hours with the packaging plasmids PsPAX2 and PMD2G by the recombinant lentivirus expression plasmid PLVX-EF1 alpha-anti-MUC 1 CAR. IRES. ZsGreen 1.

FIG. 6 shows the result of the calculation of GFP positivity of green fluorescent protein and virus titer after the concentrated MUC1.28.BB. z CA lentivirus fluid is transduced into 293T17 cells by flow assay.

Figure 7 is the flow test of the green fluorescent protein GFP positive rate of T cells after transduction of T cells by the novel muc1.28.bb.z CAR recombinant lentivirus.

FIG. 8 shows the expression level of MUC1 in breast cancer cell lines detected by Western blot.

FIG. 9 FIG. 10 is the observation of the binding capacity of the novel MUC1.28.BB.z CAR-T cells to HCC1954 cells highly expressed by MUC1 and MB231 and Hs578T cells lowly expressed by HCC70 and MUC1.

FIG. 11 shows that the LDH method detection shows that the novel MUC1.28.BB.z CAR-T cell has high killing effect on breast tumor cells with high MUC1 expression.

Detailed Description

For a better understanding of the nature of the present invention, reference should be made to the following detailed description taken in conjunction with the accompanying drawings.

Design of MUC1.28.BB.z CAR gene, construction and identification of recombinant lentiviral eukaryotic expression plasmid

(A) Design of MUC1.28.BB.z CAR fusion gene

The structure of the novel anti-MUC1 chimeric antigen receptor has a fusion gene structure of EcoRI-Kozak sequence-Ig kappa signal peptide-VL-linker-VH-CD 8 hinge-CD28 transmembrane region-CD 28 intracellular region-CD 137/4-1BB-CD3 zeta-BamH I. Wherein the sequences of Ig kappa signal peptide, hinge of CD8 gene, transmembrane region and intracellular region of CD28 gene, CD137 gene and CD3 zeta gene are all referred to genebank (NCBI). VL and VH are the variable region light chain and variable region heavy chain sequence portions of the humanized MUC1 antibody scFv (see figure 2). The muc1.28.bb.z CAR fusion gene was chemically synthesized by sumizia jingzhi biotechnology limited.

(B) Construction of lentivirus expression plasmids

The DNA fragment of the MUC1.28.BB. zCAR fusion gene is inserted into a PLVX-EF1 alpha-IRES. ZsGreen lentivirus expression plasmid after double digestion by EcoRI and BamHI, and the generated PLVX-EF1 alpha-anti-MUC 1 CAR. IRES. ZsGreen plasmid is identified by double digestion of EcoR I and BamH I and then confirmed by sequencing by Jinzhi Biotech Co., Ltd, Suzhou (see FIG. 4).

Preparation of recombinant lentivirus

(a) Packaging of MUC1.28.BB.z CAR recombinant lentiviruses

Inoculation of 7X10 in a 10cm Petri dish⁶293T17 cells were cultured overnight in DMEM medium containing 1% penicillin-streptomycin and 8% fetal bovine serum at 37 ℃ in 5% CO 2. After 16-24 hours, transfection was performed when the coverage of 293T17 cells in 10cm dishes reached 70-80%. 6mL of penicillin-streptomycin-free culture medium was replaced 2 hours before transfection, and the plasmid: packaging plasmid: the mass ratio of the enveloped plasmid is 4: 3: 1, adding recombinant lentivirus expression plasmids PLVX-EF1 alpha-anti-MUC 1 CAR. IRES. ZsGreen1, PsPAX2 and PMD2G into a 1.5mL centrifuge tube, adding a transfection reagent Polyethyleneimine (PEI) (plasmid: PEI 1: 3), mixing uniformly, dropwise adding the mixture into a 10cm dish, and culturing at 37 ℃ and 5% CO2 overnight. The medium was changed 16 hours after transfection and 5mL of fresh medium was added.

