CN109666651B - Secretory Lewis-Y targeting CAR-T cell - Google Patents
Secretory Lewis-Y targeting CAR-T cell Download PDFInfo
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- CN109666651B CN109666651B CN201910075310.2A CN201910075310A CN109666651B CN 109666651 B CN109666651 B CN 109666651B CN 201910075310 A CN201910075310 A CN 201910075310A CN 109666651 B CN109666651 B CN 109666651B
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Abstract
The invention discloses a secretory type CAR-T cell targeting Lewis-Y, which comprises a CAR-T cell targeting Lewis-Y and secreting IL12 cytokines; CAR-T cells targeting Lewis-Y and secreting PD-1 antibodies. The invention also discloses a preparation method of the secretory-targeted Lewis-Y CAR-T cell and application of the CAR-T cell in preparing anti-tumor cell therapeutic drugs. The PD-1 antibody secreted by the CAR-T cells is specifically combined with PD-1 on the surfaces of the T cells, blocks the inhibition reaction of PD-1/PD-L1 signals on the activity of the T cells, and is beneficial to effectively inhibiting the growth of the tumor cells by the CAR-T for a long time; the IL12 cytokine secreted by the CAR-T cell can act on the immunosuppressive cell in the tumor-suppressive immune microenvironment, reduce the immunosuppressive capability of the immunosuppressive cell, and further enhance the killing effect of the CAR-T cell.
Description
Technical Field
The invention relates to the fields of immunology, cell biology and molecular biology, in particular to a CAR-T cell which can secrete IL12 or PD-1 antibody and target Lewis-Y, and further relates to a preparation method for preparing the cell by using a lentiviral vector and application of the cell.
Background
The publication Cancer Statistics in China,2015 of 2016 shows that there are 429.2 ten thousand new tumor cases and 281.4 ten thousand death cases predicted in China in 2015. Among the cancers with highest mortality are: lung cancer, bronchus cancer, stomach cancer, liver cancer, esophagus cancer and colorectal cancer account for three-fourths of all cancer deaths. According to the latest published data of WHO, the tumor death number in China accounts for about 23 percent, which is second only to cardiovascular diseases. Along with the change of living habits caused by factors such as environmental pollution, urbanization and the like brought by the aging promotion of population in China and the industrialization process, the tumor prevalence rate and death rate of residents in China are obviously improved. Traditional methods of tumor treatment, including surgery, radiation therapy, chemotherapy, and targeted therapy, all have their limitations. The operation is mainly aimed at early tumor patients, and has poor effect on late and metastatic tumor patients; the clinical utility of radiotherapy and chemotherapy is relatively limited and the toxic and side effects are larger; although the targeting treatment has obvious effect and quick response, the targeting treatment is easy to generate drug resistance. Tumor immunotherapy is a new treatment means after surgery, radiotherapy and chemotherapy, especially the rising of immune checkpoint monoclonal antibody treatment and CAR-T cell immunotherapy, and the curative effect of immunotherapy is gradually confirmed. In 2013, journal of Science has reviewed tumor immunotherapy as the first of ten scientific breakthroughs in the year. For month 1 of 2016, the united states developed a cancer "lunar plan" based on immunotherapy and targeted therapy development.
In 1985, rosenberg SA first published the results of a clinical study of LAK in combination with IL-2 for the treatment of malignant melanoma. Thereafter, several protocols such as first generation lymphokine activated killer cell therapy (Lymphokine Activated Killer, LAK), second generation cytokine induced killer cell therapy (Cytokine Induced Killer cell, CIK), third generation tumor infiltrating lymphocyte therapy (Tumor Infiltrating Lymphocytes, TIL), fourth generation cytotoxic T cell therapy (Cytotoxic T Lymphocytes, CTL), and fifth generation chimeric antigen receptor T cell therapy (Chimeric Antigen Receptor-modified T cells, CAR-T) were sequentially evolved on this basis.
Chimeric antigen receptor T cells (CAR-T cells): the CAR-T cell treatment is to transfect a chimeric antigen receptor CAR in vitro of a T lymphocyte of a subject by utilizing a genetic engineering technology to form effector T cells with specific recognition capability to specific antigens, and then to infuse the effector T cells back into the subject after in vitro amplification, wherein the infuse CAR-T cells recognize specific tumor cell surface antigens in a high-efficiency specific non-MHC restriction mode, and kill the tumor cells. CARs typically encode a scFv comprising a single chain antibody to bind to a tumor cell surface specific antigen; also linked to an intracellular signaling domain, typically a CD28 or/and 4-1BB costimulatory molecule and a CD3ζ intracellular signaling domain, mediate T cell activation. In 2017, the FDA approved the marketing of CD 19-targeted 4-1 BB-based second generation CAR-T products.
The Lewis Y antigen, also known as CD174, one of the members of the family of CD174 antigens belonging to the blood group antigens (blood group antigen, BGA) ABH and Lewis antigens, is H2 dimeric fucose (. Alpha.1-2 galactose. Beta.1-4 acetylglucosamine), and the CD174 and ABH antigens are closely related, and they are regulated by different genes, and are produced by common precursor cells. In 2001, lewis Y was formally designated as CD174. As a blood group antigen, CD174 plays a role in cell recognition, differentiation, growth regulation, and is significantly altered during the course of benign-malignant transformation. A study of Wakabayashi et al on 46 liver cancer patients found that 20 of the tumor cells had CD174 expression (43.5%), and that the CD174 expression rate was higher in patients with AFP >200ng/ml than in patients with AFP <200 ng/ml. Immunohistochemical detection of the surgically resected specimens of breast cancer patients shows that the expression of CD174 is higher for later breast cancer patients than for earlier breast cancer patients, and the study of Inufusa and the like shows that the 5-year survival rate of CD174 positive patients is obviously lower than that of negative patients. In lung cancer patients, there is also found to be high expression of CD174. Tanaka et al studied 236 patients with non-small cell lung cancer, 179 positive for CD174 expression, at 75.8%. Mehdi et al found that a certain degree of CD174 positive expression was detected in 234 patient surgical samples in 260 non-small cell lung cancer patients at I, II.
Scott et al published 2000 a humanized CD174 antibody hu3S193, and administration of hu3S193 antibody to mice xenografted with MCF-7 was found to inhibit tumor growth in mice. In a phase II clinical study with CD174 mab (hu 3S 193) to treat refractory ovarian, fallopian tube and primary peritoneal cancers, 31 subjects received mab treatment of hu3S193, demonstrating the safety of hu3S193 mab targeting CD174 antigen, the major side effects observed were fatigue, nausea and anaphylaxis.
Westwood et al found that when anti-CD174CAR-T cells were co-cultured with CD174 antigen-positive expressing tumor cells, the T cells could kill lysed tumor cells, and the water-soluble CD174 antigen in serum did not affect the activity of the CAR-T cells. In vivo experiments in mice, westwood, peinert et al found that CAR-T cells constructed based on hu3S193 could kill tumors in mice and thereby inhibit tumor growth. Ritche et al completed a phase I clinical trial with anti-CD174CAR-T cells for treating acute myelogenous leukemia patients, 4 subjects received 1.3X10 9 No toxic side effects of grade 3 or grade 4 were found, demonstrating that CAR-T of anti-CD174 is safe for clinical treatment.
