CN117736336A - BiTE-secreting CAR-T cells and uses thereof - Google Patents

BiTE-secreting CAR-T cells and uses thereof Download PDF

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CN117736336A
CN117736336A CN202211150670.2A CN202211150670A CN117736336A CN 117736336 A CN117736336 A CN 117736336A CN 202211150670 A CN202211150670 A CN 202211150670A CN 117736336 A CN117736336 A CN 117736336A
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万晓春
刘茂玄
陈有海
王琪
许晨光
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Shenzhen Institute of Advanced Technology of CAS
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Abstract

The invention discloses a BiTE-secreting CAR-T cell and application thereof, in particular discloses a BiTE-secreting chimeric antigen receptor, which comprises a leader peptide, a nano antibody targeting mesothelin, an extracellular hinge region, a transmembrane region, an intracellular signal region, a P2A connecting peptide, an Ig kappa leader peptide, a NKG2D ligand-targeting protein, a connecting peptide and a CD 3-targeting single-chain antibody which are sequentially connected from an amino end to a carboxyl end, and further discloses the BiTE-secreting CAR-T cell as a T cell for expressing the chimeric antigen receptor. The CAR-T cells secreting the BiTE can promote the killing effect of the T cells, reduce the recurrence rate of the CAR-T cells for treating tumors, improve the treatment effect, find a new treatment method and an optimal treatment target for solid tumors, and improve the curative effect of the current CAR-T cell therapy on triple negative breast cancer.

Description

BiTE-secreting CAR-T cells and uses thereof
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to a CAR-T cell secreting BiTE and application thereof.
Background
Tumors such as triple negative breast cancer have the characteristics of high invasiveness, easiness in transfer and recurrence, poor prognosis and the like, and compared with other types of breast cancer, the core symptom of difficult treatment is that an effective treatment target is lacking at present. The current surgical treatment has a large limitation, and only the target tumor block is resected, so that the healing effect is achieved. However, for the spread tumor, the surgical excision does not solve the problem well, and the best curative effect is possible only by combining other methods for the treatment. More current tumor treatments rely on chemoradiotherapy and have no suitable effective therapeutic targets and treatment schemes. However, no suitable targeting drug for treating solid tumor cancer exists at present, and only systemic chemotherapy can be adopted. Docetaxel (DTX), for example, belongs to taxane antitumor drugs, and can inhibit the mitosis process of tumor cells by promoting microtubule polymerization in M phase, thereby achieving the purpose of antitumor. However, the ubiquitous resistance of tumor cells to chemotherapeutic agents can result in primary or secondary DTX resistance that can lead to chemotherapy failure, affecting patient prognosis. The other reported anthracyclines, mTOR inhibitors, src amino acid kinase inhibitors and vascular endothelial growth factor inhibitors all report a certain curative effect on triple negative breast cancer, but have no conclusion on the long-term survival effect of patients. Currently, tumors, especially solid tumors, still lack definite therapeutic methods and effective drugs.