(b) Concentration and purification of MUC1.28.BB.z CAR recombinant lentivirus

(1) Collecting recombinant lentivirus and primary purification

After 24 hours of fluid exchange, the first viral supernatant was collected in a 50mL centrifuge tube and 5mL of fresh medium was added to the transfected 293T17 cells. The virus supernatant was centrifuged at 3000rpm for 10 minutes at low speed to remove cell debris, filtered through a 0.45 μm low protein adsorption filter to remove impurities, and stored at 4 ℃ for virus concentration. After 24 hours, a second viral supernatant was collected and treated as described above.

(2) Ultracentrifugation concentration and further purification of recombinant lentiviruses

The virus solution thus treated was put into an ultracentrifuge tube containing 20% sucrose solution (sucrose: virus solution: 1:4),82700g was centrifuged at 4 ℃ for 2 hours. The supernatant was discarded and the viral pellet was resuspended in 1/100 volumes of RPMI-1640 medium. The concentrated virus solution was transferred to a 1.5mL centrifuge tube and centrifuged at 14000rpm for 1 minute for further purification. The supernatant is then aspirated and used to transduce cells, or stored in aliquots at-80 ℃.

(c) Titer determination of muc1.28.bb.z CAR recombinant lentivirus

24-hole culture plate with 1.5X10 per hole⁵293T17 cells, 500uL medium (DMEM medium +8% fetal bovine serum +1% penicillin-streptomycin). A total of 8 wells were set, 1 well was used as pre-transduction cell count, 1 well was used as untransduced control well, and the remaining 6 wells were used as Vector and muc1.28.bb.z CAR virus-transduced multiple dilution wells, respectively. And (3) performing transduction when the cell coverage rate is 30-40%, changing the liquid 24 hours after the transduction, observing the expression of the green fluorescent protein GFP under a fluorescence microscope 72 hours after the transduction, and selecting a dilution multiple hole with the green fluorescent protein GFP expression positive rate of 5-20% for flow detection.

Titers were calculated as (P × N)/(D × V). T: titer (TU/ml), P: GFP positive cells%, N: number of cells per well at transduction, D: dilution factor of added virus, V: diluted virus volume added per well.

Preparation of novel MUC1.28.BB.z CAR-T cells

Isolation of human Peripheral Blood Mononuclear Cells (PBMC)

200mL of peripheral blood of healthy volunteers was collected by a disposable blood collection bag (Shandongwei GaoJi medical Polymer products, Ltd.). The mixture was dispensed into 50mL centrifuge tubes, 50mL and 700g per tube, centrifuged for 20 minutes to remove the plasma as much as possible, and PBS was added to 50 mL. Adding 25mL of Ficoll lymphocyte separation solution into a centrifuge tube, adding 25mL of blood diluted by PBS, centrifuging for 15 minutes at 900g, and centrifuging to slowly rise and fall. After centrifugation, the tunica albuginea mononuclear cells are sucked into a new centrifuge tube, PBS is added for centrifugation, and the cells are washed for 500g and 10 minutes. One part of the cell pellet was resuspended in RPMI-1640 culture medium, the other part was resuspended in freezing medium (DMSO: FBS ═ 1: 9), and the pellet was stored at-80 ℃ overnight and then transferred to liquid nitrogen.

② activation and amplification of human peripheral blood T lymphocytes

(1) Magnetic bead coating of activated T cells

mu.L of CD2-Biotin, 100. mu.L of CD3-Biotin, 100. mu.L of CD28-Biotin (May. Tian, Germany, and whirlwind Co.) were added to a 2mL centrifuge tube and mixed well (the final antibody concentration was 10. mu.g/mL, which is the optimal concentration for activating T cells). The beads were vortexed and resuspended thoroughly, 500. mu.L of beads (1X 10)⁸) To this solution, 200. mu.L of a prepared buffer (buffer: fetal bovine serum, EDTA in PBS (pH 7.2) to give a final concentration of 0.5% fetal bovine serum and 2mM EDTA, mixing well and storing at 4 ℃) was added, and the volume was adjusted to 1 mL. The antibody was fully coated by shaking at 4 ℃ for 2 hours. The product is taken out after 2 hours and can be stored at 4 ℃ for 4 months.