At present, the CAR structure for treating the CD174 positive tumor is a second-generation CAR structure, although CAR-T cells of the structure have strong antigen recognition binding capacity, the CAR-T cells cannot be well immersed into tumor tissues due to the complex tumor microenvironment of solid tumors, and the CAR-T cells can be inhibited due to the combination of the CAR-T cells and PD-L1 molecules on the surfaces of the tumor cells, so that the recognition and killing capacity of the second-generation CAR-T on the tumor cells is limited. The CAR-T cell adopts a fourth-generation CAR structure, and can secrete cytokines such as IL12 or antibodies of PD-1 while recognizing CD174, the secreted cytokines such as IL12 can strengthen the self killing function of the CAR-T cell, and other immune cells such as DC cells are recruited and simultaneously kill tumor cells; the secreted PD-1 antibody can block the combination of the PD-L1 molecule on the surface of the tumor cell and the CAR-T cell, so that the inhibition effect of the PD-L1 on the CAR-T cell is relieved, and the technical scheme can improve the killing effect of the CAR-T cell on the tumor cell.
Disclosure of Invention
One of the technical problems to be solved by the invention is to provide a secretory Lewis-Y targeting CAR-T cell. The CAR-T cell expresses Lewis-Y-CAR and simultaneously secretes cytokines or antibodies, so that the immunosuppression of tumor microenvironment can be better overcome, and the killing capacity of the CAR-T cell to tumor cells is enhanced.
The second technical problem to be solved by the invention is to provide a preparation method of the secretory targeting Lewis-Y CAR-T cell, which can prepare the cell which has cell killing toxicity and can express the cytokine or antibody at a high level.
The third technical problem to be solved by the invention is to provide the application of the secretory-targeted Lewis-Y CAR-T cell in preparing an anti-tumor cell therapeutic drug.
In order to solve the technical problems, the invention adopts the following technical scheme:
in a first aspect of the invention, there is provided a secreted Lewis-Y targeting CAR-T cell comprising the following cells:
t cells expressing Lewis-Y-CAR while secreting antibodies;
a T cell expressing the Lewis-Y-CAR while secreting a cytokine.
In the present invention, the term Lewis-Y-CAR refers to a Lewis-Y chimeric antigen receptor comprising a scFv sequence targeting Lewis-Y.
scFv: single-chain antibody fragment, a single chain antibody fragment, is known.
In the invention, the term Lewis-Y-scFv refers to an anti-Lewis-Y single-chain antibody fragment, and the nucleotide sequence after codon optimization is shown as SEQ ID NO. 1; the nucleotide sequence is used on a lentiviral expression vector, a retroviral vector, an adenoviral expression vector, an adeno-associated viral expression vector or other types of expression vectors, preferably a lentiviral expression vector.
As a preferred embodiment of the present invention, the antibody-secreting T cells expressing Lewis-Y-CAR are preferably PD-1 antibodies.
As a preferred embodiment of the present invention, the cytokine-secreting T cells expressing Lewis-Y-CAR are preferably IL12 cytokines.
As a second aspect of the present invention, there is provided a method for preparing the secretory Lewis-Y targeted CAR-T cell, comprising the steps of:
(1) Isolating PBMCs from peripheral blood provided by the donor;
(2) Activating and sorting CD3 positive T cells from the PBMC;
(3) Constructing a lentiviral plasmid skeleton carrying Lewis-Y-CAR and antibody or cytokine genes to obtain a recombinant lentiviral plasmid;
(4) Transfecting the recombinant lentiviral plasmid and auxiliary plasmid carrying the Lewis-Y-CAR and antibody or cytokine genes obtained in the step (3) into host cells to prepare a recombinant lentiviral vector capable of infecting T cells;
(5) Transducing the recombinant lentiviral vector obtained in step (4) into the activated T cells selected in step (2) to produce CAR-T cells expressing Lewis-Y-CAR and secreting antibodies or cytokines;
(6) Culturing the cells obtained in the step (5) in vitro;
(7) Amplifying the cells obtained in step (6) in large numbers;
(8) Collecting CAR-T cells expressing the Lewis-Y-CAR that secrete antibodies or cytokines at the same time.
The basic design of Chimeric Antigen Receptor (CAR) comprises a tumor-associated antigen binding region (Single chain antibody Fragment, scFv), an extracellular hinge region, a transmembrane region and an intracellular signaling region.
The extracellular tumor associated antigen binding region of the Lewis-Y-CAR in the step (3) of the invention is a Lewis-Y single chain antibody (scFv) for binding to the Lewis-Y protein, and the c-myc epitope tag, the CD8 Hinge chimeric receptor Hinge and the CD8 Transmembrane chimeric receptor Transmembrane region are sequentially connected in series.
The intracellular signaling region of the Lewis-Y-CAR in step (3) of the present invention comprises a region encoding a co-stimulatory factor selected from the group consisting of 4-1BB, CD28, CD27, OX40, CD30, CD40, PD-1, ICOS, LIGHT, B7-H3, a ligand that specifically binds CD83, ICAM-1, HVEM (LIGHTR), CD160, lymphocyte function-associated antigen-1 (LFA-1), IL2Rα, CD103, CD11B, CD11c, TRANCE/RANKL, SLAMF4 (CD 244,2B 4), CD69, SLAM (SLAMF 1, CD150, IPO-3) or any combination of more.
As a preferred technical scheme of the invention, the costimulatory factor region is CD28-4-1BB; the intracellular signaling region is CD28-4-1BB-CD3 zeta.
In the step (3), the sequence obtained after codon optimization is scFv region sequence, and the nucleotide sequence is shown as SEQ ID NO. 1;
as a preferred technical scheme of the invention, in the step (3), the recombinant lentiviral plasmid is a recombinant replication-defective lentiviral plasmid, can integrate exogenous fragments into host genes, is disposable, cannot replicate and proliferate, and is safe and reliable; the recombinant lentiviral plasmid is a second-generation or third-generation lentiviral transgenic plasmid.
As a preferred technical scheme of the invention, in the step (3), the recombinant lentiviral plasmid is preferably a third generation lentiviral transgenic plasmid, and the third generation lentiviral skeleton plasmid vector used in the invention is pCDH-CMV-MCS-EF1-copGFP (vast organism, P0980), and as shown in figure 1, the third generation lentiviral plasmid comprises ampicillin resistance gene AmpR sequence, prokaryotic replicon pUC Ori sequence, viral replicon SV40 Ori sequence, RSV promoter, lentivirus 5Terminal LTR, lentivirus 3Terminal Self-activating LTR, gag cis element, RRE cis element, env cis element, cPPT cis element and eHBV WPRE enhanced woodchuck virus post-transcriptional regulatory element.
As a preferred technical scheme of the invention, in the step (3), the recombinant lentiviral plasmid is a lentiviral transgenic plasmid targeting Lewis-Y antigen, and the recombinant lentiviral plasmid sequentially comprises a CMV promoter sequence, a Lewis-Y single chain antibody (scFv), a Flag, a CD8 Transmembrane chimeric receptor Transmembrane region, an intracellular signaling region, an NFAT promoter sequence and an IL12 cytokine sequence, wherein the nucleotide sequence of the NFAT promoter is shown as SEQ ID NO.7, and the nucleotide sequence of the IL12 cytokine is shown as SEQ ID NO. 8; the recombinant lentiviral vector described in step (5) expresses a Lewis-Y targeting chimeric antigen receptor on the surface of a T cell (or NK cell) while being capable of secreting IL12 cytokine.
