CN112111014A - Antioxidant chimeric antigen receptor and immune cell - Google Patents

Antioxidant chimeric antigen receptor and immune cell Download PDF

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CN112111014A
CN112111014A CN202011001854.3A CN202011001854A CN112111014A CN 112111014 A CN112111014 A CN 112111014A CN 202011001854 A CN202011001854 A CN 202011001854A CN 112111014 A CN112111014 A CN 112111014A
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廖文强
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Abstract

The invention discloses an antioxidant chimeric antigen receptor and an immune cell, belonging to the technical field of molecular biology, wherein the chimeric antigen receptor comprises an extracellular region, the extracellular region comprises a spacer region, and the amino acid sequence of the Core Hinge region of the spacer region is Core Hinge-Y1: CDKTYTCPPCP or Core Hinge-Y2: CDKTSTCPPCP are provided. The anti-oxidation chimeric antigen receptor is obtained by replacing histidine with positive polarity at the core hinge region of the chimeric antigen receptor with neutral uncharged serine or tyrosine; the data prove that the chimeric antigen receptor and the immune cell have the advantages of oxidation resistance, stronger tumor cell killing effect or anti-tumor effect.

Description

Antioxidant chimeric antigen receptor and immune cell
Technical Field
The invention relates to the technical field of molecular biology, in particular to an antioxidant chimeric antigen receptor and an immune cell.
Background
With the continuous deterioration of global environmental factors, the morbidity and mortality of malignant tumors are on the rise no matter in developed countries or developing countries, and the malignant tumors become the biggest harm to human life health. In 2020, there are about 550 ten thousand new cancer cases each year in China, with 400 thousands of deaths.
The chimeric antigen receptor immune cell is a novel accurate targeting immune cell for killing tumors, has a good effect on killing tumors by optimizing and improving in recent years, is a very promising mode and can be accurately, quickly and efficiently used. Chimeric Antigen Receptor immune cells include CAR-T, CAR-NK, CAR-B, and CAR-MP (CAR-macrophage), which is a Chimeric Antigen Receptor (CAR) -based immune cell. By in vitro gene transfer techniques, gene sequences encoding specific chimeric antigen receptors are transduced into immune cells to generate tumor-specific T cells that can bind to a target antigen.
CAR-T cells are currently effective in treating hematological tumors, such as yescatta for acute B lymphoblastic leukemia and for refractory/relapsed non-hodgkin's lymphoma, which are marketed in the united states. However, immune cells such as CAR-T cells are far less effective than hematological tumors in killing solid tumors. It may be associated with the Tumor Microenvironment (TME) of solid tumors affecting CAR-T extracellular structural stability and integrity, such as local Oxygen enrichment (ROS) degrading the space of CAR, disrupting CAR-T binding to antigens on the Tumor cell surface, thereby depriving CAR-T of its attacking effect on Tumor cells. Hypoxia (hypoxia) is a major feature of the solid tumor microenvironment, resulting in local ROS enrichment and promotion of tumor cell growth.
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention provides an antioxidant chimeric antigen receptor and an immune cell, which improve the stability by improving the antioxidant property of the chimeric antigen receptor and the immune cell.
The invention discloses an antioxidant chimeric antigen receptor, which comprises an extracellular region, wherein the extracellular region comprises a spacer region, and the amino acid sequence of a Core Hinge region of the spacer region is Core Hinge-Y1: CDKTYTCPPCP (SEQ ID NO: 3) or Core Hinge-Y2: CDKTSTCPPCP (SEQ ID NO: 4).
Preferably, the chimeric antigen receptor of the present invention further comprises a transmembrane region and an intracellular region, wherein the extracellular region further comprises a single-chain antibody, the transmembrane region and the single-chain antibody are connected at two ends of the spacer region, the transmembrane region is connected with the intracellular region, and the spacer region is a constant-chain fragment of immunoglobulin G.
Preferably, the spacer has the gene sequence IgG HCH3-Y1 cDNA (SEQ ID NO: 6) or IgG HCH3-Y2 cDNA (SEQ ID NO: 7).