The CAR-T targeting anti-tumor cell immune technology is characterized in that chimeric antigen receptors comprising an antibody for recognizing a specific target of cancer, a hinge region, a transmembrane region, an intracellular signal region (immune receptor tyrosine activation motif, ITAM) and a co-stimulatory molecule CD28 and CD137 (4-1 BB) conduction domain of the intracellular signal region are expressed in a lentiviral vector, and the vector is transfected into autologous T cells, so that the modified CAR-T cells have targeting, can specifically recognize and kill cells expressing the specific antigen, and can proliferate and activate in vivo. There are a number of CAR-T studies currently in use in the treatment of solid tumors. For example, li et al published a report that their in vitro experiments demonstrated that EphA 2-targeted CAR-T cells could lyse EphA 2-positive lung cancer cells. Louis et al treated 19 neuroblastomas with GD 2-targeted CAR-T cells, with complete remission obtained in 3 patients and no serious adverse effects. Beatty et al in 2018 also reported that some refractory metastatic pancreatic cancer patients received mesothelin-targeted CAR-T cell therapy. Recently, gianpiretro led team successfully screened a suitable target for CAR-T cells in solid tumor therapy-B7-H3, the team successfully constructed B7-H3.CAR-T cells co-activated with 4-1BB, and the construction was demonstrated in mouse experiments to increase the activity of T cells, and the efficacy of tumor immunotherapy; in addition, researchers find that the T cell can also reduce the expression quantity of PD-1 in the cell, and has better effect of treating tumors positive to PD- (L) 1. No side effect is detected in the subsequent mouse experiments, and the safety is guaranteed to a certain extent. In addition, there are a variety of CAR-T that are clinically treated for triple negative breast cancers, such as CAR-T targeting MUC1, c-Met, ROR1, mesothelin, NKG2D, and the like. The NKG2D CAR-T has been reported to be used in the study of the triple-negative breast cancer, and the data show that the ligand of the NKG2D is widely expressed on the triple-negative breast cancer cell line, and the NKG2D CAR-T has good killing effect on the triple-negative breast cancer cell line in vitro experiments and in-vivo experiments of mice. In addition, the Mesothelin target is specifically and highly expressed in triple negative breast cancer, and researchers report that the Mesothelin-targeted CAR-T cells have a certain curative effect on the triple negative breast cancer. Although CAR-T cell therapy has achieved great success in the treatment of hematological cancers, it has not been effective for solid tumors. One of the main reasons is the antigenic heterogeneity of solid tumors, which leads to easy antigen escape of single-target CAR-T, thereby generating drug resistance to the single-antigen-target CAR-T and improving the recurrence rate of tumors.
Disclosure of Invention
In order to solve the defects of single antigen target CAR-T in the prior art in tumor treatment, the invention aims to provide a CAR-T cell secreting BiTE and application thereof.
Mesothelin (Mesothelin) is a tumor-associated antigen that is overexpressed in most malignant pleural mesothelioma, pancreatic cancer, ovarian cancer, and some lung cancers. Although mesothelin has limited expression in normal tissues, it is expressed at low levels on normal peritoneal, pleural and pericardial mesothelial surfaces. In addition, mesothelin is a target point of endogenous immune response in pleural mesothelioma, ovarian cancer and pancreatic cancer, and the target point is better in immunogenicity. Clinical trials targeting mesothelin-overexpressed tumors using antibody-based strategies have shown preliminary safety and potential efficacy, with only serositis, a toxicity of the targeted extra-tumor antigen determined to be dose-dependent, this target safety is excellent. More importantly, the Mesothelin target is specifically and highly expressed in triple negative breast cancer rather than non-triple negative breast cancer, and the high expression and the progress of the triple negative breast cancer are positively correlated, so that the Mesothelin target can be used as a potential CAR-T target of the triple negative breast cancer, and the safety of the target in a human body is good.
NKG2D protein is a C-type lectin-like glycoprotein, widely expressed in human CD8 + T cells, NK cells, gamma delta T cells and CD4 + T cell subpopulation surface. NKG2D is an activated receptor and plays an important role in the activation of NK cells. However, it cannot directly activate T cells, but only provides a co-stimulatory signal to participate in T cell activation. The NKG2D receptor can recognize ligand proteins with various structures, and is mainly divided into MHC-I molecule related molecules A/B, namely MICA/MICB ligand proteins and MHC-I molecule related molecules 6 UL16 binding proteins, namely ULBP1-6 ligand proteins. Since two major classes of ligands for NKG2D are generally expressed little or very poorly on normal cells, but expression regulation is mainly elevated at the surface of various tumor cells or at the time of viral infection, it is considered to be an expression of stress response in the body. When the NKG2D protein on the immune cells in vivo is combined with the NKG2D ligand cells which express the related NKG2D, the immune cells can be rapidly activated to trigger a series of effect reactions, thereby exerting the cytoxic effect,eventually killing the cells. The NKG2D CAR has better killing power and targeting effect on tumor cells which highly express the ligand, such as prostate cancer, ovarian cancer, lymphoma and the like. In addition, studies have demonstrated that NKG2DCAR can recognize tumor vascular endothelial cells in the tumor microenvironment in addition to tumor cells.
