CN117402231B - Receptor capable of spontaneously transmitting IL-21 signal and application thereof - Google Patents
Receptor capable of spontaneously transmitting IL-21 signal and application thereof Download PDFInfo
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- CN117402231B CN117402231B CN202311716900.1A CN202311716900A CN117402231B CN 117402231 B CN117402231 B CN 117402231B CN 202311716900 A CN202311716900 A CN 202311716900A CN 117402231 B CN117402231 B CN 117402231B
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to a receptor capable of spontaneously transmitting IL-21 signals and application thereof, belonging to the technical field of biology. The invention provides a receptor capable of spontaneously transmitting IL-21 signals, which comprises an extracellular segment with an amino acid sequence shown as SEQ ID NO.1, a transmembrane segment with an amino acid sequence shown as SEQ ID NO.2 and/or an intracellular segment with an amino acid sequence shown as SEQ ID NO. 3. The research shows that when the receptor is expressed on T cells, the receptor can spontaneously dimerize on the surfaces of the T cells, and the IL-21 signaling pathway is spontaneously activated through the intracellular segment structure of the IL-21Rα, so that the engineering T cells expressing the receptor can obtain stronger viability, tumor infiltration capacity, anti-apoptosis capacity and anti-tumor function, simultaneously avoid side effects caused by injecting exogenous IL-21 cytokines, and have great application prospects in the field of tumor treatment.
Description
Technical Field
The invention relates to a receptor capable of spontaneously transmitting IL-21 signals and application thereof, belonging to the technical field of biology.
Background
Adoptive cellular immunotherapy (adoptive cell transfer therapy, ACT) is a method of treating tumors by infusing ex vivo activated and expanded autologous or allogeneic immune effector cells into patients, and is suitable for patients with hypoimmunity, such as patients with impaired immune cell numbers and functions after high dose chemotherapy, radiotherapy, bone marrow transplantation, and viral infection, especially patients with tumors of the blood/immune system.
Cells used in adoptive cellular immunotherapy can be divided into two classes. The first category is tumor antigen non-specific immune cells, including lymphokine activated killer cells (lymphokine activated killer, LAK), cytokine mediated killer cells (cytokine induced killer, CIK) and natural killer cells (natural killer cell, NK), which are mainly obtained by peripheral blood separation and proliferation stimulation in vitro, and show a certain effect in tumor treatment, but since the adoptively infused cells have no antigen specificity, their antitumor effect is limited. The second category is tumor antigen specific T cells, including tumor-infiltrating lymphocytes (tumor infiltrating lymphocytes, TIL), T Cell Receptor (TCR) genetically engineered T cells (TCR-T), chimeric antigen receptor therapy (chimeric antigen receptor, CAR) genetically engineered T cells (CAR-T), the main sources of such cells being solid tumor tissue obtained by surgical excision, infiltrating lymph nodes and cancerous chest/ascites, wherein tumor-infiltrating lymphocytes have tumor antigen recognition specificity and have a stronger anti-tumor effect than tumor antigen non-specific immune cells, but not all patients are able to isolate and expand enough tumor-infiltrating lymphocytes, severely limiting their clinical application in tumor therapy (see literature: zhang R, zhang Z, liu Z, wei D, wu X, bian H, chen Z Adoptive cell transfer therapy for hepatocellular carcinoma. Front med 2019 feb;13 (1): 3-11.). Thus, T cell receptor genetically engineered T cells and chimeric antigen receptor therapeutic genetically engineered T cells are becoming research hotspots for adoptive immunotherapy of tumors.
However, unlike non-solid tumors such as hematological tumors, a broad inhibitory tumor immune microenvironment exists around solid tumors such as liver cancer, and this corresponding immune microenvironment can significantly inhibit the sustained survival and killing ability of T cells in vivo, inducing the occurrence of T cell depletion (see literature: newick K, O' Brien S, moon E, albelda SM. CAR T Cell Therapy for Solid Tumors. Annu Rev Med. 2017 Jan 14;68:139-152.). Therefore, how to make genetically engineered T cells able to overcome the inhibition of tumor microenvironment within solid tumors is an unavoidable problem of genetically engineered T cells in clinical solid tumor therapeutic applications.
Cytokines such as IL-2, IL-7, IL-15 and IL-21 play a very important role in the production, development, maturation, proliferation and differentiation of T cells. Among them, IL-21 and IL-2, IL-7 and IL-15 are similar, and belong to a member of The gamma receptor factor family, mainly derived from CD4 positive T cells and NKT cells (see literature: leonard WJ, lin JX, O' Shea JJ. The. Gamma. C Family of Cytokines: basic Biology to Therapeutic Ramisitives. Immunity. 2019 Apr 16;50 (4): 832-850.). IL-21 has been shown to have a crucial role in the control of chronic viral infections in maintaining the proliferative capacity and effector function of T cells in the body during long-term chronic infections (see, e.g., elsaesser H, sauer K, brooks DG. IL-21 is required to control chronic viral infection. Science 2009 Jun 19;324 (5934): 1569-72.). Thus, there are studies on the application of IL-21 to T cell immune cell therapy, solving the problems of genetically engineered T cells in clinical solid tumor therapy by cytokine combination strategy, and it was concluded that IL-21 signal is essential for the antitumor function of the organism in solid tumors, and that providing IL-21 signal into solid tumors can promote the effector function of CD8 positive T cells in solid tumors, enhancing the antitumor effect thereof (see literature: cui C, wang J, fagerberg E, chen PM, connolly KA, damo M, cheung JF, mao T, askari AS, chen S, fitzgerald B, foster GG, eisenbarth SC, zhao H, craf J, jo NS. Neantigen-driven B cell and CD 4T follicular helper cell collaboration promotes anti-tumor CD 8T cell responses cell 1 Dec 9;184 (25): 6101-6118.6118. However, the anti-tumor effect achieved by directly infusing IL-21 into a tumor patient in a practical clinic is still limited, and autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, type I diabetes and the like may be caused (see documents: spolski, R., leonard, W. Intereukin-21: a double-edged sword with therapeutic potential Nat Rev Drug Discov, 379-395 (2014): https:// doi. Org/10.1038/nrd 4296), which limit the clinical application of IL-21 in the treatment of tumors, especially solid tumors, by combining genetically engineered T cells.
Disclosure of Invention
In order to solve the problems, the invention provides a receptor capable of spontaneously transmitting IL-21 signals, which comprises an extracellular segment with an amino acid sequence shown as SEQ ID NO.1, a transmembrane segment with an amino acid sequence shown as SEQ ID NO.2 and/or an intracellular segment with an amino acid sequence shown as SEQ ID NO. 3.