As a preferred technical scheme of the invention, in the step (3), the recombinant lentiviral plasmid is a lentiviral transgenic plasmid targeting Lewis-Y antigen, the recombinant lentiviral plasmid sequentially comprises a CMV promoter sequence, a Lewis-Y single chain antibody (scFv), a Flag, a CD8 Transmembrane chimeric receptor Transmembrane region, an intracellular signal transduction region, an EF1 alpha promoter sequence and a PD-1 antibody sequence, the EF1 alpha promoter nucleotide sequence is shown as SEQ ID NO.9, the IL2SS nucleotide sequence is shown as SEQ ID NO.10, and the Anti-PD-1scFv, namely, the PD-1 antibody nucleotide sequence is shown as SEQ ID NO. 11; the recombinant lentiviral vector described in step (4) expresses a Lewis-Y targeting chimeric antigen receptor on the surface of T cells (or NK cells) while being capable of expressing PD-1 antibodies.
As a preferable technical scheme of the invention, in the step (3), the signal peptide of the PD-1 in the lentiviral plasmid is replaced by the strong secretion signal peptide IL2SS, so that the exocrine capacity of the PD-1 can be obviously improved.
As a preferred embodiment of the present invention, the host cell in step (5) is a 293FT or 293T cell.
In a third aspect of the invention, there is provided the use of a secretory Lewis-Y targeted CAR-T cell as described above in the manufacture of an anti-tumour cell therapeutic agent. The cell can be used for preparing an anti-tumor cell therapeutic drug, in particular a cell therapeutic drug targeting Lewis-Y positive tumor stem cells; a mixed preparation can be prepared, which comprises the above-mentioned CAR-T cell expressing Lewis-Y-CAR and secreting PD-1 antibody and the medicine mixed in different proportions by the CAR-T cell expressing Lewis-Y-CAR and secreting IL12 cytokine.
Compared with the prior art, the invention has the following beneficial effects:
the present invention is a CAR-T cell that can secrete a cytokine IL12 or PD-1 blocking antibody. The PD-1 signal peptide is replaced by the strong secretion signal peptide IL2SS, so that the exocrine capacity of the PD-1 can be obviously improved, the IL12 is beneficial to the activation of T cells and NK cells, and the anti-tumor capacity of the T cells is enhanced. The PD-1 antibody secreted by the CAR-T cells can be combined with the PD-1 molecules on the surfaces of the T cells, so that the combination of the PD-L1 receptor on the surfaces of the tumor cells and the PD-1 on the surfaces of the T cells is blocked, and the anti-tumor capability of the T cells is fully exerted.
The co-stimulatory factor or combination of factors used in the invention can lead T cells to be continuously activated and proliferated, cell factors to be continuously secreted, and enhance the effect of killing tumor cells and immunological memory.
Drawings
FIG. 1 is a schematic diagram of pCDH-CMV-MCS-EF1-copGFP backbone vector plasmids in examples 1 and 2 of the present invention;
FIG. 2 is a diagram showing the construction of a lentiviral backbone plasmid SV305 carrying a Lewis-Y-CAR and cytokine IL12 gene according to example 1 of the present invention;
FIG. 3 shows the helper plasmid pRSV-Rev according to examples 1 and 2 of the present invention;
FIG. 4 shows the helper plasmid pCMV-VSV-G according to examples 1 and 2 of the present invention;
FIG. 5 shows the helper plasmid pMDLg-pPRE according to examples 1 and 2 of the present invention;
FIG. 6 is a graph showing the results of ELISA assays for IL12 secretion in CAR-T cells expressing Lewis Y-CAR and secreting IL12 as described in example 1 of the present invention;
FIG. 7 is a diagram showing the construction of a lentiviral backbone plasmid SV306 carrying the Lewis-Y-CAR and PD-1 antibody genes described in example 2 of the present invention;
FIG. 8 is a graph showing the results of a secretion ELISA assay for PD-1 antibodies in CAR-T cells expressing a Lewis Y-CAR and a PD-1 antibody as described in example 2 of the present invention;
FIG. 9 is a plot of killing efficiency of Lewis Y-CAR expressing IL 12-secreting CAR-T cells and CD133-CAR expressing PD-1 antibody-secreting CAR-T cells against target cells at different effective target ratios in example 4 of the present invention.
Detailed Description
The present invention is further described below with reference to examples and drawings, and the specific examples of the invention should not be construed as limiting the scope of the invention. Modifications to the disclosure of the invention may be made in the materials, methods, and reaction conditions, all of which are intended to fall within the spirit and scope of the invention.
The experimental procedure, which does not address the detailed conditions in the examples below, is generally in accordance with the conditions recommended by the manufacturer. Percentages are by weight unless otherwise indicated. The experimental materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified.
Although any methods and materials similar or equivalent to those described herein can be used in the embodiments of the present invention, the preferred materials and methods are described herein.
Example 1 CAR-T cells expressing Lewis-Y-CAR while secreting IL12 were prepared.
The preparation method of the CAR-T cell expressing the Lewis-Y-CAR and secreting IL12 simultaneously comprises the following steps:
1. PBMCs were isolated from peripheral blood supplied from the donor.
1. 80-100mL of human peripheral blood is collected by an anticoagulant tube or a blood collection bag (the peripheral blood is fully mixed with an anticoagulant by shaking during collection), and PBMC are separated in a sterile cell preparation workshop. Transferring peripheral blood into a 50ml centrifuge tube, centrifuging at room temperature of 800g for 15 minutes, sucking upper yellow autologous plasma into a new 50ml centrifuge tube, inactivating at 56 ℃ for 30 minutes, and placing at-20 ℃ for later use; adding equal volume of PBS into the cell layer at the lower layer, and mixing the mixture upside down;
2. sucking 15ml lymphocyte separation liquid (Ficoll-PaquePlus, GE) at the bottom of a new 50ml centrifuge tube, slowly adding 30ml diluted blood along the tube wall to the upper layer of the lymphocyte separation liquid, placing the centrifuge tube in a centrifuge, centrifuging at 450g in a slowly rising and slowly falling way for 25 minutes, no break;
3. after centrifugation, carefully sucking the white membrane layer cells above the lymphocyte separation liquid, transferring the white membrane layer cells into a new 50ml centrifuge tube, adding 50ml PBS for cleaning, and slowly lifting and slowly lowering the centrifugal liquid to centrifuge for 10min;
4. discarding the supernatant, and reserving cell sediment at the bottom of the centrifuge tube; adding 50ml of PBS (phosphate buffer solution) and 160g of PBS again, slowly lifting and slowly lowering, and centrifuging for 15min;
5. discarding the supernatant; finally, adding PBS (phosphate buffer solution) 300g, slowly lifting and slowly lowering, centrifuging for 10min, and discarding the supernatant to obtain the PBMC cells.
2. CD3 positive T cells were activated and sorted from PBMC.
Sorting kit according to CD3 positive T cells (EasySep TM Human T Cell Isolation Kit product number: #17951, vendor: STEMCELL) sorting C in (5) PBMCD3 positive T cells, specifically as follows:
1. cell density was adjusted to 5X 10 7 cells/mL, adding the cells to a 5mL cell freezing tube;
2. adding Isolation Cocktail ul/mL of the separating liquid, uniformly mixing, and standing for 5 minutes;
3. the rapidrays are used by a vortex instrument TM Swirling the reagent for 30 seconds to uniformly mix the particles, adding the particles into cells according to the proportion of 40ul/mL, and uniformly mixing;
4. PBS is added to 2.5mL and blown up and down for 2-3 times, and the mixture is placed on a magnetic frame for 3 minutes;
5. and (3) tilting the magnetic rack to transfer the liquid to a new cell culture dish, so as to obtain the sorted CD3 positive T cells.