The "BiTE" technique, a Bispecific T-cell engager, uses the VH-VL segment of two-segment specific antibodies as two "binding arms," one arm designed to bind CD3 to bind and activate T cells, and the other arm to tumor antigens.
In order to reduce the recurrence rate of the CAR-T cells for treating tumors and improve the treatment effect, the invention utilizes the modern genetic engineering technology to express the Mesothelin VHH on human killer T cells and secrete 'BiTE', prepares Mesothelin CART-BiTE cells, and returns the Mesothelin CART-BiTE cells to a patient, so that the cancer cells can be killed efficiently and specifically, and the invention can be used for improving the treatment effect of the current immune cell therapy on the tumors.
The specific technical scheme of the invention is as follows:
the first aspect of the present invention provides a chimeric antigen receptor secreting BiTE, comprising a leader peptide, a nanobody targeting mesothelin, an extracellular hinge region, a transmembrane region, an intracellular signal region, a P2A linker peptide, an Ig kappa leader peptide, a protein targeting NKG2D ligand, a linker peptide, a single chain antibody targeting CD3, linked in sequence from amino terminus to carboxy terminus.
Further, the mesothelin-targeting nanobody comprises an amino acid sequence shown as SEQ ID NO. 1;
the protein targeting the NKG2D ligand comprises an amino acid sequence shown as SEQ ID NO. 3;
the CD3 targeting single chain antibody comprises an amino acid sequence shown as SEQ ID NO. 5.
Further, the leader peptide is selected from the group consisting of one or more of a CD8 a signal peptide, a GM-CSF signal peptide, a CD28 signal peptide, a CD4 signal peptide, a CD5 signal peptide, a CD134 signal peptide, and a CD137 signal peptide;
the extracellular hinge region is selected from the group consisting of one or more of a CD8 a hinge region, a CD28 hinge region, a CD4 hinge region, a CD5 hinge region, a CD134 hinge region, a CD137 hinge region, and an ICOS hinge region;
the transmembrane region is selected from one or more of a CD3 transmembrane region, a CD4 transmembrane region, a CD8 transmembrane region and a CD28 transmembrane region;
the intracellular signaling region is selected from the group consisting of a combination of one or more of a 4-1BB signaling region, a CD3 zeta signaling region, an ICOS signaling region, a CD27 signaling region, an OX40 signaling region, a CD28 signaling region, an IL1R1 signaling region, a CD70 signaling region, and a TNFRSF19L signaling region;
preferably, the leader peptide is selected from the group consisting of CD8 a signal peptide;
preferably, the extracellular hinge region is selected from the group consisting of a CD8 a hinge region;
preferably, the transmembrane region is selected from the group consisting of a CD8 transmembrane region;
preferably, the intracellular signal region is selected from the group consisting of 4-1BB and CD3 zeta, and the 4-1BB and CD3 zeta are linked in sequence.
In a second aspect the invention provides a nucleotide sequence encoding said chimeric antigen receptor.
In a third aspect the invention provides an expression vector comprising said nucleotide sequence.
Further, the expression vector is selected from a lentiviral expression vector, a retroviral expression vector, or an adenoviral expression vector.
In a fourth aspect the invention provides a CAR-T cell that secretes BiTE, said cell being a T cell expressing said chimeric antigen receptor.
In a fifth aspect the invention provides a pharmaceutical composition comprising said expression vector or said BiTE-secreting CAR-T cell.
The sixth aspect of the invention provides the use of a BiTE-secreting CAR-T cell in the manufacture of a medicament for the treatment of a tumor.
Further, the tumor is a mesothelin high-expression tumor;
preferably, the tumor is a solid tumor;
preferably, the tumor comprises a pleural mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, breast cancer.
Preferably, the tumor is a triple negative breast cancer.