In one embodiment of the invention, the receptor is formed by sequentially connecting an extracellular segment with an amino acid sequence shown as SEQ ID NO.1, a transmembrane segment with an amino acid sequence shown as SEQ ID NO.2 and an intracellular segment with an amino acid sequence shown as SEQ ID NO. 3.
The invention also provides a nucleic acid molecule encoding a receptor as described above that spontaneously transmits an IL-21 signal.
In one embodiment of the invention, the nucleotide sequence of the nucleic acid molecule encoding the extracellular segment is shown in SEQ ID NO.4, the nucleotide sequence of the nucleic acid molecule encoding the transmembrane segment is shown in SEQ ID NO.5, and the nucleotide sequence of the nucleic acid molecule encoding the intracellular segment is shown in SEQ ID NO. 6.
The invention also provides a recombinant plasmid carrying the nucleic acid molecule.
In one embodiment of the invention, the recombinant plasmid further carries a nucleic acid molecule encoding an AFP-TCR; the AFP-TCR refers to the alpha and beta chains of T cell receptors that target alpha-fetoprotein (AFP).
In one embodiment of the invention, the amino acid sequence of the AFP-TCR is shown in SEQ ID NO. 7.
In one embodiment of the invention, the nucleotide sequence of the nucleic acid molecule encoding the AFP-TCR is shown in SEQ ID NO. 8.
In one embodiment of the invention, the nucleic acid molecule is linked to a nucleic acid molecule encoding an AFP-TCR by a nucleic acid molecule encoding a self-cleaving peptide.
In one embodiment of the invention, the self-cleaving peptide comprises a 2A peptide; the 2A peptide includes P2A peptide, T2A peptide, E2A peptide and/or F2A peptide.
In one embodiment of the invention, the amino acid sequence of the P2A peptide is shown in SEQ ID NO. 9.
In one embodiment of the invention, the vector of the recombinant plasmid is a lentiviral expression vector, a retroviral expression vector, or an adenoviral expression vector.
In one embodiment of the invention, the recombinant plasmid vector is a pCDH-EF1 a lentiviral expression vector.
In one embodiment of the invention, the nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO. 10.
The invention also provides a host cell transfected with the recombinant plasmid; alternatively, the genome of the host cell has the nucleic acid molecule described above integrated.
In one embodiment of the invention, the host cell is co-transfected with the recombinant plasmid described above and a lentiviral packaging plasmid; alternatively, the genome of the host cell has integrated therein a nucleic acid molecule as described above and a nucleic acid molecule encoding an AFP-TCR; the AFP-TCR refers to the alpha and beta chains of T cell receptors that target alpha fetoprotein.
In one embodiment of the invention, the amino acid sequence of the AFP-TCR is shown in SEQ ID NO. 7.
In one embodiment of the invention, the nucleotide sequence of the nucleic acid molecule encoding the AFP-TCR is shown in SEQ ID NO. 8.
In one embodiment of the invention, the nucleic acid molecule is linked to a nucleic acid molecule encoding an AFP-TCR by a nucleic acid molecule encoding a self-cleaving peptide.
In one embodiment of the invention, the self-cleaving peptide comprises a 2A peptide; the 2A peptide includes P2A peptide, T2A peptide, E2A peptide and/or F2A peptide.
In one embodiment of the invention, the amino acid sequence of the P2A peptide is shown in SEQ ID NO. 9.
In one embodiment of the invention, the host cell is a HEK-293T cell, a Jurkat cell, an NK cell or a primary human T cell.
The invention also provides a recombinant virus, the genome of which carries the nucleic acid molecule.
In one embodiment of the invention, the genome of the virus also carries a nucleic acid molecule encoding an AFP-TCR; the AFP-TCR refers to the alpha and beta chains of T cell receptors that target alpha fetoprotein.
In one embodiment of the invention, the amino acid sequence of the AFP-TCR is shown in SEQ ID NO. 7.
In one embodiment of the invention, the nucleotide sequence of the nucleic acid molecule encoding the AFP-TCR is shown in SEQ ID NO. 8.
In one embodiment of the invention, the nucleic acid molecule is linked to a nucleic acid molecule encoding an AFP-TCR by a nucleic acid molecule encoding a self-cleaving peptide.
In one embodiment of the invention, the self-cleaving peptide comprises a 2A peptide; the 2A peptide includes P2A peptide, T2A peptide, E2A peptide and/or F2A peptide.
In one embodiment of the invention, the amino acid sequence of the P2A peptide is shown in SEQ ID NO. 9.
In one embodiment of the invention, the recombinant virus expresses the above nucleic acid molecule using a lentivirus as a vector.
In one embodiment of the invention, the recombinant virus expresses the above nucleic acid molecule as well as a nucleic acid molecule encoding an AFP-TCR using a lentivirus as a vector.
In one embodiment of the present invention, the method for preparing a recombinant virus comprises: culturing host cells co-transfected with the recombinant plasmid and the lentiviral packaging plasmid to obtain a culture solution; the recombinant virus is isolated from the culture solution.
The invention also provides a genetically engineered T cell carrying the nucleic acid molecule or the receptor spontaneously transmitting IL-21 signal.
In one embodiment of the invention, the genetically engineered T cell further carries a nucleic acid molecule encoding an AFP-TCR, or the genetically engineered T cell further carries an AFP-TCR; the AFP-TCR refers to the alpha and beta chains of T cell receptors that target alpha fetoprotein.
In one embodiment of the invention, the amino acid sequence of the AFP-TCR is shown in SEQ ID NO. 7.
In one embodiment of the invention, the nucleotide sequence of the nucleic acid molecule encoding the AFP-TCR is shown in SEQ ID NO. 8.
In one embodiment of the invention, the nucleic acid molecule is linked to a nucleic acid molecule encoding an AFP-TCR by a nucleic acid molecule encoding a self-cleaving peptide.
In one embodiment of the invention, the self-cleaving peptide comprises a 2A peptide; the 2A peptide includes P2A peptide, T2A peptide, E2A peptide and/or F2A peptide.
In one embodiment of the invention, the amino acid sequence of the P2A peptide is shown in SEQ ID NO. 9.
In one embodiment of the present invention, the method for preparing a genetically engineered T cell comprises: and (3) infecting the T cells by using the recombinant viruses to obtain genetically engineered T cells.