3. The Lewis-Y-scFv codons were optimized.
The preparation of a Lewis-Y Chimeric Antigen Receptor (CAR), namely the Lewis-Y-CAR, carries out codon optimization on scFv fragments to ensure that the scFv fragments are easier to express in human cells, and the sequence after the codon optimization is the nucleotide sequence shown as SEQ ID NO. 1.
4. Constructing a lentiviral plasmid carrying a Lewis-Y-CAR and cytokine IL12 gene to obtain a recombinant lentiviral plasmid.
Construction of recombinant lentiviral backbone plasmid (SV 305, see fig. 2): the CAR structure secreting IL12 is synthesized and cloned on pCDH-CMV-MCS-EF 1-copGGFP skeleton plasmid (vast organism, P0980, see figure 1), and the SV305 skeleton plasmid is obtained after sequencing correctly, and CMV promoter-Lewis-YscFv-Flag-CD 8TM-CD28-4-1BB-CD3 zeta-NFAT promoter-IL12 is serially connected, wherein the nucleotide sequence after optimization of Lewis-YscFv is shown as SEQ ID NO.1, flag nucleotide sequence is shown as SEQ ID NO.2, CD8TM nucleotide sequence is shown as SEQ ID NO.3, CD28 nucleotide sequence is shown as SEQ ID NO.4, 4-1BB nucleotide sequence is shown as SEQ ID NO.5, CD3 zeta nucleotide sequence is shown as SEQ ID NO.6, NFAT promoter nucleotide sequence is shown as SEQ ID NO.7, and IL12 nucleotide sequence is shown as SEQ ID NO. 8.
5. And (3) infecting 293T cells with the recombinant lentiviral plasmid carrying the Lewis-Y-CAR and cytokine IL12 genes obtained in the step four to prepare the lentivirus capable of infecting the T cells.
1. The 293T cells are used for working cells of lentivirus packaging after being subjected to secondary culture for 2-3 generations after being resuscitated, and the total number of times of passage is not more than 10 generations.
2. Liposome transfection (10 cm dish for example).
1) The 293T cells are inoculated into a culture dish with the diameter of 10cm the day before transfection, and the quantity of the inoculated cells is preferably such that the cell growth on the day of transfection reaches 90-95% fusion;
2) On the day of transfection, the culture solution is removed, and 5ml of culture solution for virus packaging is added;
3) Preparation of DNA-Lipofectamine2000 complex:
A. a sterile 5mL centrifuge tube was prepared, and 1.5mL serum-free Opti-MEM culture medium, helper plasmids (pRSV-Rev (adedge, # 12253), pCMV-VSV-G (adedge, # 8454), pMDLg-pRRE (adedge, # 12251), FIGS. 3-5, respectively, and lentiviral expression plasmid (SV 305) were added and thoroughly blown and mixed;
B. preparing another sterile 5mL centrifuge tube, adding 1.5mL serum-free Opti-MEM culture solution and 68 mu L Lipofectamine2000, gently stirring, mixing, and incubating at room temperature for 10min;
after c.30 minutes, adding the serum-free Opti-MEM culture solution containing plasmid DNA into the serum-free Opti-MEM culture solution containing Lipofectamine2000, gently stirring and mixing, and incubating for 30 minutes at room temperature;
D. the DNA-Lipofectamine2000 complexes were added drop by drop to 293T cells and the dishes were gently shaken back and forth to mix the complexes. Placing at 37deg.C 5% CO 2 Culturing in a saturated humidity incubator.
3. And (5) collecting and concentrating the recombinant lentiviral vector.
1) The culture broth containing the virus was collected 48 hours after transfection. Sucking the culture solution into a sterile 50mL centrifuge tube with a cover, and adding 8mL of culture solution for virus packaging into cells capable of producing viruses;
2) Centrifuging at 3000rpm for 15 minutes at 4 ℃, centrifuging the virus supernatant at a low speed, removing cell debris, and recovering the supernatant;
3) The culture broth containing the virus was collected 72 hours after transfection. The mixture was centrifuged at 3000rpm at 4℃for 15 minutes, and the virus supernatant was centrifuged at a low speed to remove cell debris and collect the supernatant. The virus supernatant collected at 48 hours and the virus supernatant collected at 72 hours were filtered with a small filter of 0.45 μm, and the filtrate was collected with a 50mL centrifuge tube;
4) Concentrating the collected virus supernatant by an ultracentrifuge, and centrifuging for 120 minutes at a temperature of 72000g/min at a temperature of 4 ℃; the viral pellet was resuspended in 500. Mu.l fresh medium, sub-packaged, frozen and the viral titer was determined from the fractions.
6. And D, transducing the recombinant lentiviral vector obtained in the step five into the T cells selected in the step two to generate the CAR-T cells which express the Lewis-Y-CAR and secrete the cytokine IL 12.
Activation of T cells
(1) Sorting T cells at a density of 0.5 to 1X 10 6 cells/ml was resuspended in KBM 581 Medium (cat No. 88-581-CM, corning) supplemented with 1% autologous plasma;
(2) Wash Dynabeads:
a) Taking Dynabeads out of the refrigerator, oscillating for 30 seconds by using a vortex instrument, and uniformly re-suspending the magnetic beads deposited at the bottom;
b) According to the number of T cells in (1), the required Dynabeads were calculated according to the ratio of Dynabeads: T=1:1 to 1:3, and Dynabeads were transferred into a new 15ml centrifuge tube using a sterile pipette, and DPBS of the same volume was added, and the mixture was blown and homogenized using a pipette. Placing the centrifuge tube on a magnetic rack, standing for 5 minutes, and sucking the liquid by using a pipette;
c) Adding 2ml of DPBS into a 15ml centrifuge tube, and repeating the washing step in b);
d) Adding 581 culture medium of 100ul to resuspend beads, adding dropwise into cell culture solution, blowing and mixing uniformly, and standing at 37deg.C, 5% CO 2 The cells were cultured in a cell incubator for 3 days.
2. The recombinant lentiviral vector transduces T cells.
(1) T cells were counted 3 days after T cell activation by trypan blue staining. The required viral load was calculated as moi=3-10: required viral amount (ml) = (MOI cell number)/viral titer (Tu/ml);
(2) Taking out slow virus from-80 deg.C refrigerator, quickly thawing in 37 deg.C water bath (without making pipe orifice deep below liquid surface);
(3) Taking out the T cell culture dish, adding polybrene to a final concentration of 6ug/ml, adding the required virus amount, lightly blowing and mixing by a liquid transfer device to uniformly disperse the aggregated cell mass, sealing the culture dish by using a sealing film, and centrifuging at 800g for 1 hour under the room temperature condition;
(4) Removing the sealing film, placing the culture dish at 37deg.C, 5% CO 2 Culturing in an incubator for 24 hours;
(5) Centrifugation at 250g for 10min at room temperature, removal of the virus-containing medium supernatant, resuspension of the cell pellet with fresh medium, transfer of the cells to a new dish and further culture.
7. And (3) culturing the cells obtained in the step (six) in vitro.
The cell mass in the culture system was gently blown daily to disperse the cells, and the cell number was counted by trypan blue staining to maintain the cell density at 1X 10 6 And each ml.
8. Amplifying the cells obtained in the step seven in a large quantity.
a) When the cell number is greater than 100X 10 6 At this time, the CAR-T cells were transferred to a cell culture bag for culture. Cells were counted by trypan blue counting every other day, maintaining cell density at 1×10 6 Individual/ml;
b) When the CAR-T cells are cultured for 10 days, part of cell culture solution can be extracted for sterile detection;
c) When the total number of cells reaches 500-1000×10 6 When the CAR-T cells are collected, the magnetic beads are removed by using a magnetic pole.
d) Taking part of cell suspension, co-culturing with target cell OVCAR-3 (Lewis Y positive), stimulating for two days by using PMA, detecting secretion of IL12 in supernatant by ELISA (see figure 6), comparing with a control group, and experimental to form functional secretion of IL12, which indicates that CAR-T cells which express Lewis-Y-CAR and secrete cytokine IL12 are successfully prepared, and culturing the cells in an enlarged mode and freezing.