The beneficial effects of the invention are as follows:
the chimeric antigen receptor Mesothelin CAR-BiTE secreting BiTE provided by the invention is a CAR which is designed by combining BiTE technology and targets a plurality of antigens of Mesothelin and NKG2DL, the Mesothelin CAR-BiTE is further expressed on human killer T cells by using a modern genetic engineering technology, mesothelin CART-BiTE cells are prepared, and are returned to a patient, so that the problems of antigen escape and the like of tumor cells can be avoided, and in addition, when the T cells secrete 'BiTE', the 'BiTE' is combined with NKG2D on the tumor cells, an intracellular signal region is activated, so that the expansion of the T cells in the patient is promoted, the killing effect is exerted, the recurrence rate of the CAR-T cells for treating tumors is reduced, the treatment effect is improved, a new treatment method and an optimal treatment target point are found for the solid CAR, and the curative effect of the current-T cell therapy on triple negative breast cancer is improved.
Drawings
Fig. 1: the construction of lentiviral plasmids is schematically shown.
Fig. 2: mesothelin CART-BiTE cell preparation procedure.
Fig. 3: positive rate of Mesothelin CART-BiTE cells.
Fig. 4: mesothelin CART-BiTE targeted killing tumor schematic.
Fig. 5: effect of Mesothelin CART-BiTE cells on killing MDA231 cells in vitro. After about 24h of killing, E: t=10: 1, a step of; after about 24 hours of killing, E: t=5: 1.
Detailed Description
For a clearer understanding of the present invention, the present invention will now be further described with reference to the following examples and drawings. The examples are for illustration only and are not intended to limit the invention in any way. In the examples, each of the starting reagent materials is commercially available, and the experimental methods without specifying the specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.
Example 1
1. Structure of chimeric antigen receptor secreting BiTE
This example provides a chimeric antigen receptor secreting BiTE that targets Mesothelin (Mesothelin) and NKG2D ligand (NKG 2 DL), denoted Mesothelin CAR-BiTE. Mesothelin CAR-BiTE comprises a leader peptide, a nanobody targeting Mesothelin, an extracellular hinge region, a transmembrane region, an intracellular signal region, a P2A linker peptide, an Ig kappa leader peptide, a protein targeting NKG2D ligand, a linker peptide, a single chain antibody targeting CD3, linked in sequence from amino to carboxy terminus.
In a specific embodiment, the leader peptide is selected from the group consisting of a CD 8. Alpha. Signal peptide, the extracellular hinge region is selected from the group consisting of a CD 8. Alpha. Hinge region, the transmembrane region is selected from the group consisting of a CD8 transmembrane region, the intracellular signal region is selected from the group consisting of 4-1BB and CD3 zeta, and the 4-1BB and CD3 zeta are linked in sequence.
Mesothelin-targeting nanobody (MSLN VHH) (amino acid sequence) SEQ ID No.1:
QVQLVQSGGGLVHPGGSLRLSCAASGIDLSLYRMRWYRQAPGKERDLVALITDDGTSYYEDSVKGRFTITRDNPSNKVFLQMNSLKPEDTAVYYCNAETPLSPVNYWGQGTQVTVS
mesothelin-targeting nanobody (MSLN VHH) (nucleotide sequence) SEQ ID No.2:
CAAGTACAACTCGTGCAAAGTGGAGGCGGATTGGTGCATCCAGGAGGGAGCCTCAGACTGTCATGCGCTGCCAGCGGCATAGATCTTTCTTTGTACCGGATGAGATGGTACAGGCAGGCGCCAGGAAAGGAGAGAGATCTCGTCGCACTGATCACCGACGATGGGACCAGCTACTACGAAGACAGTGTCAAGGGCCGGTTCACAATCACCAGAGACAACCCCAGCAACAAGGTGTTTCTCCAAATGAACAGCCTTAAACCAGAGGACACCGCCGTGTATTATTGCAACGCAGAGACACCTCTGTCTCCTGTAAACTACTGGGGGCAGGGAACTCAGGTGACCGTGAGC protein targeting NKG2D ligand (NKG 2D) (amino acid sequence) SEQ ID No.3:
VTRQMCIYTNPTSCNEIYGKFSSAYLACDGKQMEIITLLNPSLISGDEWQWSGNTPIHVLGMWHYSKVLKLLDQDEKSYVKLLSANQSMCSAQSEYWNKSEDFFQYCNNKYCIWNKPCPGCYSETLPIQVEQNFLSNLFVASWI
protein targeting NKG2D ligand (NKG 2D) (nucleotide sequence) SEQ ID No.4:
GTGACCAGGCAGATGTGTATTTATACAAACCCTACTTCATGCAATGAGATTTACGGAAAATTCAGTAGCGCCTATCTCGCCTGCGATGGCAAGCAAATGGAGATCATTACCCTCTTGAACCCAAGTCTGATTTCCGGGGATGAATGGCAATGGTCAGGAAATACCCCCATTCACGTGCTGGGCATGTGGCATTACTCCAAGGTGCTTAAGCTTCTGGATCAGGACGAGAAAAGCTACGTAAAACTCTTGTCTGCTAATCAATCTATGTGTAGCGCTCAGAGCGAGTACTGGAATAAATCTGAAGATTTCTTCCAGTATTGTAACAATAAGTACTGTATTTGGAACAAACCTTGTCCCGGCTGTTATTCTGAGACCTTGCCTATTCAGGTGGAGCAGAACTTCTTGTCCAACCTGTTCGTGGCCTCCTGGATT
CD 3-targeting single chain antibody (amino acid sequence) SEQ ID No.5:
DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK
CD 3-targeting single chain antibody (nucleotide sequence) SEQ ID No.6:
GATATTAAACTGCAACAGAGCGGAGCCGAACTGGCCAGACCTGGTGCATCCGTGAAAATGTCATGCAAGACAAGCGGCTATACCTTCACCAGGTACACCATGCACTGGGTCAAACAGAGACCTGGACAGGGGCTGGAATGGATTGGTTACATTAATCCCTCCCGAGGGTATACAAATTATAATCAGAAATTCAAAGACAAGGCTACTCTGACCACCGATAAATCCTCATCCACCGCCTACATGCAGTTGTCCTCCCTGACCTCCGAGGATTCAGCCGTCTATTACTGCGCCAGGTACTATGACGACCACTACTGTCTGGACTACTGGGGACAGGGTACCACGCTGACTGTTTCATCAGTGGAGGGAGGATCCGGCGGTTCAGGAGGTAGCGGAGGTTCAGGAGGTGTGGACGACATTCAGTTGACACAGTCTCCAGCCATTATGTCTGCAAGCCCAGGGGAAAAAGTCACGATGACCTGCAGGGCGTCCTCAAGTGTCTCTTATATGAACTGGTATCAGCAGAAATCAGGTACCAGCCCCAAGAGGTGGATATACGACACATCCAAGGTGGCGAGCGGCGTGCCTTACAGGTTTAGTGGGTCCGGCTCCGGTACGTCTTATAGCCTTACTATCAGTAGTATGGAAGCTGAAGATGCCGCTACCTATTACTGCCAGCAGTGGAGCTCCAACCCCCTGACTTTTGGGGCCGGCACTAAGCTCGAATTGAAA
leader (nucleotide sequence) SEQ ID No.7:
ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCC
extracellular hinge region (hinge) (nucleotide sequence) SEQ ID No.8:
ACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATC
transmembrane region (TM) (nucleotide sequence) SEQ ID No.9:
TACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGT
intracellular costimulatory region (4-1 BB) (nucleotide sequence) SEQ ID NO.10:
CGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTG
intracellular signal transduction region (cd3ζ) (nucleotide sequence) SEQ ID No.11:
CGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG
P2A connecting peptide (nucleotide sequence) SEQ ID NO.12:
GCAACAAACTTCTCTCTGCTGAAACAAGCCGGAGATGTCGAAGAGAATCCTGGACCG
ig kappa leader peptide (nucleotide sequence) SEQ ID NO.13:
ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGAC
linker peptide (nucleotide sequence) SEQ ID No.14:
GGAGGAGGAAGT
construction of Mesothelin CAR.BiTE lentiviral plasmid
This example further provides a method for constructing a Mesothelin CAR BiTE lentiviral plasmid, the construction of which is schematically shown in figure 1, the vector of which is derived from NL4-3 clone of HIV. The internal structure of HIV has been disrupted to the greatest extent to remove its pathogenicity, so the vector retains only a portion of the conserved regions of HIV. The Mesothelin CAR-BiTE recognition domain structure was incorporated into the SIN vector, plus elongation factor 1 a (EF 1 a) as promoter. In addition, vesicular stomatitis virus glycoprotein (VSV-G) capsids are employed for viral packaging. To ensure safety, VSV-GDNA and vector DNA are co-transferred into HEK293T cells only when the viral vector is produced using different plasmids. VSV-G capsids assist in the adhesion of lentiviral vectors to cell membranes and maintain lentiviral infectivity. The method comprises the following specific steps:
(1) Construction of pCDH recombinant plasmid containing CAR coding Gene
The coding gene of the CAR was inserted into pCDH vector and located after elongation factor 1α (EF 1 α) of the vector. When the coding gene of the CAR is inserted into the pCDH vector, a start codon (such as ATG) can be added to the 5 'end of the coding gene of the CAR-and a stop codon (such as TAA) can be added to the 3' end. Then transferring into competent cells DH5 alpha of the escherichia coli, and carrying out positive clone PCR identification and sequencing identification. And (3) detecting and sequencing by using a PCR product gel electrophoresis to identify the size and sequence of the fragment meeting the purpose, and successfully constructing the pCDH recombinant plasmid, namely the recombinant expression plasmid.