The invention also provides the application of the receptor spontaneously transmitting IL-21 signals or the nucleic acid molecules or the recombinant plasmids or the host cells or the recombinant viruses or the genetically engineered T cells in preparing tumor therapeutic drugs.
In one embodiment of the invention, the tumor comprises a solid tumor and/or a non-solid tumor.
In one embodiment of the invention, the solid tumor comprises liver cancer, melanoma, lymphoma, lung cancer, colorectal cancer and/or pancreatic cancer; the non-solid tumor comprises a hematological tumor and/or a malignant lymphoma; the hematological neoplasm includes leukemia and/or myeloma; the leukemia includes acute leukemia and/or chronic leukemia; the myeloma includes single myeloma and/or multiple myeloma.
The technical scheme of the invention has the following advantages:
the invention provides a receptor capable of spontaneously transmitting IL-21 signals, which comprises an extracellular segment with an amino acid sequence shown as SEQ ID NO.1, a transmembrane segment with an amino acid sequence shown as SEQ ID NO.2 and/or an intracellular segment with an amino acid sequence shown as SEQ ID NO. 3. The research shows that when the receptor capable of spontaneously transmitting IL-21 signals is expressed on T cells, the receptor can spontaneously dimerize on the surfaces of the T cells, and the IL-21 signal path is spontaneously activated through the intracellular segment structure of IL-21R alpha, so that the engineering T cells expressing the receptor can obtain stronger viability, tumor infiltration capacity, anti-apoptosis capacity and anti-tumor function, simultaneously avoid side effects caused by injecting exogenous IL-21 cytokines, and have great application prospects in the tumor treatment field. In addition, when a cytokine is used to enhance the function of T cells, it is necessary to infuse a free IL-21 cytokine into a patient, and this method has a limited antitumor effect and brings about a serious side effect (for example, autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, type I diabetes, etc.) which limits the clinical application of IL-21 in combined immune cell therapy, and the receptor spontaneously transmitting IL-21 signal does not depend on exogenous IL-21, and can exert a corresponding effect of enhancing the function of T cells by coexpression with TCR or CAR on the surface of T cells.
Drawings
Fig. 1: effects of different cytokine combinations on the results of co-culture of AFP-TCR-T with HepG 2. In fig. 1, a is a schematic diagram of a multiple round of repeated co-culture experiments of AFP-TCR-T and target cells HepG2, B is the tumor cell killing rate (n=4) of control T (Mock T) or AFP-TCR-T after 36h co-culture with HepG2 in the presence of different combinations of cytokines measured by LDH detection kit, C is the IFN- γ level (n=3) in the co-culture supernatant of control T or AFP-TCR-T cells after 36h co-culture with HepG2 in the presence of different combinations of cytokines measured by ELISA detection kit of IFN- γ.
Fig. 2: effect of exogenous IL-21 on anti-tumor function of AFP-TCR-T in liver cancer engrafting tumor mouse model. In fig. 2, a is an experimental flow chart for verifying the effect of IL-21 on promoting anti-tumor function in T cells, B is the tumor volume change (n=5) of each group of mice after HepG2 subcutaneous tumor implantation and mock T or AFP-TCR-T cell adoptive therapy, C is tumor tissue isolated from each group of tumor-bearing mice at the end of the experiment, tumor weights of each group of mice are shown (n=5), D is the percentage of TCR-T in total lymphocytes (mouse and human lymphocyte) in the peripheral blood of mice at days 1, 14, 28 after TCR-T adoptive therapy (n=5).
Fig. 3: construction of an AFP-TCR-T engineered IL-21 receptor that spontaneously transmits IL-21 signaling and verification of its spontaneous IL-21 signaling. In fig. 3, a is a schematic diagram of native IL-21 receptor and engineered IL-21 receptor, B is a schematic diagram of AFP-TCR-T and AFP-TCR-T lentiviral vector expressing engineered IL-21 receptor, C is the result of expression of AFP-TCR and engineered IL-21 receptor (CD 34 extracellular segment) after infection of Jurkat cells with two lentiviruses in B, measured by flow cytometry, D is the phosphorylation level of STAT3 in cells after infection of Jurkat cells with two lentiviruses in B, measured by Western Blot quantification, E is the expression of AFP-TCR and engineered IL-21 receptor (CD 34 extracellular segment) in human AFP-TCR-T and TCR-T expressing engineered IL-21 receptor, measured by flow cytometry, F is the phosphorylation level of STAT3 in human AFP-TCR-T and AFP-TCR-T expressing engineered IL-21 receptor, measured by flow cytometry, i.e. the percentage of phosphorylated STAT3 positive cells in two cells and the average strength of phosphorylated STAT3 in two cells (n=3).
Fig. 4: effects of different TCR-T cells on target cells HepG 2. In fig. 4, a is a microscopic white light pattern of AFP-TCR-T or AFP-TCR-T expressing an engineered IL-21 receptor (IL-21R-TCR-T) after repeated co-culture with HepG2 cells for 36 hours, B is a tumor cell killing rate (n=4) of AFP-TCR-T or AFP-TCR-T expressing an engineered IL-21 receptor after repeated co-culture with HepG2 for 36 hours as measured by LDH detection kit, C is an IFN- γ level of supernatant after repeated co-culture of AFP-TCR-T expressing an engineered IL-21 receptor with HepG2 for 36 hours (n=4) as measured by ELISA detection kit for IFN- γ.
Fig. 5: antitumor properties of different TCR-T cells in a mouse model of liver cancer transplantation tumor. In fig. 5, a is tumor volume change (n=5) in each group of mice after HepG2 subcutaneous tumor implantation and mock T, AFP-TCR-T or AFP-TCR-T cells expressing the engineered IL-21 receptor, B is tumor weight (n=5) after separation of tumor tissue of each group of mice at the end of the experiment, C is the percentage of TCR-T in total lymphocytes (mice and human lymphocytes) in the peripheral blood of the mice on days 1, 14, 28 after AFP-TCR-T or AFP-TCR-T cells expressing the engineered IL-21 receptor after adoptive treatment (n=5), D is the number of TCR-T in the tumors of the AFP-TCR-T group and IL-21R-TCR-T group on day 7 after adoptive treatment (n=5), E is the percentage of TCR-T in the peripheral blood of the mice and the total T cells in the tumors on day 7 after adoptive treatment (n=5), and the positive result of TCR-T in the peripheral blood flow of the mice and IL-21R-T group after adoptive treatment (t=7 and T-21R-T positive result of the T in the peripheral blood of mice and IL-21R-T group after adoptive treatment).