9. CAR-T cells expressing Lewis-Y-CAR and secreting cytokine IL12 were collected.
Example 2 preparation of CAR-T cells expressing Lewis-Y-CAR and secreting PD-1 antibodies
The preparation method of the CAR-T cell expressing the Lewis-Y-CAR and secreting the PD-1 antibody simultaneously comprises the following steps:
1. PBMCs were isolated from peripheral blood supplied from the donor.
1. 80-100mL of human peripheral blood is collected by an anticoagulant tube or a blood collection bag (the peripheral blood is fully mixed with an anticoagulant by shaking during collection), and PBMC are separated in a sterile cell preparation workshop. Transferring peripheral blood into a 50ml centrifuge tube, centrifuging at room temperature of 800g for 15 minutes, sucking upper yellow autologous plasma into a new 50ml centrifuge tube, inactivating at 56 ℃ for 30 minutes, and placing at-20 ℃ for later use; adding equal volume of PBS into the cell layer at the lower layer, and mixing the mixture upside down;
2. sucking 15ml lymphocyte separation liquid (Ficoll-Paque Plus, GE) at the bottom of a new 50ml centrifuge tube, slowly adding 30ml diluted blood to the upper layer of the lymphocyte separation liquid along the tube wall, placing the centrifuge tube in a centrifuge, centrifuging at 450g in a slowly rising and slowly falling way for 25 minutes, no break;
3. after centrifugation, carefully sucking the white membrane layer cells above the lymphocyte separation liquid, transferring the white membrane layer cells into a new 50ml centrifuge tube, adding 50ml PBS for cleaning, and slowly lifting and slowly lowering the centrifugal liquid to centrifuge for 10min;
4. discarding the supernatant, and reserving cell sediment at the bottom of the centrifuge tube; adding 50ml of PBS (phosphate buffer solution) and 160g of PBS again, slowly lifting and slowly lowering, and centrifuging for 15min;
5. discarding the supernatant; finally, adding PBS (phosphate buffer solution) 300g, slowly lifting and slowly lowering, centrifuging for 10min, and discarding the supernatant to obtain the PBMC cells.
2. CD3 positive T cells were activated and sorted from PBMC.
Sorting kit according to CD3 positive T cells (EasySep TM Human T Cell Isolation Kit product number: #17951, vendor: stemcel l) sorting (5) CD3 positive T cells in PBMCs, specifically as follows:
1. cell density was adjusted to 5X 10 7 cells/mL, adding the cells to a 5mL cell freezing tube;
2. adding Isolation Cocktail ul/mL of the separating liquid, uniformly mixing, and standing for 5 minutes;
3. the rapidrays are used by a vortex instrument TM Swirling the reagent for 30 seconds to uniformly mix the particles, adding the particles into cells according to the proportion of 40ul/mL, and uniformly mixing;
4. PBS is added to 2.5mL and blown up and down for 2-3 times, and the mixture is placed on a magnetic frame for 3 minutes;
5. and (3) tilting the magnetic rack to transfer the liquid to a new cell culture dish, so as to obtain the sorted CD3 positive T cells.
3. The Lewis-Y-scFv codons were optimized.
The preparation of a Lewis-Y Chimeric Antigen Receptor (CAR), namely the Lewis-Y-CAR, carries out codon optimization on scFv fragments to ensure that the scFv fragments are easier to express in human cells, and the sequence after the codon optimization is the nucleotide sequence shown as SEQ ID NO. 1.
4. Constructing a lentiviral plasmid skeleton carrying Lewis-Y-CAR and PD-1 antibody genes to obtain a recombinant lentiviral plasmid.
Construction of recombinant lentiviral backbone plasmid (SV 306, see FIG. 7): the CAR structure containing PD-1 antibody is synthesized and cloned on pCDH-CMV-MCS-EF 1-copGGFP skeleton plasmid (vast organism, P0980, see figure 1), after sequencing, SV306 skeleton plasmid is obtained, according to which CMV promoter-Lewis-YscFv-Flag-CD 8TM-CD28-4-1BB-CD3 zeta-EF 1-IL2SS-Anti-PD-1scFv is connected in series, wherein the nucleotide sequence after Lewis-YscFv optimization is shown as SEQ ID NO.1, flag nucleotide sequence is shown as SEQ ID NO.2, CD8TM nucleotide sequence is shown as SEQ ID NO.3, CD28 nucleotide sequence is shown as SEQ ID NO.4, 4-1BB nucleotide sequence is shown as SEQ ID NO.5, CD3 zeta nucleotide sequence is shown as SEQ ID NO.6, IL2SS nucleotide sequence is shown as SEQ ID NO.10, and Anti-PD-1scFv nucleotide sequence is shown as SEQ ID NO. 11.
5. And (3) infecting 293T cells with the recombinant lentiviral plasmid carrying the Lewis-Y-CAR and PD-1 antibody genes obtained in the step (IV) to prepare the recombinant lentiviral vector capable of infecting the T cells.
1. The 293T cells are used for working cells of lentivirus packaging after being subjected to secondary culture for 2-3 generations after being resuscitated, and the total number of the secondary culture times is not more than 10 generations;
2. liposome transfection (10 cm Petri dish for example)
1) The 293T cells are inoculated into a culture dish with the diameter of 10cm the day before transfection, and the quantity of the inoculated cells is preferably such that the cell growth on the day of transfection reaches 90-95% fusion;
2) On the day of transfection, the culture solution is removed, and 5ml of culture solution for virus packaging is added;
3) Preparation of DNA-Lipofectamine2000 complex:
A. a sterile 5mL centrifuge tube was prepared, and 1.5mL serum-free Opti-MEM culture medium, helper plasmids (pRSV-Rev (adedge, # 12253), pCMV-VSV-G (adedge, # 8454), pMDLg-pRRE (adedge, # 12251), FIGS. 3-5, respectively, and lentiviral expression plasmids (SV 306, FIG. 7) were added and thoroughly blown and mixed;
B. preparing another sterile 5mL centrifuge tube, adding 1.5mL serum-free Opti-MEM culture solution and 68 mu L Lipofectamine2000, gently stirring, mixing, and incubating at room temperature for 10min;
after c.30 minutes, adding the serum-free Opti-MEM culture solution containing plasmid DNA into the serum-free Opti-MEM culture solution containing Lipofectamine2000, gently stirring and mixing, and incubating for 30 minutes at room temperature;
D. the DNA-Lipofectamine2000 complexes were added drop by drop to 293T cells and the dishes were gently shaken back and forth to mix the complexes. Placing at 37deg.C 5% CO 2 Culturing in a saturated humidity incubator.