(2) Activation of human peripheral blood T cells (to stimulate 1X 10)⁶Example of PBMC cell

Taking 5 μ L (5X 10) of coated magnetic beads⁵Individually) and pre-prepared basal RPMI-1640, horizontally centrifuged for 300g for 5min, washed twice and then resuspended in 100. mu.L of T cell culture medium (GTT551 medium +10% fetal bovine serum +300 IU/mLIL-2). Will be 1 × 10⁶The PBMC cells and the magnetic beads 2:1 are fully and uniformly mixed, inoculated into a 24-hole culture plate and cultured in an incubator at 37 ℃ for 2 days.

③ detection of positive rate of recombinant lentivirus transduced human peripheral blood T cell and MUC1.28.BB.z CAR

(1) Optimization of recombinant lentivirus transduction

After 5-8 days after the recombinant lentivirus transduces human peripheral blood T cells with different MOI values (5, 10, 15, 20, 25), the green fluorescent protein GFP positive rate of the cells is detected by flow.

(2) Transduction of human peripheral blood T cells by recombinant lentiviruses

The freshly prepared or frozen concentrated virus was taken, added to T cell culture wells which had been activated for two days at an MOI value of 20, and polybrene was added to a final concentration of 8. mu.g/mL at 1200. sup. 1500g, centrifuged at 37 ℃ for 2 hours, and then placed in an incubator. The solution was changed after 16 hours, and 24 hours after the first transduction, human peripheral blood T cells were transduced with the recombinant lentivirus twice under the same conditions as the first transduction.

(3) Detection of human peripheral blood T cell MUC1.28.BB.z CAR positivity

5-8 days after the lentivirus transduces the human peripheral blood T cells, the positive rate of the green fluorescent protein GFP of the T cells is detected by flow cytometry.

Four, detection of efficacy of novel MUC1.28.BB.z CAR-T cells in killing breast tumor cells in vitro

1. Novel MUC1.28.BB.z CAR-T cell and target cell co-culture detection binding capacity

Vector-T (i.e., empty Vector virus-infected T cells), anti-MUC1 CAR-T (MUC1.28.BB. z CAR virus-infected T cells) were co-cultured with target cells HCC1954 (HER 2 positive breast cancer cells highly expressing MUC1), HCC70 (triple negative breast cancer cells highly expressing MUC1), MB231 (triple negative breast cancer cells lowly expressing MUC1), Hs578T cells (triple negative breast cancer cells in which MUC1 expression was not detected), and MCF10A (normal mammary epithelial cells), respectively, for 18 hours, and the binding ability of MUC1.28.BB. z-T cells to the target cells was observed using an inverted phase contrast microscope.

2. LDH method for detecting killing effect of MUC1.28.BB.z CAR-T cells on target cells

The method comprises the steps of inoculating target cells in a 96-well culture plate, setting a cell-free culture solution (a background empty control well), untreated control cells (a sample control well), untreated subsequent lysed cells (a sample maximum enzyme activity control well) and an experimental group (different effect-target ratio), and detecting the killing effect of MUC1.28.BB.z CAR-T cells on tumor cells under the condition of different effect-target ratios by using a cytoxicity LDH Assay Kit of Dongnren chemical technology Co., Ltd.

The experimental results are as follows:

FIG. 1 anti-MUC1 antibody (HzMUC1) did not recognize MUC1 protein in peripheral blood of pancreatic cancer patients. MUC1 high-expression breast cancer cell strain ZR75-1 to obtain cell lysate; lane 1, normal human peripheral blood serum; lane 2, serum from pancreatic cancer patients. (A) The expression of MUC1-N terminal protein was detected by Western blotting using an anti-MUC 1-NT antibody commonly used in the research field. (B) Immunoprecipitation was performed using anti-MUC 1-NT antibody and HzMUC1 antibody, and Western blotting was performed using anti-MUC 1-NT antibody to examine the recognition of free MUC1 protein in human peripheral blood by anti-MUC1 antibody.