Fig. 6: effect of hepatoma tumor stimulation on expression of AFP-TCR-T which remodels the IL-21 receptor. In fig. 6, a is the percentage of cell subsets of different degrees of differentiation defined by CD45RO and CD62L in CD8 positive subsets of AFP-TCR-T and AFP-TCR-T expressing the modified IL-21 receptor, detected by flow cytometry, after 36h co-culturing AFP-TCR-T and AFP-TCR-T expressing the modified IL-21 receptor with HepG2, i.e. the proportion of highly expressed PD-1 cells in the CD8 positive TCR-T subset and the average fluorescence intensity of PD-1 in the two groups (n=3), and C is the viability of cells of the AFP-TCR-T and AFP-TCR-T expressing the modified IL-21 receptor after 36h co-culturing AFP 8 positive subsets of aftcr-T and aftcr-T expressing the modified IL-21 receptor, detected by flow cytometry, i.e. the percentage of apoptosis in CD8 positive TCR-T and afnet positive subsets in the two groups (aaxin=3).
Detailed Description
The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.
The following examples do not identify specific experimental procedures or conditions, which may be followed by procedures or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.
Example 1: effect of exogenous IL-21 on T cell receptor engineered T cell (AFP-TCR-T) anti-tumor function targeting alpha fetoprotein antigen
1. Experimental method
1.1 construction and amplification of AFP-TCR-T
PBS buffer (available from Gibco) was used at a volume ratio of 1:1 diluting whole blood of a healthy person (from a southern hospital volunteer) to obtain diluted whole blood; diluted whole blood and lymphocyte isolates Percol (from Stemcell) were mixed in a volume ratio of 2:1, and centrifuging at 800g for 25min; after centrifugation, sucking the white membrane layer by a Pasteur pipe to obtain Peripheral Blood Mononuclear Cells (PBMC); washing peripheral blood mononuclear cells twice by using PBS buffer solution, and sorting T cells in the peripheral blood mononuclear cells by using magnetic beads; t cells were grown at 2X 10 6 The inoculum size of each was inoculated into 2mL of a medium containing 10% (v/v) fetal bovine serum (from Gibco), 10% (w/v, g/100 mL) penicillin-streptomycin diabody (from Gibco), 20ng/mL of fineCytokine IL-2 (from PEPLOTECH) and 200ng/mL anti-CD3/CD2 (from Stemcell) in RPMI1640 medium (from Gibco) were incubated at 37℃for 48h; after the cultivation is completed, the culture medium is prepared to contain 2×10 6 Cell culture broth of individual T cells lentiviral particles expressing AFP-TCR (see patent application publication No. US 2018/0327773 A1) were inoculated into cell culture broth at a multiplicity of infection with moi=1 for 72h at 37 ℃; after infection, centrifuging the cell culture solution at 450g for 5min, discarding the supernatant, replacing a fresh culture medium, and continuously culturing at 37 ℃ for 7 days to obtain the cell culture solution containing AFP-TCR-T cells, wherein 10ng/mL of cytokine IL-2 (purchased from PeproTech) is additionally added to the fresh culture medium on the basis of the original culture medium; centrifuging cell culture solution containing AFP-TCR-T cells at 450g for 5min, and taking precipitate, and re-suspending with RPMI1640 medium to cell concentration of 1×10 7 AFP-TCR-T cell suspension was obtained at each mL.
1.2 in vitro Co-culture experiments with AFP-TCR-T and HepG2
HepG2 cells (available from Saiborin Biotechnology Co., ltd.) were used at 1X 10 7 The inoculum size of the animals was inoculated into 25cm of DMEM high-sugar medium (available from Gibco) supplemented with 5mL of 10% (v/v) fetal bovine serum 2 Culturing in a bottom area cell culture flask at 37 ℃ for 72h; after the completion of the culture, the medium in the cell culture flask was discarded, 1mL of 0.25% pancreatin (purchased from Gibco) was added to the cell culture flask, and incubated at 37℃for 10min to digest HepG2 cells; after the digestion was completed, 3mL of DMEM high-sugar medium containing 10% (v/v) fetal bovine serum was added to the cell culture flask to terminate the digestion; after termination of digestion, the cell culture broth was centrifuged at 1000rpm for 5min, and the pellet was resuspended in DMEM complete medium (available from Corning Corp.) to a cell concentration of 1X 10 7 Each mL, hepG2 cell suspension was obtained.
HepG2 cell suspensions were 1X 10 per well 6 The inoculum size of the HepG2 cells was inoculated into a 96-well plate, and then cultured at 37℃for 24 hours; after the culture, the HepG2 cells in the 96-well plate are divided into four groups, namely an IL-2 group, an IL-2+IL-21 group, an IL-2+IL-7 group and an IL-2+IL-15 group; after the end of the grouping, AFP-TCR-T was performed using non-engineered T cells as control (control T)Cell suspension at 5X 10 per well 5 Inoculum size of each TCR positive subpopulation was inoculated into 96 well plates and co-cultured with four HepG2 cells at 37 ℃ for 36h; after the co-culture is finished, each well of AFP-TCR-T cells is taken out (a pipetting gun is used for light blowing, suspension cells can be blown into a supernatant, and then culture supernatant is taken out), and the AFP-TCR-T cells are added into four groups of HepG2 cells which are newly placed in the 96-well plate and incubated for 24 hours at 37 ℃ for co-culture, before co-culture, 10ng/mL of IL-2 (purchased from PeproTech company) is added into a co-culture system, 10ng/mL of IL-2 and 20ng/mL of IL-7 (purchased from PeproTech company) are added into the co-culture system from IL-2+IL-21 group, 10ng/mL of IL-2 and 20ng/mL of IL-15 (purchased from PeproTech company) are added into the co-culture system from IL-2+IL-15 group, and 10ng/mL of IL-2 and 20ng/mL of IL-21 (purchased from PeproTech company) are added into the co-culture system; co-culturing is carried out for three times, after each round of co-culturing is finished, the co-culture supernatant of each hole is sucked, the killing rate of AFP-TCR-T on HepG2 cells is calculated through an LDH detection kit (purchased from Promega), the change of the killing rate of the AFP-TCR-T after different cytokine combinations are added is compared, and the IFN-gamma secretion level in the co-culture supernatant of the AFP-TCR-T and the HepG2 is detected through an ELISA detection kit (purchased from Biolegend); wherein, the calculation formula of the killing rate of the AFP-TCR-T to the HepG2 cell is as follows: killing rate = (co-culture well absorbance-pure HepG2 well absorbance-pure T cell well absorbance + pure medium well absorbance)/(maximum release well absorbance-pure HepG2 well absorbance). The experimental results are shown in FIG. 1.