(2) Recombinant lentivirus construction
The pCDH-CAR recombinant plasmid obtained above, envelope plasmid pMD2G and packaging plasmid (pMDLg/pRRE and pRSV-REV) are co-transfected into cultured HEK293T cells through liposome transfection reagent Lipofectamine 3000, and the recombinant lentivirus is obtained after 48 hours by centrifugation.
3. Preparation method of Mesothelin CART-BiTE cells
Preparation of CAR-T cells secreting BiTE, T cells can be transfected with lentiviral vectors, retroviral vectors, adenoviral vectors, etc. expressing the chimeric antigen receptor Mesothelin CAR-BiTE. When the lentiviral vector, the retrovirus vector and the adenovirus vector expressing the chimeric antigen receptor are prepared, transfection methods such as liposome and calcium phosphate can be adopted. The preparation of the Mesothelin CART-BiTE cells by the recombinant lentivirus constructed above is described below, and the preparation flow is shown in FIG. 2, and specifically includes the steps of:
a) Isolation of PBMC (peripheral blood mononuclear cells)
PBMCs are derived from autologous venous blood, autologous bone marrow, umbilical cord blood, placental blood, and the like. Preferably from fresh peripheral blood or bone marrow collected one month after surgery and one month after chemoradiotherapy of cancer patients.
Drawing the blood of the patient and sending the sample to a blood separation chamber; collecting peripheral blood mononuclear cells, and taking middle-layer cells after Ficoll centrifugal separation; after washing with PBS, PBMC were obtained.
b) Isolation of antigen-specific T lymphocytes by immunomagnetic bead method
Adding a basic culture medium without serum into the PBMC to prepare cell suspension; adding CD3/CD28 immunomagnetic beads according to the ratio of the magnetic beads to the cells being 1:1, and incubating for 1-2 hours at room temperature; screening cells incubated with the magnetic beads by using a magnet; washing with PBS, and removing immunomagnetic beads to obtain CD3 positive T lymphocytes.
c) Method for preparing antigen-specific T lymphocyte by virus transfection method
Co-culturing the CD3 positive T lymphocyte obtained by immunomagnetic bead separation method in step (2) with the recombinant lentivirus, counting cells and changing liquid on the 3 rd day of culture, and adjusting cell concentration to 0.5X10 6 Inoculating and culturing the strain in a single/mL mode; on day 5 of culture, the cell state was observed, and if the cell density was increased, the diluted cell concentration was 0.5X10 6 Cell activity was measured at each mL and culture was continued. Cells were collected after expansion culture to days 9-11, and expression of chimeric antigen receptor was detected by flow cytometry, and the results are shown in FIG. 3.