The results show that: free MUC1-N terminus was present in the serum of pancreatic cancer patients, and ZR75-1 cells expressed MUC1-N terminus (A). The results of immunoprecipitation experiments showed that anti-MUC 1-NT antibody bound to MUC1 expressed by ZR75-1 cells and MUC1 in the serum of pancreatic cancer patients, whereas anti-MUC1 antibody bound only to MUC1 expressed by ZR75-1 cells and did not bind to MUC1(B) in the serum of pancreatic cancer patients.

FIG. 5 comparison of visible light and fluorescent light microscopic cells (100X) after cotransfection of 293T17 cells 16, 40, 64 hours with the packaging plasmid, recombinant lentivirus plasmid pLVX-anti-MUC1 CAR. IRES. ZsGreen 1. Wherein, the 1 st and 3 rd columns are visible light field pictures, the 2 nd and 4 th columns are fluorescence field pictures, the 1 st and 2 nd columns are the same field of view, the 3 rd and 4 th columns are the same field of view, and Bar is 200 um.

Figure 6 flow cytometry detects the green fluorescent protein GFP positivity and calculation of virus fluid titer after concentrated muc1.28.bb.z CAR lentivirus fluid transduction of 293T17 cells. In the first row 293T17 cells were infected with 0.7. mu.L Vector virus and the number of cells counted before transduction was 3.5X10⁵The GFP positive rate was 27.9%, so the titer calculation result was 1.4X10⁸Tu/mL, second row infected 293T-17 cells with 0.7. mu.L of MUC1.28.BB. z CAR Virus, and the number of cells counted before transduction was 3.5X10⁵The GFP positive rate is 19.3%, so the titer calculation result is 9.6x10⁷Tu/mL。

Figure 7 novel muc1.28.bb.z CAR recombinant lentivirus transduced T cells followed by flow detection of the GFP positive rate of muc1.28.bb.z CAR-T cells. The GFP positive rate of Vector-T cells was 38%, and the GFP positive rate of MUC1.28.BB. z CAR-T cells was 30%.

Figure 9 binding capacity of novel MUC1.28.bb.z CAR-T cells to HCC1954 cells (HER 2 positive breast cancer cells highly expressing MUC1), MB231 cells (triple negative breast cancer cells lowexpressing MUC1) was observed (200X). Vector-T cells and MUC1.28.BB.z CAR-T cells were cultured at 2:1 and 4:1 and HCC1954, MB231 cells were co-cultured for 18 hours and photographed by inverted phase contrast microscope observation. The effective target ratio is the ratio of the number of T cells to the number of tumor target cells. The 1 st column shows the co-culture photographs of Vector-T and HCC1954, the 2 nd column shows the co-culture photographs of MUC1.28.BB. z CAR-T and HCC1954, the 3 rd column shows the co-culture photographs of Vector-T and MB231, and the 4 th column shows the co-culture photographs of MUC1.28.BB. z CAR-T and MB 231. The first horizontal alignment target ratio is 2:1, the second horizontal alignment target ratio is 4:1, and the scale is 100 μm.

The results show that: inverted phase contrast microscope observation proves that MUC1.28.BB.z CAR-T cells can be combined with MUC1 high-expression target cells HCC1954 to form a cluster relatively tightly, but are not combined with MUC1 low-expression cells MB231 to form a cluster obviously.

Figure 10 observation of the binding capacity of novel MUC1.28.bb.z CAR-T cells to HCC70 cells (triple negative breast cancer cells highly expressing MUC1), Hs578T cells (triple negative breast cancer cells not detecting MUC1 expression) (200X). Vector-T cells and MUC1.28.BB.z CAR-T cells were cultured at 8:1 and HCC70, Hs578T cells were co-cultured for 18 hours and photographed by inverted phase contrast microscope observation. The upper left is the photograph of co-culture of Vector-T with HCC70, the upper right is the photograph of co-culture of MUC1.28.BB. z CAR-T cells with HCC1954, the lower left is the photograph of co-culture of Vector-T with Hs578T, and the lower right is the photograph of co-culture of MUC1.28.BB. z CAR-T cells with Hs 578T. The effective target ratio is 8:1, and the scale is 100 μm.