1.3 in vivo anti-tumor experiments in NPG mice
Subcutaneous injection of 1×10 in each immunodeficient NPG mouse (immunodeficient NPG mice purchased from beverToyota biotechnology limited) using a 1mL syringe 6 Constructing a liver cancer transplantation tumor mouse model by using the HepG2 cells; wait for tumor formation to a volume of 35.+ -.15 mm 3 After that, mice were divided into four groups, which were experimental group A (TCR-T), experimental group B (TCR-T+IL-21), control group A (Mock T) and control group B (Mock T+IL-21), respectively; after the end of the grouping, mice of experimental group a and experimental group B were infused with 5×10 drug solution by tail vein using 1mL syringe 6 Individual TCRsAFP-TCR-T cells of the positive subpopulation, mice of control group A and control group B were infused with 5X 10 by tail vein 6 The same number of unmodified T cells as the experimental group, four groups of mice were simultaneously infused with 5 μg of IL-2 recombinant protein (purchased from Peprotech), and the experimental group B and the control group B were infused with an additional 10 μg of IL-21 recombinant protein (purchased from Peprotech); peripheral blood of each group of mice was collected through the limbic vein at days 1, 7, and 14 after the infusion, centrifuged at 6000rpm for 1min, the supernatant was discarded, erythrocytes were lysed using EL Buffer (QIAGEN), and the lysate was examined by flow cytometry to obtain the ratio of TCR-T in the total lymphocytes of each group of mice; the volume of each group of mice tumors is continuously measured by using a vernier caliper, wherein the calculation formula of the tumor volume is as follows: tumor volume (mm) 3 ) =length x width x height/2; at the end of the experiment, each group of mice was sacrificed by cervical dislocation, the mice tumors were isolated, tumor weights were weighed and recorded. The experimental results are shown in FIG. 2.
2. Experimental results
2.1, IL-21 can be used as potential target for enhancing the anti-tumor function of T cells
The results in FIG. 1 show that the AFP-TCR-T cells after IL-2 addition significantly decreased tumor cell killing during the second and third rounds of co-culture, and that the addition of IL-7, IL-15, and IL-21 increased the killing capacity of AFP-TCR-T cells for tumor cells during the second and third rounds of co-culture, whereas the AFP-TCR-T cells with IL-21 addition had the strongest killing capacity for tumor cells. The results in FIG. 1 show that the AFP-TCR-T cells supplemented with IL-21 released significantly higher IFN-gamma levels during co-culture with tumor cells than IL-2 alone, IL-2+IL-7 and IL-2+IL-15, especially during the second and third rounds of co-culture. In summary, the results in FIG. 1 indicate that IL-21 can be one of the potential targets for enhancing the anti-tumor function of T cells, and has important application value in the field of T cell therapy.
2.2. 2 IL-21 enhances the anti-tumor function of T cells in vivo
The results in FIG. 2B demonstrate that the in vivo infusion of AFP-TCR-T cells into mice is effective in slowing down tumor growth compared to the control group, while the combination of IL-21 can further enhance the in vivo antitumor capacity of TCR-T. The results in FIG. 2 show that infusion of AFP-TCR-T cells significantly reduced tumor weight in mice at the end of the experiment, and that IL-21 combination further enhanced the anti-tumor effect of TCR-T in vivo. The results in FIG. 2, D, show that the TCR-T ratio in the blood of mice from the IL-21 group in combination with TCR-T was significantly higher than that from the TCR-T group alone, at day 7 and day 14 after adoptive therapy, indicating that IL-21 was able to enhance the survival of AFP-TCR-T cells in mice. Taken together, the results in FIG. 2 demonstrate that IL-21 can enhance the anti-tumor capacity of T cells in vivo, further illustrating the value of IL-21 in the field of T cell therapy.
Example 2: construction of AFP-TCR-T (IL-21R-TCR-T) expressing engineered IL-21 receptor and verification of intracellular signaling and antitumor function
1. Experimental method
1.1 construction of AFP-TCR-T (IL-21R-TCR-T) expressing an engineered IL-21 receptor and verification of intracellular Signal
1.2.1 lentiviral particle construction for expression of IL-21R-TCR
Adding 4 mug of lentiviral vector AFP-TCR-pCDH-EF1 alpha (see patent application text with publication number of US 2018/0327773 A1) and 0.5 mug of endonuclease BamHI (purchased from NEB) into 20 mug of digestion buffer (purchased from NEB), reacting for 60min in a water bath kettle at 37 ℃ and then running gel to recover linear DNA to obtain double digested lentiviral vector AFP-TCR-pCDH-EF1 alpha (AFP-TCR refers to alpha chain and beta chain of T cell receptor of targeting alpha fetoprotein, the amino acid sequence of AFP-TCR is shown as SEQ ID NO.7, and the nucleotide sequence of nucleic acid molecule for encoding AFP-TCR is shown as SEQ ID NO. 8); synthesizing an altered IL-21 receptor (formed by sequentially connecting an extracellular segment with an amino acid sequence shown as SEQ ID NO.1, a transmembrane segment with an amino acid sequence shown as SEQ ID NO.2 and an intracellular segment with an amino acid sequence shown as SEQ ID NO. 3), wherein the nucleotide sequence of a nucleic acid molecule for encoding the extracellular segment is shown as SEQ ID NO.4, the nucleotide sequence of a nucleic acid molecule for encoding the transmembrane segment is shown as SEQ ID NO.5, the nucleotide sequence of a nucleic acid molecule for encoding the intracellular segment is shown as SEQ ID NO. 6), and connecting gene fragments for encoding P2A peptide (with an amino acid sequence shown as SEQ ID NO. 9) at the 3' -end of the gene fragments to obtain a target gene; connecting a target gene with a double-digested lentiviral vector AFP-TCR-pCDH-EF1 alpha by using T4 DNA ligase (purchased from NEB) to obtain a connecting product; the ligation product was transformed into E.coli competent cells HB101 (purchased from Takara) to give a transformed product; the transformation products were streaked on LB agar plate medium (available from Thermo Fisher) containing 50. Mu.g/mL ampicillin (Amp), incubated at 37℃for 24h, and single colonies were picked; inoculating a single colony to 3mL of LB liquid medium (purchased from Thermo Fisher), and culturing at 37 ℃ for 24 hours to obtain bacterial liquid; and (3) sequencing the recombinant plasmid in the extract bacterial liquid, and obtaining the coexpression vector pCDH-EF1 alpha-IL-21R-TCR of the modified IL-21 receptor and AFP-TCR which spontaneously transmit IL-21 signals after successful sequencing (the nucleotide sequence of the coexpression vector pCDH-EF1 alpha-IL-21R-TCR is shown as SEQ ID NO. 10).