3. Collection and concentration of recombinant lentiviral vectors
1) The culture broth containing the virus was collected 48 hours after transfection. Sucking the culture solution into a sterile 50mL centrifuge tube with a cover, and adding 8mL of culture solution for virus packaging into cells capable of producing viruses;
2) Centrifuging at 3000rpm for 15 minutes at 4 ℃, centrifuging the virus supernatant at a low speed, removing cell debris, and recovering the supernatant;
3) The culture broth containing the virus was collected 72 hours after transfection. The mixture was centrifuged at 3000rpm at 4℃for 15 minutes, and the virus supernatant was centrifuged at a low speed to remove cell debris and collect the supernatant. The virus supernatant collected at 48 hours and the virus supernatant collected at 72 hours were filtered with a small filter of 0.45 μm, and the filtrate was collected with a 50mL centrifuge tube;
4) Concentrating the collected virus supernatant by an ultracentrifuge, and centrifuging for 120 minutes at a temperature of 72000g/min at a temperature of 4 ℃; the viral pellet was resuspended in 500ul fresh medium, sub-packaged, frozen and the virus titer was determined from the fractions.
6. And D, transducing the recombinant lentiviral vector obtained in the step five into T cells selected in the step two to generate CAR-T cells which express the Lewis-Y-CAR and secrete PD-1 antibodies.
Activation of T cells
(1) Sorting T cells at a density of 0.5 to 1X 10 6 cells/ml was resuspended in KBM 581 Medium (cat No. 88-581-CM, corning) supplemented with 1% autologous plasma;
(2) Wash Dynabeads:
a) Taking Dynabeads out of the refrigerator, oscillating for 30 seconds by using a vortex instrument, and uniformly re-suspending the magnetic beads deposited at the bottom;
b) According to the number of T cells in (1), the required Dynabeads were calculated according to the ratio of Dynabeads: T=1:1 to 1:3, and Dynabeads were transferred into a new 15ml centrifuge tube using a sterile pipette, and DPBS of the same volume was added, and the mixture was blown and homogenized using a pipette. Placing the centrifuge tube on a magnetic rack, standing for 5 minutes, and sucking the liquid by using a pipette;
c) Adding 2ml of DPBS into a 15ml centrifuge tube, and repeating the washing step in b);
d) Adding 581 culture medium of 100ul to resuspend beads, adding dropwise into cell culture solution, blowing and mixing uniformly, and standing at 37deg.C, 5% CO 2 Culturing in a cell incubator for 3 days;
2. transduction of T cells with recombinant viral vectors
(1) T cells were counted 3 days after T cell activation by trypan blue staining. The required viral load was calculated as moi=3-10: required viral load (ml) = (MOI cell number)/viral titer (Tu/ml).
(2) Taking out slow virus from-80 deg.C refrigerator, quickly thawing in 37 deg.C water bath (without making pipe orifice deep below liquid surface);
(3) Taking out the T cell culture dish, adding polybrene to a final concentration of 6ug/ml, adding the required virus amount, lightly blowing and mixing by a liquid transfer device to uniformly disperse the aggregated cell mass, sealing the culture dish by using a sealing film, and centrifuging at 800g for 1 hour under the room temperature condition;
(4) Removing the sealing film, placing the culture dish at 37deg.C, 5% CO 2 Culturing in an incubator for 24 hours;
(5) Centrifugation at 250g for 10min at room temperature, removal of the virus-containing medium supernatant, resuspension of the cell pellet with fresh medium, transfer of the cells to a new dish and further culture.
7. And (3) culturing the cells obtained in the step (six) in vitro.
The cell mass in the culture system was gently blown daily to disperse the cells, and the cell number was counted by trypan blue staining to maintain the cell density at 1X 10 6 And each ml.
8. Amplifying the cells obtained in the step seven in a large quantity.
a) When the cell number is greater than 100X 10 6 At the time, transferring the CAR-T cells into a cell culture bag for culture; cells were counted by trypan blue counting every other day, maintaining cell density at 1×10 6 Individual/ml;
b) When the CAR-T cells are cultured for 10 days, part of cell culture solution can be extracted for sterile detection;
c) When the total number of cells reaches 500-1000×10 6 When the CAR-T cells are collected, the magnetic beads are removed by using the magnetic poles;
d) After taking part of the cell suspension and adding BFA for 24 hours, ELISA detects the secretion of PD-1 in the supernatant (see figure 8), and compared with a control group, the experimental group secretes PD-1, which shows that the CAR-T cells expressing Lewis-Y-CAR and secreting PD-1 antibodies are successfully prepared, and the cells are subjected to expansion culture and frozen.
EXAMPLE 3 targeting Lewis-Y-CAR-T cells secreting IL12 or PD-1 antibodies for immunotherapy
1. Detecting whether tumor tissue cells of a tumor patient highly express Lewis-Y by an immunohistochemical method, wherein the Lewis-Y has high expression in various tumors, such as lung cancer and gastric cancer, and all tumor patients which highly express Lewis-Y antigen can be treated by adopting targeted Lewis-Y-CAR-T cells capable of secreting IL12 or PD-1 antibodies;
2. collecting peripheral blood of a patient and separating T cells;
CAR-T cell preparation CAR-T cells were prepared in GMP workshops. After activation of isolated T cells with CD3/CD28 beads, T cells were transduced with lentiviruses at moi=3-10 and CAR-T cells expanded in vitro;
after the CAR-T cells are cultured in vitro to meet the number of cells for treatment, extracting part of the T cells, performing sterile detection, performing phenotype detection on the T cells, and enabling the T cells to be used for treatment after meeting the release standard of the CAR-T cells for clinical treatment;
5. the CAR-T cells are reinfused back to the patient by intravenous infusion.
In vitro killing verification of Lewis-Y-CAR-expressing IL 12-secreting CAR-T cells and Lewis-Y-CAR-expressing PD-1 antibody-secreting CAR-T cells prepared in example 4
The CAR-T cells expressing Lewis-Y-CAR and secreting IL12 at the same time, and the CAR-T cells expressing Lewis-Y-CAR and secreting PD-1 antibody at the same time, and the CAR-T cells expressing Lewis-Y-CAR were cultured separately.
Collection of target cells Raji 4X 10 5 cells and effector cells (CAR-T cells) were each 3X 10 6 centrifuging for 10min, slowly lifting and lowering, and discarding supernatant; respectively re-suspending target cells and effector cells with 1ml PBS solution, centrifuging for 10min, slowly lifting and lowering, and discarding supernatant; repeating the process once; effector cells were resuspended in 700. Mu.l medium (AIM-V medium+10% FBS) and target cells were resuspended in 2ml medium (1640 medium+10% FBS).
Setting experimental holes with effective target ratios of 1:1, 5:1, 10:1 and 20:1, and setting control groups with 3 compound holes at 37 ℃ and 5% CO 2 Culturing in an incubator for 2 hours; 500g, centrifuging for 5min, and centrifuging by a slow-lifting slow-lowering flat plate; mu.l of the supernatant from each well was taken into a new 96-well plate, and 50. Mu.l of substrate solution (working in the dark) was added to each well and incubated at room temperature for 15min in the dark; 50 μl of stop solution was added to each well, and absorbance at 490nm was measured by an ELISA reader. Under different effective target ratio conditions, the CAR-T cells expressing the Lewis-Y-CAR and secreting IL12 simultaneously, the CAR-T cells expressing the Lewis-Y-CAR and secreting PD-1 antibodies obviously have higher killing efficiency in Raji target cells than the CAR-T cells expressing the Lewis-Y-CAR,when the effective target ratio is 20:1, the CAR-T cells expressing the Lewis-Y-CAR and secreting IL12 simultaneously prepared by the invention have 58% and 64% of the tumor killing capacity of the CAR-T cells expressing the Lewis-Y-CAR and secreting PD-1 antibodies simultaneously (see figure 9).