The results show that: inverted phase contrast microscope observation proves that MUC1.28.BB.z CAR-T cells can be tightly combined with a MUC1 high-expression target cell HCC70 to form a cluster, but are not obviously combined with MUC1 negative cells Hs578T to form a cluster.

The detection of the LDH method in figure 11 shows that the novel MUC1.28.BB.z CAR-T cell has high killing effect on breast tumor cells with high MUC1 expression. Vector-T cells and MUC1.28.BB.z CAR-T cells were cultured at 4:1 and 8:1 in co-culture with HCC1954, MB231, MCF10A cells for 18 hours (upper panel), or at a ratio of 4: 1-effect target ratio was co-cultured with Hs578T, HCC70 cells for 18 hours (lower panel), and then the efficacy of T-cell killing tumor cells was quantitatively determined using the LDH method. HCC 1954: HER2 positive breast cancer cells highly expressing MUC 1; HCC 70: triple negative breast cancer cells highly expressing MUC 1; MB 231: TNBC-type breast cancer cells that underexpress MUC 1; hs 578T: TNBC-type breast cancer cells in which MUC1 expression was not detected; MCF10A normal mammary epithelial cells; **: p < 0.01, x: p is less than 0.001.

The results show that: the killing efficiency of target cells is obvious (70-80% killing rate) after the MUC1.28.BB.z CAR-T cells are co-cultured with the MUC1 high-expression target breast cancer cells HCC1954 and HCC70 cells, but the killing efficiency of the target cells is not obvious after the co-culture with MB231 with low MUC1 expression, a MUC1 negative Hs578T breast cancer cell strain and normal breast epithelial cells MCF 10A. In addition, Vector-T cells have no obvious killing effect on breast cancer cell lines with low and high MUC1 expression and normal breast epithelial cells.

The skilled person should understand that: although the invention has been described in terms of the above specific embodiments, the inventive concept is not limited thereto and any modification applying the inventive concept is intended to be included within the scope of the patent claims.

The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may occur to those skilled in the art without departing from the principle of the invention, and are considered to be within the scope of the invention.

Sequence listing

<110> Wenzhou university of medical science

<120> novel chimeric antigen receptor targeting MUC1 on tumor cell surface and preparation method of MUC1 chimeric antigen receptor T cell

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Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr

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Cys Ala Ser Ser Thr Ala Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu

245 250 255

Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro

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Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys

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

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

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Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys

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

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Claims

1. A novel chimeric antigen receptor targeting a tumor cell surface antigen MUC1 is characterized in that the novel chimeric antigen receptor targeting the tumor cell surface antigen MUC1 consists of a single-chain antibody scFv which is positioned outside a cell and binds to a tumor cell surface antigen MUC1, a transmembrane region and an activation motif of a T cell positioned inside the cell, a novel fusion gene anti-MUC1-scFv-CD28-CD137/4-1BB-CD3 zeta is constructed, and the sequence of the novel chimeric antigen receptor is shown as SEQ ID NO: 1 is shown.

2. The method for preparing the novel chimeric antigen receptor T cell targeted to MUC1 on the surface of tumor cells according to claim 1, comprising the following steps:

(2) preparing a novel anti-MUC 1CAR recombinant lentivirus;

(3) preparation of novel MUC1.28.BB.z CAR-T cells.