HEK293T cells (from the China academy of sciences typical culture Collection Committee cell Bank/Stem cell Bank) were cultured at 5X 10 6 The inoculum size of the animals was inoculated into 150cm of DMEM medium (available from Gibco) supplemented with 20mL of a medium containing 10% (v/v) fetal bovine serum and 1% (w/v, g/100 mL) penicillin-streptomycin diab (available from Gibco) 2 After cell culture dishes, third generation lentiviral packaging plasmid (purchased from Addgene) and co-expression vector pCDH-EF1 alpha-IL-21R-TCR were transfected into HEK293T cells using PEI transfection (PEI transfection Methods see: longo PA, kavran JM, kim MS, leahy DJ. Transient mammalian cell transfection with Polyethylenimine (PEI). Methods enzymes ol. 2013; 529:227-40); after 8h transfection at 37℃fresh medium was changed and cultivation continued for 48h at 37 ℃; after the culture is finished, collecting culture supernatant, centrifuging at 10000g at a high speed for 6h, and taking out precipitate to obtain the slow virus particles for expressing IL-21R-TCR.
1.2.2 verification of IL-21R-TCR infection efficiency in Jurkat cell lines
The Jurkat cell line (available from Saibuten Biotechnology Co.) was resuspended to a cell concentration of 5X 10 using RPMI 1640 medium (available from Gibco) containing 10% (v/v) fetal bovine serum (available from Gibco) 5 And (3) one mL to obtain JSuspension of urkat cells; jurkat cell suspensions were plated at 5X 10 per well 4 The inoculum size of the individual Jurkat cells was inoculated into a 96-well plate, and after adding lentiviral particles expressing AFP-TCR and lentiviral particles expressing IL-21R-TCR to the 96-well plate in an amount of 20. Mu.L of virus liquid per well, respectively, culturing was carried out at 37 ℃; after 48h of incubation, the expression of IL-21R (CD 34) and AFP-TCR on the surface of Jurkat cells was examined by flow cytometry; after 72h incubation, the cell proteins of Jurkat cells were extracted by Blue Loading Buffer Pack (purchased from Cell Signaling Technology) and the expression level of phosphorylated STAT3 in Jurkat cells infected with AFP-TCR and IL-21R-TCR lentiviruses was detected by western blotting. The detection result is shown in FIG. 3.
1.2.3 construction of IL-21R-TCR-T and verification of intracellular STAT3 phosphorylation Signal
PBS buffer (available from Gibco) was used at a volume ratio of 1:1 diluting whole blood of a healthy person (from a southern hospital volunteer) to obtain diluted whole blood; diluted whole blood and lymphocyte isolates Percol (from Stemcell) were mixed in a volume ratio of 2:1, and centrifuging at 800g for 25min; after centrifugation, sucking the white membrane layer by a Pasteur pipe to obtain Peripheral Blood Mononuclear Cells (PBMC); washing peripheral blood mononuclear cells twice by using PBS buffer solution, and sorting T cells in the peripheral blood mononuclear cells by using magnetic beads; t cells were grown at 2X 10 6 The inoculum size of the individuals was inoculated into 2mL of RPMI1640 medium (from Gibco) containing 10% (v/v) fetal bovine serum (from Gibco), 10% (w/v, g/100 mL) penicillin-streptomycin diab (from Gibco), 20ng/mL cytokine IL-2 (from PEPLOTECH), and 200ng/mL anti-CD3/CD2 (from Stemcell), and cultured at 37℃for 48 hours; after the cultivation is completed, the culture medium is prepared to contain 2×10 6 Cell culture broth of individual T cells, lentiviral particles expressing IL-21R-TCR were inoculated into the cell culture broth at a multiplicity of infection with moi=1 for 72h at 37 ℃; after infection, centrifuging the cell culture solution at 450g for 5min, discarding the supernatant, replacing a fresh culture medium, and continuously culturing at 37 ℃ for 7 days to obtain the cell culture solution containing IL-21R-TCR-T cells, wherein the fresh culture medium is additionally added with 10ng/mL of cytokine IL-2 (purchased from PeproTech) on the basis of the original culture medium; by flow cytometryDetecting the expression of CD34 and AFP-TCR on the surface of T cells after infection of IL-21R-TCR virus by using an instrument, centrifuging a cell culture solution containing IL-21R-TCR-T cells at 450g for 5min, and dividing the sediment into two parts; one aliquot of pellet was resuspended in RPMI1640 medium to a cell concentration of 1X 10 6 Obtaining IL-21R-TCR-T cell suspension at a concentration of one/mL; another pellet was resuspended to a concentration of 1X 10 in RPMI1640 medium containing 10% (v/v) fetal bovine serum 6 After each mL, the cells were incubated at 37℃for 3 hours, and after the completion of the incubation, the level of intracellular phosphorylated STAT3 in the CD 8-positive TCR-T subgroup was detected by flow cytometry. The detection experiment results are shown in figure 3.
1.2 in vitro and in vivo anti-liver cancer effect verification and in vitro phenotype detection of IL-21R-TCR-T
1.2.1 in vitro Co-culture experiments with IL-21R-TCR-T
HepG2 cells (available from Saiborin Biotechnology Co., ltd.) were used at 2X 10 6 The inoculum size of the animals was inoculated into 25cm of DMEM high-sugar medium (available from Gibco) supplemented with 5mL of 10% (v/v) fetal bovine serum 2 Culturing in a bottom area cell culture flask at 37 ℃ for 72h; after the completion of the culture, the medium in the cell culture flask was discarded, 1mL of 0.25% pancreatin (purchased from Gibco) was added to the cell culture flask, and incubated at 37℃for 10min to digest HepG2 cells; after the digestion was completed, 3mL of DMEM high-sugar medium containing 10% (v/v) fetal bovine serum was added to the cell culture flask to terminate the digestion; after termination of digestion, the cell culture broth was centrifuged at 1000rpm for 5min, and the pellet was resuspended in DMEM complete medium (available from Corning Corp.) to a cell concentration of 2X 10 6 Each mL, hepG2 cell suspension was obtained.