The invention also provides an immune cell expressing the chimeric antigen receptor.
Preferably, the immune cell comprises a CAR-T cell, CAR-NK cell, CAR-B cell, or CAR-MP cell.
Preferably, the immune cells are used to kill tumor cells.
Preferably, the immune cells are used to kill solid tumor cells or hematological tumor cells.
Preferably, the preparation method of the immune cell comprises:
synthesizing cDNA of chimeric antigen receptor;
preparing a lentiviral plasmid from the cDNA;
preparing lentiviral particles from the lentiviral plasmid;
obtaining mononuclear cells and performing pre-culture;
transfecting the lentivirus particles into the pre-cultured mononuclear cells, and culturing to obtain immune cells.
Preferably, the method for preparing the lentivirus particles comprises the following steps:
culture of 6X10 Using DMEM Medium containing 10% fetal bovine serum6A HEK293T cell;
when the cell degree of polymerization reaches about 80% on the next day, the cell is stabilized for 2-3 hours after the cell is changed into a culture solution without 10% FBS and antibiotics;
cloning the cDNA of the CAR into pCCL/MNDU3-X shuttle plasmid to obtain a lentiviral plasmid containing the CAR sequence: the pCCL-CAR plasmid is prepared by respectively adding the pCCL-CAR plasmid with the final concentration of 6.9 mug/ml, the pMDLg-pRRE plasmid with the final concentration of 3.4 mug/ml, the pMD2.G plasmid with the final concentration of 2 mug/ml and the pRSV-Rev plasmid with the final concentration of 1.71 mug/ml into a culture solution, and uniformly mixing to obtain a plasmid culture solution;
adding 35 mu g of polyethyleneimine with the concentration of 1 mu g/mu l into 1 ml of DMEM culture solution, and uniformly mixing to obtain a polyethyleneimine culture solution;
culturing polyethyleneimine, adding the polyethyleneimine into material culture, uniformly mixing, and standing at room temperature for 20 minutes to obtain a first transfection solution;
adding the first transfection solution into the stabilized HEK293T cells for transfection;
after 4 hours of transfection, the culture solution was changed to DMEM containing 10% fetal bovine serum;
after culturing for 48 hours, collecting cell culture solution, and filtering with a 0.22 μm filter membrane;
centrifuging the filtrate at 26000rpm and 4 deg.C for 2 hr, and collecting precipitate;
resuspend the pellet with XVivo15 whole medium to obtain lentiviral particles.
Preferably, the method of obtaining immune cells comprises:
taking 50ml of peripheral blood, and performing density gradient centrifugation on Ficoll lymphocyte separation liquid to obtain mononuclear cells;
the mononuclear cell is expressed at 2x106Cell density of/ml in X-VIVO 15 whole culture medium, 37 deg.C, 5% CO2Culturing for 2 hours, and collecting suspension cells;
suspension cells at 1.2 × 106Inoculating the cells with the density of/ml into a culture bottle, adding CD3/CD28 magnetic beads according to the ratio of the cells to the magnetic beads being 1:3, and adding interleukin 2 with the final concentration of 100U/ml for culturing overnight;
adding the lentivirus particles for transfection;
after cell transfection, the cells were cultured for 5 days, and the magnetic beads were removed by a magnetic holder to obtain immune cells.
Compared with the prior art, the invention has the beneficial effects that:
the anti-oxidation chimeric antigen receptor is obtained by replacing histidine with positive polarity at the core hinge region of the chimeric antigen receptor with neutral uncharged serine or tyrosine; the data prove that the chimeric antigen receptor and the immune cell have the advantages of oxidation resistance, stronger tumor cell killing effect or anti-tumor effect.