3. The method for preparing a novel chimeric antigen receptor T cell targeting tumor cell surface antigen MUC1 according to claim 2, wherein the step (1) of designing MUC1.28.BB. z CAR gene, constructing and identifying recombinant lentiviral eukaryotic expression plasmid comprises the following steps:

(A) design of muc1.28.bb.z CAR fusion gene: the fusion gene structure is EcoRI-Kozak sequence-Ig kappa signal peptide-VL-linker-VH-CD 8 hige-CD 28 transmembrane region-CD 28 intracellular region-CD 137/4-1BB-CD3 zeta-BamH I, wherein the Ig kappa signal peptide, the hige of CD8 gene, the transmembrane region and intracellular region of CD28 gene, the sequences of CD137/4-1BB gene and CD3 zeta gene are all referred to Genebank (NCBI), and VL and VH are variable region light chain and variable region heavy chain sequence parts in humanized MUC1 antibody scFv;

(B) construction of lentivirus expression plasmid: the MUC1.28.BB. zCAR fusion gene DNA fragment is inserted into a PLVX-EF1a-IRES. ZsGreen lentivirus expression plasmid after double enzyme digestion by EcoRI and BamHI, and the generated PLVX-EF1a-anti-MUC1 CAR. IRES. ZsGreen plasmid is verified by sequence after double enzyme digestion identification by EcoR I and BamH I.

4. The method for preparing a novel chimeric antigen receptor T cell targeting tumor cell surface antigen MUC1 according to claim 2, wherein the step (2) of preparing the novel anti-MUC 1CAR recombinant lentivirus comprises the following steps:

(a) packaging of muc1.28.bb.z CAR recombinant lentivirus: spreading 7X10 in 10cm culture dish⁶293T17 cells are transfected by DMEM medium containing 1% penicillin-streptomycin and 8% fetal calf serum at 37 ℃ and 5% CO2 overnight after 16-24h and when the coverage rate of the 293T17 cells in a 10cm dish reaches 70-80%, the cells are replaced by 6mL of the penicillin-streptomycin-free culture solution 2h before transfection, and plasmids are packaged according to target plasmids: the mass ratio of the enveloped plasmid is 4: 3: 1, adding recombinant lentivirus expression plasmids PLVX-EF1a-anti-MUC1 CAR. IRES. ZsGreen1, PsPAX2 and PMD2G into a 1.5mL centrifuge tube, adding a transfection reagent Polyethyleneimine (PEI), and adding a plasmid: PEI =1: 3, after fully and uniformly mixing, dropwise adding the mixture into a 10cm dish, culturing overnight at 37 ℃ under 5% CO2, changing the culture solution 16 hours after transfection, and adding 5ml of fresh culture solution;

(b) concentration and purification of muc1.28.bb.z CAR recombinant lentivirus: after 24h of liquid change, collecting the first virus supernatant in a 50mL centrifuge tube, adding 5mL of fresh culture solution in transfected 293T17 cells, centrifuging the virus supernatant at 3000rpm for 10min at low speed to remove cell debris, filtering with a 0.45um low protein adsorption filter membrane to remove impurities, storing at 4 ℃, concentrating the virus to be performed, collecting the second virus supernatant after 24h for the same treatment as above, adding the virus solution after the primary treatment into an ultracentrifuge tube containing 20% of sucrose solution, and adding sucrose: centrifuging the virus solution =1:4,82700g at 4 ℃ for 2h, discarding the supernatant, resuspending the virus pellet in RPMI-1640 culture medium with 1/100 virus stock solution volume, transferring the concentrated virus solution into a 1.5mL centrifuge tube, centrifuging at 14000rpm for 1 minute, further purifying, and sucking the supernatant to be used for transducing cells or subpackaging and storing at-80 ℃ for storage;

(c) titer determination of muc1.28.bb.z CAR recombinant lentivirus: laying 7X10 in each hole of 24-hole culture plate⁴293T-17 cells and 500uL culture solution are arranged in 8 holes in total, wherein 1 hole is used for counting the cells before transduction, 1 hole is used as a non-transduced control hole, the other 6 holes are respectively used as multiple dilution holes for Vector and MUC1.28.BB.z CAR virus transduction, transduction is carried out when the cell coverage rate is 30-40%, the solution is changed 24h after transduction, the expression of green fluorescent protein GFP is observed under a fluorescence microscope 72h after transduction, and the dilution multiple holes with the green fluorescent protein GFP expression positive rate of 5-20% are selected for flow detection.