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Sequence listing
<110> Souzhou Maoxing biotechnology Co., ltd
<120> a secretory Lewis-Y targeting CAR-T cell, and preparation method and application thereof
<160> 11
<170> SIPOSequenceListing 1.0
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atggagttcg ggctgagatg ggtgtttctg gtggccatcc tgaaggacgt gcagtgcgag 60
gtgcagctgg tggagagcgg ggggggagtg gtgcagcctg gaaggagcct gagactgagc 120
tgtagcacaa gcggctttac cttttctgat tattacatgt actgggtgag gcaggccccc 180
ggaaagggcc tggagtgggt ggcttatatg tccaacgtgg gcgccattac agactacccc 240
gacacagtga agggcagatt caccattagc cgggacaaca gcaaaaacac actgttcctg 300
cagatggaca gcctgagacc cgaggacacc ggcgtgtact tctgcgccag aggcaccaga 360
gacggcagct ggttcgccta ctggggccag ggaacacccg tgaccgtgag cagcggcggc 420
ggaggaagcg gaggaggagg aagcggcgga ggaggcagcg acatccagat gacccagagc 480
cctagcagtc tgagtgccag cgtgggcgac agagtgacca tcacctgcag aagcagtcag 540
agaatcgtgc acagcaacgg aaatacatac ctggagtggt accagcagac acccggcaaa 600
gcccccaaac tgctgatcta caaggtgagc aatagattca gcggcgtgcc cagcagattc 660
agcggaagcg gaagcggcac cgacttcacc ttcactatca gcagcctgca gcccgaagat 720
atcgcaacct actactgctt ccaggggagc cacgtgccat tcaccttcgg ccagggaacc 780
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gattacaagg acgacgatga caag 24
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ttcgtccccg tgttcctgcc tgccaagcca acaactaccc ctgctccacg accacctact 60
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gctggaggag cagtgcacac ccgaggcctg gacttcgcat gcgatatcta catttgggca 180
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aggagcaaac gctcccgact gctgcattcc gactacatga acatgacacc tcggagacca 60
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<210> 6
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<212> DNA
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agagtgaagt tcagccggag cgccgatgca ccagcatatc agcagggaca gaatcagctg 60
tacaacgagc tgaatctggg caggcgcgag gaatatgacg tgctggataa gcgacgagga 120
cgggaccccg aaatgggagg aaaacccaga aggaagaacc ctcaggaggg gctgtataat 180
gaactgcaga aagacaagat ggctgaggca tacagcgaaa ttggaatgaa aggagagcgc 240
cgacggggga agggacacga tgggctgtac cagggactgt caaccgccac taaagatacc 300
tacgacgcac tgcacatgca ggctctgccc ccaaga 336
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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ggaggaaaaa ctgtttcata cagaaggcgg gaggaaaaac tgtttcatac agaaggcggg 60
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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attttgacac ccccataata tttttccaga attaacagta taaattgcat ctcttgttca 60
agagttccct atcactctct ttaatcacta ctcacagtaa cctcaactcc tgccacaacc 120
ggtatgtgtc accagcagtt ggtcatctct tggttttccc tggtttttct ggcatctccc 180
ctcgtggcca tatgggaact gaagaaagat gtttatgtcg tagaattgga ttggtatccg 240
gatgcccctg gagaaatggt ggtcctcacc tgtgacaccc ctgaagaaga tggtatcacc 300
tggaccttgg accagagcag tgaggtctta ggctctggca aaaccctgac catccaagtc 360
aaagagtttg gagatgctgg ccagtacacc tgtcacaaag gaggcgaggt tctaagccat 420
tcgctcctgc tgcttcacaa aaaggaagat ggaatttggt ccactgatat tttaaaggac 480
cagaaagaac ccaaaaataa gacctttcta agatgcgagg ccaagaatta ttctggacgt 540
ttcacctgct ggtggctgac gacaatcagt actgatttga cattcagtgt caaaagcagc 600
agaggctctt ctgaccccca aggggtgacg tgcggagctg ctacactctc tgcagagaga 660
gtcagagggg acaacaagga gtatgagtac tcagtggagt gccaggagga cagtgcctgc 720
ccagctgctg aggagagtct gcccattgag gtcatggtgg atgccgttca caagctcaag 780
tatgaaaact acaccagcag cttcttcatc agggacatca tcaaacctga cccacccaag 840
aacttgcagc tgaagccatt aaagaattct cggcaggtgg aggtcagctg ggagtaccct 900
gacacctgga gtactccaca ttcctacttc tccctgacat tctgcgttca ggtccagggc 960
aagagcaaga gagaaaagaa agatagagtc ttcacggaca agacctcagc cacggtcatc 1020
tgccgcaaaa atgccagcat tagcgtgcgg gcccaggacc gctactatag ctcatcttgg 1080
agcgaatggg catctgtgcc ctgcaggggc ggaggcggaa gcggaggcgg aggaagcggc 1140
ggtggcggca gcagaaacct ccccgtggcc actccagacc caggaatgtt cccatgcctt 1200
caccactccc aaaacctgct gagggccgtc agcaacatgc tccagaaggc cagacaaact 1260
ctagaatttt acccttgcac ttctgaagag attgatcatg aagatatcac aaaagataaa 1320
accagcacag tggaggcctg tttaccattg gaattaacca agaatgagag ttgcctaaat 1380
tccagagaga cctctttcat aactaatggg agttgcctgg cctccagaaa gacctctttt 1440
atgatggccc tgtgccttag tagtatttat gaagacttga agatgtacca ggtggagttc 1500
aagaccatga atgcaaagct tctgatggat cctaagaggc agatctttct agatcaaaac 1560
atgctggcag ttattgatga gctgatgcag gccctgaatt tcaacagtga gactgtgcca 1620
caaaaatcct cccttgaaga accggatttt tataaaacta aaatcaagct ctgcatactt 1680
cttcatgctt tcagaattcg ggcagtgact attgatagag tgatgagcta tctgaatgct 1740
tcctaa 1746
<210> 9
<211> 546
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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aaggatctgc gatcgctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc 60
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aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc 180
gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac 240
acagctgaag cttcgagggg ctcgcatctc tccttcacgc gcccgccgcc ctacctgagg 300
ccgccatcca cgccggttga gtcgcgttct gccgcctccc gcctgtggtg cctcctgaac 360
tgcgtccgcc gtctaggtaa gtttaaagct caggtcgaga ccgggccttt gtccggcgct 420
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gcctac 546
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<211> 60
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacgaactcg 60
<210> 11
<211> 2196
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
atggagttct ggttgtcatg ggtctttctg gtagctattc ttaagggagt acagtgtcag 60
gtgcagctgg tgcagagcgg cgtggaagtg aaaaaaccgg gcgcgagcgt gaaagtgagc 120
tgcaaagcga gcggctatac ctttaccaac tattatatgt attgggtgcg ccaggcgccg 180
ggccagggcc tggaatggat gggcggcatt aacccgagca acggcggcac caactttaac 240
gaaaaattta aaaaccgcgt gaccctgacc accgatagca gcaccaccac cgcgtatatg 300
gaactgaaaa gcctgcagtt tgatgatacc gcggtgtatt attgcgcgcg ccgcgattat 360
cgctttgata tgggctttga ttattggggc cagggcacca ccgtgaccgt gagcagcgcg 420
agcaccaaag gcccgagcgt gtttccgctg gcgccgtgca gccgcagcac cagcgaaagc 480
accgcggcgc tgggctgcct ggtgaaagat tattttccgg aaccggtgac cgtgagctgg 540
aacagcggcg cgctgaccag cggcgtgcat acctttccgg cggtgctgca gagcagcggc 600