HepG2 cell suspensions were 1X 10 per well 5 The inoculum size of the HepG2 cells was inoculated into a 96-well plate, and then cultured at 37℃for 24 hours; after the culture is finished, the HepG2 cells in the 96-well plate are divided into two groups, wherein the two groups are respectively a TCR-T group and an IL-21R-TCR-T group; after the end of the grouping, the TCR-T group had the AFP-TCR-T cell suspension at 5X 10 per well 5 Inoculum size of each TCR Positive subset was seeded into 96-well plates and IL-21R-TCR-T group was seeded with IL-21R-TCR-T cell suspension at 5X 10 per well 5 Inoculum size of each TCR positive subpopulation was inoculated into 96 well plates and co-cultured with HepG2 cells at 37 ℃ for 36h; after co-cultivation is finishedTaking out AFP-TCR-T cells and IL-21R-TCR-T cells from each well (a pipetting gun is used for light blowing on a 96-well plate, suspended cells can be blown into a supernatant, and then culture supernatant is taken out), and respectively adding the AFP-TCR-T cells and the IL-21R-TCR-T cells into a new round of HepG2 cells which are in the 96-well plate and are incubated at 37 ℃ for 24 hours for co-culture; co-culturing is performed for three times, after each round of co-culturing is finished, killing of the AFP-TCR-T cells and the IL-21R-TCR-T cells on the HepG2 cells is observed through a microscope under a white light mode, culture supernatants of each hole are sucked, killing rates of the IL-21R-TCR-T and the common AFP-TCR-T on the HepG2 cells are calculated through an LDH detection kit (purchased from Promega), and IFN-gamma secretion levels in the culture supernatants of the IL-21R-TCR-T and the common AFP-TCR-T and the HepG2 are detected through an ELISA detection kit (purchased from Biolegend) of IFN-gamma; the calculation formula of the killing rate of IL-21R-TCR-T and common AFP-TCR-T on HepG2 cells is as follows: killing rate = (co-culture well absorbance-pure HepG2 well absorbance-pure T cell well absorbance + pure medium well absorbance)/(maximum release well absorbance-pure HepG2 well absorbance). The experimental results are shown in FIG. 4.
1.2.2 in vivo NPG mouse anti-tumor experiments with IL-21R-TCR-T
Subcutaneous injection of 1×10 in each immunodeficient NPG mouse (immunodeficient NPG mice purchased from beverToyota biotechnology limited) using a 1mL syringe 6 Constructing a liver cancer transplantation tumor mouse model by using the HepG2 cells; wait for tumor formation to a volume of 35.+ -.15 mm 3 After completion of the grouping, mice were divided into three groups, namely, experiment group A (TCR-T), experiment group B (IL-21R-TCR-T) and control group ((control T)), and after completion of the grouping, mice of experiment group A were infused with 5X 10 by tail vein using 1mL syringe 6 IL-21R-TCR-T cells of the individual TCR-positive subpopulations were infused with 5X 10 by tail vein into mice of experimental group B 6 AFP-TCR-T cells of the individual TCR positive subpopulations were infused by tail vein into mice of the control group with the same number of unmodified T cells as the experimental group, and three groups of mice were simultaneously infused with 5 μg of human IL-2 recombinant protein (available from Peprotech); peripheral blood of each group of mice was collected by eye vein at 1, 7, and 14 days after infusion, centrifuged at 6000rpm for 1min, and the supernatant was discarded, and EL Buffer (QIAGEN) was usedLysing erythrocytes, and detecting the lysate by flow cytometry to obtain the proportion of TCR-T in the total lymphocytes of each group of mice; the volume of each group of mice tumors is continuously measured by using a vernier caliper, wherein the calculation formula of the tumor volume is as follows: tumor volume (mm) 3 ) =length x width x height/2; at the experimental end point, each group of mice is sacrificed by cervical dislocation, the tumors of the mice are separated, and the weights of the tumors are weighed and recorded; on day 7 after infusion, mice were sacrificed by cervical dislocation, mice tumors were isolated and weighed, after which the tumor tissue dissociation kit (purchased from Miltenyi) was used to isolate the mice intratumoral cells and the number of TCR positive subpopulations and the proportion of TCR positive subpopulations to mouse lymphocytes were detected by flow cytometry. The experimental results are shown in FIG. 5.
1.2.3 comparison of in vitro phenotypic characteristics of IL-21R-TCR-T and ordinary AFP-TCR-T
HepG2 cell suspensions were 1X 10 per well 5 The inoculum size of the HepG2 cells was inoculated into a 96-well plate, and then cultured at 37℃for 24 hours; after the culture is finished, the HepG2 cells in the 96-well plate are divided into two groups, wherein the two groups are respectively a TCR-T group and an IL-21R-TCR-T group; after the end of the grouping, the TCR-T group was used to control the AFP-TCR-T cell suspension at 5X 10 per well using a 96-well plate without HepG2 cells 5 Inoculum size of each TCR Positive subset was seeded into 96-well plates and IL-21R-TCR-T group was seeded with IL-21R-TCR-T cell suspension at 5X 10 per well 5 Inoculum size of each TCR positive subpopulation was inoculated into 96 well plates and co-cultured with HepG2 cells at 37 ℃ for 36h; after the co-culture is finished, taking out AFP-TCR-T cells and IL-21R-TCR-T cells from each well (a pipetting gun is used for light blowing on a 96-well plate, suspended cells can be blown into a supernatant, and then culture supernatants are taken out), and respectively adding the AFP-TCR-T cells and the IL-21R-TCR-T cells into a new round of HepG2 cells which are in the 96-well plate and are incubated at 37 ℃ for 24 hours for co-culture; co-cultivation was performed three times, and after each round of co-cultivation was completed, the expression of memory phenotype markers CD45RO and CD62L, depletion marker PD-1, apoptosis-related markers Annexin V and 7-AAD (antibodies were all purchased from Biolegend) of the double positive sub-populations of CD8 and TCR in IL-21R-TCR-T and common AFP-TCR-T after co-cultivation with HepG2 was examined by flow cytometry, and the examination results were analyzed using FCS Express 6.0 software. The experimental results are shown in FIG. 6.