5. The method for preparing a novel chimeric antigen receptor T cell targeting tumor cell surface antigen MUC1 according to claim 2, wherein the step (3) of preparing a novel MUC1.28.BB.z CAR-T cell comprises the following steps:

isolation of human Peripheral Blood Mononuclear Cells (PBMCs): drawing 200mL of peripheral blood, subpackaging the peripheral blood into 50mL centrifuge tubes, centrifuging each tube for 50mL and 700g for 20min to remove plasma as much as possible, adding PBS to 50mL, adding 25mL of Ficoll lymphocyte separation liquid into the centrifuge tube, adding 25mL of blood diluted by PBS for 900g, centrifuging for 15min, slowly ascending and descending for centrifugation, sucking tunica albuginea mononuclear cells into a new centrifuge tube after centrifugation, adding PBS for centrifugally washing the cells for 500g and 10min, resuspending a part of cell sediment by RPMI-1640 culture solution, and resuspending the other part by freezing solution, wherein DMSO is FBS =1: 9, resuspending, subpackaging and storing at-80 ℃, and transferring into liquid nitrogen after overnight;

activation and expansion of human peripheral blood T lymphocytes: adding 100ml of CD2-Biotin, 100ml of CD3-Biotin and 100ml of CD28-Biotin into a 2ml centrifuge tube, fully mixing, whirling the magnetic beads, fully suspending the magnetic beads, and taking 500ml of magnetic beads, namely 1 multiplied by 10⁸Adding 200ml of prepared buffer solution, shaking at 4 deg.C for 2 hr to fully coat antibody, taking out after 2 hr, storing at 4 deg.C for 4 months, and taking coated 5ml of magnetic beads 5 × 10⁵Mixing with prepared basal RPMI-1640, horizontally centrifuging for 5min at 300g, washing twice, resuspending magnetic beads with 100 μ L of T cell culture solution, and mixing with the supernatant⁶Fully and uniformly mixing PBMC cells and magnetic beads 2:1Inoculating into 24-well culture plate, 37^oC, culturing for 2 days in an incubator;

③ the detection of positive rate of T cell of peripheral blood of human transduced by recombined slow virus and MUC1.28.BB. z CAR: after the recombinant lentivirus transduces human peripheral blood T cells by different MOI values for 5-8 days, detecting the GFP positive rate of the cells by flow; adding the freshly prepared or frozen concentrated virus into the T cell culture wells which have been activated for two days according to the MOI value of 20, and adding 1500g of polybrene 1200-1500g with the final concentration of 8mg/ml^oAnd C, centrifuging for 2h, then placing in an incubator, changing the liquid after 16h, carrying out secondary transduction on the human peripheral blood T cells by the recombinant lentivirus 24h after the first transduction, and detecting the positive rate of the Green Fluorescent Protein (GFP) of the T cells by flow cells after 5-8 days of the transduction of the human peripheral blood T cells by the lentivirus under the same conditions of the first transduction.

6. The method for preparing a novel chimeric antigen receptor T cell targeting tumor cell surface antigen MUC1 of claim 4, wherein in the step (c) of measuring the titer of MUC1.28.BB. z CAR recombinant lentivirus, DMEM medium +8% fetal bovine serum +1% penicillin-streptomycin is used as the culture medium.

7. The method of claim 5, wherein the optimal concentration of activated T cells in the activation and expansion of T lymphocytes in human peripheral blood is 10mg/ml antibody final concentration.

8. The method for preparing a novel chimeric antigen receptor T cell targeting tumor cell surface antigen MUC1 of claim 5, wherein the buffer solution for activating and amplifying human peripheral blood T lymphocytes is prepared by adding fetal bovine serum and EDTA into PBS with pH =7.2 to make the final concentration of the fetal bovine serum 0.5% and the final concentration of the EDTA 2mM, mixing them thoroughly and storing at 4 ℃.

9. The method of claim 5, wherein the step of activating and amplifying T lymphocytes in human peripheral blood comprises the step of preparing a culture solution of T cells from GTT551 culture solution +10% fetal bovine serum +300 IU/mLIL-2.