Drawings
FIG. 1 is a schematic diagram of the construction of a chimeric antigen receptor according to the present invention;
FIG. 2 is a schematic composition of a chimeric antigen receptor;
FIG. 3 is a flow chart of a method for preparing immune cells;
FIG. 4 is a flow chart of a method for preparing lentiviral particles;
FIG. 5 is a flow chart of a method of obtaining immune cells;
FIG. 6 is a graph showing the results of an oxidation resistance test in one example;
FIG. 7 is a graph showing the results of the antioxidant test in examples
FIG. 8 is a graph showing the results of detection of viable cells after the tumor killing test of example one;
FIG. 9 is a graph showing the results of detection of viable cells after the tumor killing test of example two;
FIG. 10 is a graph showing the results of TNF α assays of example one;
FIG. 11 is a graph showing the results of IFN γ detection in the first example;
FIG. 12 is a graph showing the results of TNF α assay of example two;
FIG. 13 is a graph showing the results of IFN γ detection in example two;
FIG. 14 is a graph of tumor model test results.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
The invention is described in further detail below with reference to the attached drawing figures:
an antioxidant chimeric antigen receptor, as shown in figure 1, comprises an extracellular region including a Spacer region (Spacer) whose core hinge region amino acid sequence is CDKTYTCPPCP (SEQ ID NO: 3) or CDKTSTCPPCP(SEQ ID NO：4)。
The HCH3 fragment of IgG of the antibody molecule conforming to the structural characteristics of the antibody is used as Spacer, due to the enrichment of ROS in the microenvironment of solid tumors, in particular the main representative molecule of ROS H2O2Possibly degrading IgG HCH in Spacer3The Core Hinge region (Core Hinge, SEQ ID NO: 1) of (A) results in loss of CAR-T extracellular region CAR structure, rendering CAR-T cells non-functional for anti-tumor.
The existing Core hinge region amino acid sequence is Core hinder: cDKTHTCPPCP (SEQ ID NO: 1) containing aspartic acid (D) negatively charged in polarity, lysine (K) and histidine (H) positively charged. These charged amino acids, especially the polar positively charged histidine (H) separated by a threonine (T) from the negatively charged aspartic acid (D) and the positively charged lysine (K), may be key amino acids for driving ROS to attack the electron transfer in the core hinge region of Spacer. The invention obtains the antioxidant chimeric antigen receptor by replacing histidine (H) with positive polarity in a core hinge region by neutral uncharged serine (S) or tyrosine (Y). Can obviously enhance the stability in ROS environment, thereby leading the CAR immune cells to still keep the killing effect on tumor cells under the condition of ROS enrichment in the tumor microenvironment.
The cDNA sequence of Core Hinger is TGT GAC AAA ACTCACACA TGC CCA CCG TGC CCA, wherein the substitution of CAC for TAC can yield cDNA of Core Hinge-Y1, but is not limited thereto, and TAT; the cDNA of Core Hinge-Y2 can be obtained by replacing CAC with TCA, but is not limited thereto, and may be TCT, TCC, TCG, AGT or AGC.
As shown in fig. 1 and 2, the chimeric antigen receptor of the present invention may further include a transmembrane region and an intracellular region, the extracellular region further includes a single chain antibody (scFv), and the spacer is connected to the transmembrane region and the single chain antibody at both ends, the transmembrane region is connected to the intracellular region, and the spacer employs a constant chain (Fc) fragment (HCH3) of immunoglobulin g (igg). Wherein the signal peptide may be IgG SP, the transmembrane region may be the CD8 α transmembrane domain, and the intracellular region may be the CD28 intracellular signal domain and the CD3 ζ intracellular signal domain.
Accordingly, the spacer of the present invention has the gene sequence IgG HCH3-Y1 cDNA (SEQ ID NO: 6) or IgG HCH3-Y2 cDNA (SEQ ID NO: 7). The spacer of the present invention replaces the CAC of the core hinge region based on the existing IgG HCH3cDNA (SEQ ID NO: 5).
The invention also provides an immune cell expressing the chimeric antigen receptor. Wherein the immune cell comprises a CAR-T cell, CAR-NK cell, CAR-B cell, or CAR-MP cell. Wherein the CAR-T cell is a chimeric antigen receptor T cell; the CAR-NK cell is a chimeric antigen receptor natural killer cell; the CAR-B cell is a chimeric antigen receptor B cell; CAR-MP (CAR-macrophage) cells are chimeric antigen receptor macrophages.