2. Experimental results
2.1 construction of IL-21R-TCR-T and intracellular Signal verification
A in FIG. 3 is a schematic representation of an engineered IL-21 receptor that spontaneously transmits IL-21 signaling. FIG. 3B is a schematic diagram of the lentiviral vectors of AFP-TCR-T and IL-21R-TCR-T. FIG. 3C shows the expression levels of both cell surface CD34 and AFP-TCR after infection of Jurkat cells with either AFP-TCR lentivirus or IL-21R-TCR lentivirus, and shows that Jurkat cells can simultaneously express CD34 and AFP-TCR after infection with IL-21R-TCR lentivirus. D in FIG. 3 is the expression level of intracellular phosphorylated STAT3 after Jurkat cells are infected with AFP-TCR lentivirus or IL-21R-TCR lentivirus, and it can be seen that intracellular STAT3 phosphorylation levels are significantly increased after Jurkat cells are infected with IL-21R-TCR lentivirus. E in FIG. 3 is the expression level of human AFP-TCR-T and IL-21R-TCR-T surface CD34 and AFP-TCR, and it can be seen that human IL-21R-TCR-T is capable of expressing both CD34 and AFP-TCR. F in FIG. 3 is a measurement of the phosphorylation levels of STAT3 in human AFP-TCR-T and IL21R-TCR-T cells, showing that the levels of STAT3 in CD8 and CD4 positive IL-21R-TCR-T cells are higher than AFP-TCR-T. Taken together, the results in FIG. 3 demonstrate that IL-21R-TCR-T can be successfully constructed and that the cells have spontaneously transmitted IL-21 signaling.
2.2, IL-21R-TCR-T has stronger anti-tumor and intratumoral infiltration capacity
A in FIG. 4 is a white light photo under the mirror after IL-21R-TCR-T and ordinary AFP-TCR-T are co-cultured with a target cell HepG2, and the IL-21R-TCR-T has stronger tumor killing capability compared with the ordinary AFP-TCR-T. B in FIG. 4 and C in FIG. 4 are the killing rate and IFN-gamma secretion level of IL-21R-TCR-T and common AFP-TCR-T after co-culture with target cell HepG2, respectively, and the killing rate and IFN-gamma secretion level of IL-21R-TCR-T on tumor cells in the second and third rounds of co-culture are significantly higher than those of common TCR-T, indicating that IL-21R-TCR-T has stronger sustained effect function.
A in FIG. 5 is a plot of tumor growth following infusion of normal AFP-TCR-T, IL-21R-TCR-T and control T cells into HepG2 tumor-bearing mice, and it is seen that infusion of IL-21R-TCR-T inhibited tumor growth more significantly than normal AFP-TCR-T. B in FIG. 5 is the tumor weight of each group of mice at the end of the experiment, and it can be seen that the tumor weight in the IL-21R-TCR-T infused mice was significantly lighter than in the normal AFP-TCR-T group. The results in FIG. 5, C, show that the TCR-T ratio in the blood of mice is significantly higher than that of the normal AFP-TCR-T group in mice infused with IL-21R-TCR-T on days 7 and 14 after the adoption. D in FIG. 5 is the number of TCR-T in the tumor of the mice on day 7 after the adoptive period, and it can be seen that the number of TCR-T in the tumor of the mice in the IL-21R-TCR-T group is significantly higher than that in the normal AFP-TCR-T group. Meanwhile, the results of E in FIG. 5 and F in FIG. 5 show that the proliferation ratio of the CD8 positive TCR-T cells in the tumor of the IL-21R-TCR-T group is significantly higher than that of the common AFP-TCR-T group on the 7 th day after the adoptive period, and the ratio change in blood is not different between the two groups, thus indicating that the CD8 positive IL-21R-TCR-T has stronger proliferation and survival ability after the tumor antigen is stimulated in the tumor microenvironment.
In summary, the results shown in FIGS. 4-5 demonstrate that IL-21R-TCR-T has greater in vivo and in vitro anti-tumor capability than conventional AFP-TCR-T.
2.3 IL-21R-TCR-T exhibits stronger stem cell-like characteristics and lower levels of depletion and apoptosis during antigen stimulation
A in FIG. 6 shows the change in memory phenotype during in vitro stimulation with repeated tumor antigens for IL-21R-TCR-T and normal AFP-TCR-T, and shows that the proportion of CD45RO negative CD62L positive subset (stem cell-like T cell subset) and CD45RO and CD62L double positive subset (central memory T cell subset) in IL-21R-TCR-T is higher than that in normal AFP-TCR-T. The results in FIG. 6B show that IL-21R-TCR-T has lower levels of expression of the depletion molecule PD-1 during stimulation with repeated tumor antigens than ordinary AFP-TCR-T. FIG. 6C shows that IL-21R-TCR-T exhibits lower Annexin V and 7-AAD ratios than conventional AFP-TCR-T during stimulation with repeated tumor antigens. Taken together, the results in FIG. 6 demonstrate that IL-21R-TCR-T is capable of maintaining more stem cell-like characteristics than conventional AFP-TCR-T during antigen stimulation, and has greater resistance to depletion and viability.
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 (9)
1. The receptor capable of spontaneously transmitting IL-21 signals is characterized by being formed by sequentially connecting an extracellular segment with an amino acid sequence shown as SEQ ID NO.1, a transmembrane segment with an amino acid sequence shown as SEQ ID NO.2 and an intracellular segment with an amino acid sequence shown as SEQ ID NO. 3.
2. A nucleic acid molecule encoding the receptor of claim 1 that spontaneously transmits an IL-21 signal.
3. The nucleic acid molecule of claim 2, wherein the nucleic acid molecule encoding the extracellular domain has a nucleotide sequence shown in SEQ ID NO.4, the nucleic acid molecule encoding the transmembrane domain has a nucleotide sequence shown in SEQ ID NO.5, and the nucleic acid molecule encoding the intracellular domain has a nucleotide sequence shown in SEQ ID NO. 6.
4. A recombinant plasmid carrying the nucleic acid molecule of claim 2 or 3.
5. The recombinant plasmid of claim 4, wherein the recombinant plasmid further carries a nucleic acid molecule encoding an AFP-TCR; the AFP-TCR refers to the alpha and beta chains of T cell receptors that target alpha fetoprotein.
6. A host cell transfected with the recombinant plasmid of claim 4 or 5; alternatively, the host cell genome is integrated with the nucleic acid molecule of claim 2 or 3.
7. A recombinant virus, wherein the genome of the recombinant virus carries the nucleic acid molecule of claim 2 or 3.
8. A genetically engineered T cell carrying the nucleic acid molecule of claim 2 or 3, or the receptor of claim 1 that spontaneously transmits IL-21 signaling.
9. Use of the receptor spontaneously transmitting IL-21 signal of claim 1 or the nucleic acid molecule of claim 2 or 3 or the recombinant vector of claim 4 or 5 or the host cell of claim 6 or the recombinant virus of claim 7 or the genetically engineered T cell of claim 8 for the preparation of a medicament for the treatment of liver cancer.
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