ctgtatagcc tgagcagcgt ggtgaccgtg ccgagcagca gcctgggcac caaaacctat 660
acctgcaacg tggatcataa accgagcaac accaaagtgg ataaacgcgt ggaaagcaaa 720
tatggcccgc cgtgcccgcc gtgcccggcg ccggaatttc tgggcggccc gagcgtgttt 780
ctgtttccgc cgaaaccgaa agataccctg atgattagcc gcaccccgga agtgacctgc 840
gtggtggtgg atgtgagcca ggaagatccg gaagtgcagt ttaactggta tgtggatggc 900
gtggaagtgc ataacgcgaa aaccaaaccg cgcgaagaac agtttaacag cacctatcgc 960
gtggtgagcg tgctgaccgt gctgcatcag gattggctga acggcaaaga atataaatgc 1020
aaagtgagca acaaaggcct gccgagcagc attgaaaaaa ccattagcaa agcgaaaggc 1080
cagccgcgcg aaccgcaggt gtataccctg ccgccgagcc aggaagaaat gaccaaaaac 1140
caggtgagcc tgacctgcct ggtgaaaggc ttttatccga gcgatattgc ggtggaatgg 1200
gaaagcaacg gccagccgga aaacaactat aaaaccaccc cgccggtgct ggatagcgat 1260
ggcagctttt ttctgtatag ccgcctgacc gtggataaaa gccgctggca ggaaggcaac 1320
gtgtttagct gcagcgtgat gcatgaagcg ctgcataacc attataccca gaaaagcctg 1380
agcctgagcc tgggcaaaga attcgaagga tccgcggccg ctgagggcag aggaagtctt 1440
ctaacatgcg gtgacgtgga ggagaatccc ggcccttccg ggatggagtt ctggttgtca 1500
tgggtctttc tggtagctat tcttaaggga gtacagtgtg aaattgtgct gacccagagc 1560
ccggcgaccc tgagcctgag cccgggcgaa cgcgcgaccc tgagctgccg cgcgagcaaa 1620
ggcgtgagca ccagcggcta tagctatctg cattggtatc agcagaaacc gggccaggcg 1680
ccgcgcctgc tgatttatct ggcgagctat ctggaaagcg gcgtgccggc gcgctttagc 1740
ggcagcggca gcggcaccga ttttaccctg accattagca gcctggaacc ggaagatttt 1800
gcggtgtatt attgccagca tagccgcgat ctgccgctga cctttggcgg cggcaccaaa 1860
gtggaaatta aacgcaccgt ggcggcgccg agcgtgttta tttttccgcc gagcgatgaa 1920
cagctgaaaa gcggcaccgc gagcgtggtg tgcctgctga acaactttta tccgcgcgaa 1980
gcgaaagtgc agtggaaagt ggataacgcg ctgcagagcg gcaacagcca ggaaagcgtg 2040
accgaacagg atagcaaaga tagcacctat agcctgagca gcaccctgac cctgagcaaa 2100
gcggattatg aaaaacataa agtgtatgcg tgcgaagtga cccatcaggg cctgagcagc 2160
ccggtgacca aaagctttaa ccgcggcgaa tgctag 2196
Claims (9)
1. A secreted Lewis-Y-targeted CAR-T cell, characterized in that said CAR-T cell is a CAR-T cell expressing a Lewis-Y-CAR and secreting a PD-1 antibody or a CAR-T cell expressing a Lewis-Y-CAR and secreting an IL12 cytokine, said Lewis-Y-CAR comprising an scFv region for recognition of a Lewis-Y antigen, the scFv region sequence being an optimized nucleotide sequence as shown in SEQ ID No. 1.
2. A method of preparing a secretory Lewis-Y targeted CAR-T cell according to claim 1, comprising the steps of:
(1) Isolating PBMCs from peripheral blood provided by the donor;
(2) Activating and sorting CD3 positive T cells from the PBMC;
(3) Constructing a lentiviral plasmid skeleton carrying Lewis-Y-CAR and antibody or cytokine genes to obtain a recombinant lentiviral plasmid;
(4) Transfecting the recombinant lentiviral plasmid and auxiliary plasmid carrying the Lewis-Y-CAR and antibody or cytokine genes obtained in the step (3) into host cells to prepare a recombinant lentiviral vector capable of infecting T cells;
(5) Transducing the recombinant lentiviral vector obtained in step (4) into the activated T cells selected in step (2) to produce CAR-T cells expressing Lewis-Y-CAR and secreting antibodies or cytokines;
(6) Culturing the cells obtained in the step (5) in vitro;
(7) Amplifying the cells obtained in step (6) in large numbers;
(8) Collecting CAR-T cells expressing the Lewis-Y-CAR that secrete antibodies or cytokines at the same time.
3. The method of claim 2, wherein in step (3), the recombinant lentiviral plasmid is a second or third generation lentiviral transgene plasmid.
4. The method of claim 2, wherein in step (3), the recombinant lentiviral plasmid is a third generation lentiviral transgene plasmid; the recombinant lentiviral plasmid is a lentiviral transgenic plasmid targeting Lewis-Y antigen and secreting IL12 cytokine, and sequentially comprises a CMV promoter sequence, lewis-Y scFv, flag, CD8 chimeric receptor transmembrane region, intracellular signal transduction region, NFAT promoter sequence and IL12 cytokine sequence; the recombinant lentiviral vector described in step (5) expresses a chimeric antigen receptor targeting the Lewis-Y antigen on the surface of a T cell while being capable of secreting IL12 cytokine.
5. The method of claim 2, wherein in step (3), the recombinant lentiviral plasmid is a third generation lentiviral transgene plasmid; the recombinant lentiviral plasmid is a lentiviral transgenic plasmid targeting Lewis-Y antigen and secreting PD-1 antibody, and sequentially comprises a CMV promoter sequence, lewis-Y scFv, flag, CD8 chimeric receptor transmembrane region, intracellular signal transduction region, EF1 alpha promoter sequence, IL2SS sequence and PD-1 antibody sequence; the recombinant lentiviral vector described in step (5) expresses a chimeric antigen receptor targeting the Lewis-Y antigen on the surface of a T cell while being capable of secreting the PD-1 antibody.
6. The method of claim 4 or 5, wherein the intracellular signaling region comprises a region encoding a co-stimulatory factor selected from the group consisting of 4-1BB, CD28, CD27, OX40, CD30, CD40, PD-1, ICOS, LIGHT, B7-H3, a ligand that specifically binds CD83, ICAM-1, HVEM (light), CD160, lymphocyte function-associated antigen-1 (LFA-1), IL2 ra, CD103, CD11B, CD11c, TRANCE/RANKL, SLAMF4 (CD 244,2B 4), CD69, SLAM (SLAMF 1, CD150, IPO-3), or any combination thereof.
7. The method of claim 4 or 5, wherein the intracellular signaling region is CD28-4-1BB-CD3 ζ.
8. The method of claim 2, wherein in step (4), there are three helper plasmids, pRSV-Rev, pCMV-VSV-G, pMDLg-pRRE, respectively, and the host cells are 293FT or 293T cells.
9. A mixed formulation comprising a Lewis-Y-CAR expressing CAR-T cell or a Lewis-Y-CAR expressing CAR-T cell that simultaneously secretes IL-12 of claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN114887080B (en) * | 2020-12-08 | 2023-10-31 | 深圳瑞吉生物科技有限公司 | Immunosuppressant of mRNA dosage form and application thereof in preparing tumor therapeutic drug |
| CN114181971A (en) * | 2021-11-22 | 2022-03-15 | 东莞市麦亘生物科技有限公司 | Preparation method and application of targeted TRBC1CAR-T cell |
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