As shown in fig. 3, the preparation method of the immune cell includes:
step 301: cDNA of the chimeric antigen receptor was synthesized. cDNA was synthesized based on the IgG HCH3-Y1 cDNA or IgG HCH3-Y2 cDNA sequences.
Step 302: lentiviral plasmids were prepared using the cDNA. The cDNA can be prepared by ligating the cDNA into a lentiviral plasmid vector, such as pCCL/MNDU3-X, by ligase.
Step 303: lentiviral particles were prepared from lentiviral plasmids. The lentivirus plasmid is transfected into a host cell for propagation to obtain a large amount of lentivirus particles.
Step 304: mononuclear cells were obtained and pre-cultured. Mononuclear cells can be induced by culture to become immune cells.
Step 305: transfecting the lentivirus particles into the pre-cultured mononuclear cells, and culturing to obtain immune cells. After transfection of lentiviral particles into monocytes, the chimeric antigen receptor of the invention is successfully expressed in immune cells as the monocytes are cultured into immune cells.
In one embodiment, as shown in fig. 4, the method for preparing the lentiviral particle comprises:
step 401: culture of 6X10 Using DMEM Medium containing 10% fetal bovine serum6And HEK293T cells. Wherein, antibiotics can be added into the culture solution to prevent bacterial contamination.
Step 402: and when the cells reach the degree of polymerization of about 80% on the next day, the cells are stabilized for 2-3 hours after being replaced by DMEM culture solution without 10% FBS and antibiotics.
Step 403: the lentiviral plasmid comprises a pCCL-CAR plasmid, and the pCCL-CAR plasmid with the final concentration of 6.9 mug/ml, the pMDLg-pRRE plasmid with the final concentration of 3.4 mug/ml, the pMD2.G plasmid with the final concentration of 2 mug/ml and the pRSV-Rev plasmid with the final concentration of 1.71 mug/ml are respectively added into a culture solution and are uniformly mixed to obtain a plasmid culture solution. Among them, pMDLg-pRRE plasmid, pMD2.G plasmid and pMDLg-pRRE plasmid are commonly used lentiviral packaging plasmids and are commercially available. pCCL/MNDU3-X is a commonly used plasmid vector, and cDNA can be ligated to pCCL/MNDU3-X vector by digestion, ligation and molecular cloning, but is not limited thereto, and other plasmid vectors or packaging plasmids may be used.
Step 404: and adding 35 mu g of polyethyleneimine with the concentration of 1 mu g/mu l into 1 ml of DMEM culture solution, and uniformly mixing to obtain the polyethyleneimine culture solution.
Step 405: and adding the polyethyleneimine culture solution into the plasmid culture solution, uniformly mixing, and standing at room temperature for 20 minutes to obtain a first transfection solution.
Step 406: the first transfection solution was added to the stabilized HEK293T cells for transfection.
Step 407: after 4 hours of transfection, the culture medium was changed to a DMEM medium containing 10% fetal bovine serum.
Step 408: after further culturing for 48 hours, the cell culture broth was collected and filtered through a 0.22 μm filter. Wherein the lentiviral particles are secreted into the cell culture medium.
Step 409: the filtrate was centrifuged at 26000rpm at 4 ℃ for 2 hours to obtain a precipitate.
Step 410: resuspend the pellet with XVivo15 whole medium to obtain lentiviral particles.
In one embodiment, a method of obtaining immune cells comprises:
step 501: 50ml of peripheral blood is taken, and the mononuclear cells are obtained by density gradient centrifugation of Ficoll lymphocyte separating medium.
Step 502: the mononuclear cell is expressed at 2x106Cell density of/ml, in X-VIVO 15Nourishing liquid, 37 ℃ and 5% CO2After 2 hours of culture, suspension cells were collected.
Step 503: suspension cells at 1.2 × 106The cells were inoculated into a flask at a cell density of 100U/ml, CD3/CD28 magnetic beads were added at a cell to magnetic bead ratio of 1:3, and interleukin 2 was added at a final concentration of 100U/ml for overnight culture. Wherein, the ratio of the cells to the magnetic beads refers to the ratio of the number of the cells to the number of the magnetic beads.
Step 504: the lentiviral particles were added for transfection.