Example 2
The schematic diagram of Mesothelin CART-BiTE cell targeted killing tumor is shown in fig. 4. After the in vitro amplification of the Mesothelin CART-BiTE cells is completed, a preliminary efficacy experiment is carried out on the Mesothelin CART-BiTE cells, and the specific method of the RTCA killing experiment is as follows:
cell killing experiments were performed using a real-time cell analyzer (xCElligence RTCA SP). 5,000 Mesothelin-overexpressing MDA-231 cells (both Mesothelin and NKG2D ligand expressed positively) were added to the E-Plate wells and incubated first before the instrument was fitted to the E-Plate wells. After about 24 hours, corresponding numbers of BiTE-secreting CAR-T cells, biTE-non-secreting CAR-T cells, and UTD cells (bead-stimulated only, non-virally infected T cell control) were added at different potency target ratios (E: T), followed by co-culture for a period (> 24 hours). And analyzing the cell killing effect according to the CI value of the real-time cell analyzer. The killing result of MDA-231 cells is shown in figure 5, and the CAR-T cells secreting BiTE have very strong killing capacity to MDA-231 cells, which indicates that BiTE is successfully secreted out and promotes the killing function of T cells.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

Claims (10)

1. A chimeric antigen receptor that secretes BiTE, comprising a leader peptide, a nanobody that targets mesothelin, an extracellular hinge region, a transmembrane region, an intracellular signaling region, a P2A linker peptide, an igκ leader peptide, a protein that targets NKG2D ligand, a linker peptide, a single chain antibody that targets CD3, linked in sequence from amino terminus to carboxy terminus.
2. The chimeric antigen receptor according to claim 1, wherein the mesothelin-targeting nanobody comprises an amino acid sequence as shown in SEQ ID No. 1;
the protein targeting the NKG2D ligand comprises an amino acid sequence shown as SEQ ID NO. 3;
the CD3 targeting single chain antibody comprises an amino acid sequence shown as SEQ ID NO. 5.
3. The chimeric antigen receptor according to claim 1, wherein the leader peptide is selected from the group consisting of one or more of a CD8 a signal peptide, GM-CSF signal peptide, CD28 signal peptide, CD4 signal peptide, CD5 signal peptide, CD134 signal peptide and CD137 signal peptide;
the extracellular hinge region is selected from the group consisting of one or more of a CD8 a hinge region, a CD28 hinge region, a CD4 hinge region, a CD5 hinge region, a CD134 hinge region, a CD137 hinge region, and an ICOS hinge region;
the transmembrane region is selected from one or more of a CD3 transmembrane region, a CD4 transmembrane region, a CD8 transmembrane region and a CD28 transmembrane region;
the intracellular signaling region is selected from the group consisting of a combination of one or more of a 4-1BB signaling region, a CD3 zeta signaling region, an ICOS signaling region, a CD27 signaling region, an OX40 signaling region, a CD28 signaling region, an IL1R1 signaling region, a CD70 signaling region, and a TNFRSF19L signaling region;
preferably, the leader peptide is selected from the group consisting of CD8 a signal peptide;
preferably, the extracellular hinge region is selected from the group consisting of a CD8 a hinge region;
preferably, the transmembrane region is selected from the group consisting of a CD8 transmembrane region;
preferably, the intracellular signal region is selected from the group consisting of 4-1BB and CD3 zeta, and the 4-1BB and CD3 zeta are linked in sequence.
4. A nucleotide sequence encoding the chimeric antigen receptor of any one of claims 1-3.
5. An expression vector comprising the nucleotide sequence of claim 4.
6. The expression vector of claim 5, wherein the expression vector is selected from a lentiviral expression vector, a retroviral expression vector, or an adenoviral expression vector.
7. A BiTE-secreting CAR-T cell, wherein said cell is a T cell expressing the chimeric antigen receptor of any one of claims 1-3.
8. A pharmaceutical composition comprising the expression vector of claim 5 or the BiTE-secreting CAR-T cell of claim 7.
9. Use of a BiTE-secreting CAR-T cell of claim 7 for the manufacture of a medicament for the treatment of a tumor.
10. The use according to claim 9, wherein the tumor is a mesothelin-highly expressed tumor;
preferably, the tumor is a solid tumor;
preferably, the tumor comprises a pleural mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, breast cancer.
Preferably, the tumor is a triple negative breast cancer.
CN202211150670.2A 2022-09-21 2022-09-21 BiTE-secreting CAR-T cells and uses thereof Pending CN117736336A (en)

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