Step 505: after cell transfection, the cells were cultured for 5 days, and the magnetic beads were removed by a magnetic holder to obtain immune cells.
Without limitation, lentiviral plasmids or immune cells can also be obtained by other methods or vectors.
Examples one and two were set up as shown in the following tables. Wherein, CD19 is B lymphocyte antigen molecule, PSMA is prostate-specific membrane antigen (prostate-specific antigen), in the first embodiment, scFv binds CD19 with CD19 target, i.e. chimeric antigen receptor of immune cell; in the second example, the scFv targets PSMA, i.e., the chimeric antigen receptor of immune cells binds PSMA.
Figure BDA0002694606560000071
Wherein, please look at the sequence table about the amino acid sequence and cDNA sequence in the table.
In the present invention, the oxidation resistance test, the tumor cell killing test and the mouse tumor model test were performed on the first example and the second example.
The method of oxidation resistance testing is shown in the table below.
Figure BDA0002694606560000072
Figure BDA0002694606560000081
H2O2Is the most important substance in the ROS environment, in H2O2The structural integrity of the chimeric antigen in CAR-T cells was observed by measuring the amount of scFv under ambient cell culture conditions. Protein L binds scFv and ELISA measures the amount of Protein L in the CAR-T cell lysate to determine the amount of scFv, and if the amount of Protein L is reduced, a reduction in the amount of scFv bound thereto is indicated. Wherein the horseradish peroxidase labeled antibody can be combined with the anti-protein L antibody. Wherein, a standard hole is arranged in the 96-well plate, and standard protein L is added into the standard hole and is used for detecting the amount of the protein L in each sample.
The results of the detection are shown in FIGS. 6 and 7, H2O2(-) is a non-oxidizing packet, H2O2(+) H of oxidized subgroup, control group (CAR CD19 and CAR PSMA)2O2The amount of oxidized fraction ProteinL was significantly lower than the non-oxidized fraction, indicating a reduced scFv content of the oxidized fraction, H2O2The integrity of the CAR is destroyed; CAR CD19Y1, CAR PSMA Y1, CARCD 19Y 2 and CAR PSMA Y2, H2O2No significant difference in the amount of Protein L was observed between the absence and presence of oxidative treatment, indicating that the integrity of the scFv of optimized group 1 and optimized group 2 of the present invention was not affected by H2O2The oxidation resistance is exhibited. Among them, fig. 6 and 7 were obtained based on the grapphad Prism 8.0 analysis software, 2Way ANOVA statistical analysis method.
The method of tumor killing test is as follows:
Figure BDA0002694606560000091
among them, XTT is a tetrazole nitrogen derivative, and a metabolic product which can be reduced to water-soluble by metabolic enzymes in living cells has an absorption peak at OD450, and the OD450 value decreases, indicating that the number of living cells is small, and the OD450 value is high, indicating that the number of living cells is large. CAR-T kills tumor cells, the number of viable cells decreases, and the OD450 value decreases, so CAR-T cell killing on tumor cells can be measured by XTT.
As shown in FIGS. 8 and 9, control group (CAR)CD19 and CAR PSMA) had significantly higher OD450 values for the oxidized fraction than the non-oxidized fraction, indicating H2O2The number of living cells in the oxidative grouping is high, and the killing effect of the oxidative grouping immune cells on tumor cells is not as good as that of non-oxidative grouping; the OD450 values of the oxidized and non-oxidized groups of CAR CD19Y1, CAR PSMA Y1, CAR CD19Y1 and CAR PSMA Y2 are not significantly different, so that the killing of tumor cells by the compounds is not influenced by H2O2Has ROS resistance or oxidation resistance. The oxidation group OD450 value of the control group is obviously higher than that of the optimization group 1 and the optimization group 2, so that the oxidation group of the control group has high living cell quantity, and the killing effect on tumor cells is obviously lower than that of the optimization group 1 and the optimization group 2.
Cytokines such as tumor necrosis factor alpha (TNF alpha) and Interferon gamma (IFN gamma) play an important role in the anti-tumor effect of CAR-T cells, so that the content of TNF alpha and IFN gamma in cell culture fluid reflects the tumor killing power. Among them, ELISA detection of cytokine is prior art, and the present invention is not described in detail. As shown in fig. 10-13, the CARCD19 and CARPSMA cells, whose TNF α and IFN γ content were significantly lower in the oxidized fraction than in the non-oxidized fraction, whereas the oxidized and non-oxidized fractions of CAR CD19Y1, CAR PSMA Y1, CAR CD19Y1, and CAR PSMA Y2, had no significant change in TNF α and IFN γ content; the oxidation cohort of the control group had a lower TNF α and IFN γ content than the oxidation cohort of the optimized group 1 and the optimized group 2. Wherein H2O2Oxidation may result in a decrease in the expression of anti-tumor factors by immune cells. Therefore, the optimized group 1 and the optimized group 2 showed good antioxidant activity and tumor cell inhibitory activity.
Mouse tumor model testing:
NOD/SCID mice, dorsal flank subcutaneous inoculation of 2x106Prostate cancer cells LNCaP, approximately 2 weeks after inoculation until tumors grow 100 cubic millimeters (mm)3) Left and right. Wherein, the length and the width of the tumor are measured by a vernier caliper, and the size of the tumor is calculated by the following formula:
V＝(a×b2)÷2
wherein V is the tumor volume, a is the tumor length, and b is the tumor width.
The 24 mice were randomly divided into 4 groups: blank control group, CAR PSMA Y1 group, and CAR PSMA Y2 group. CAR PSMA group, CAR PSMA Y1 group, and CAR PSMA Y2 group were injected with the corresponding immune cells, respectively, and the blank control group was injected with an equal volume of saline.
Tumor size was measured for each mouse on days 4, 8, 12, 17, 21, 24, 26, 29 and 31, respectively, and averaged for each group. The blank control group died on day 26, and the results are shown in fig. 14. Tumor growth was significantly lower in the carpma group than in the blank control group; the effects of the CAR PSMA Y1 group and the CAR PSMA Y1 group on tumor inhibition are equivalent and are obviously better than those of the CAR PSMA group. Therefore, the chimeric antigen receptor and the immune cell of the invention have good solid tumor inhibition effect.
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Liangwenqiang
<120> an antioxidant chimeric antigen receptor and immune cell
<130> NP201621
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 11
<212> PRT
<213> unknown (unknown)
<400> 1
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
1 5 10
<210> 2
<211> 33
<212> DNA
<213> unknown (unknown)
<400> 2
tgtgacaaaa ctcacacatg cccaccgtgc cca 33
<210> 3
<211> 11
<212> PRT
<213> unknown (unknown)
<400> 3
Cys Asp Lys Thr Tyr Thr Cys Pro Pro Cys Pro
1 5 10
<210> 4
<211> 11
<212> PRT
<213> unknown (unknown)
<400> 4
Cys Asp Lys Thr Ser Thr Cys Pro Pro Cys Pro
1 5 10
<210> 5
<211> 366
<212> DNA
<213> unknown (unknown)
<400> 5
gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagggca gccccgagaa 60
ccacaggtgt acaccctgcc cccatcccgg gatgagctga ccaagaacca ggtcagcctg 120
acctgcctgg tcaaaggctt ctatcccagc gacatcgccg tggagtggga gagcaatggg 180
cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgtcgg ctccttcttc 240
ctctacagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt cttctcatgc 300
tccgtgatgc atgaggctct gcacaaccac tacacgcaga agagcctctc cctgtctccg 360
ggtaaa 366
<210> 6
<211> 366
<212> DNA
<213> unknown (unknown)
<400> 6
gagcccaaat cttgtgacaa aacttacaca tgcccaccgt gcccagggca gccccgagaa 60
ccacaggtgt acaccctgcc cccatcccgg gatgagctga ccaagaacca ggtcagcctg 120
acctgcctgg tcaaaggctt ctatcccagc gacatcgccg tggagtggga gagcaatggg 180
cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgtcgg ctccttcttc 240
ctctacagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt cttctcatgc 300
tccgtgatgc atgaggctct gcacaaccac tacacgcaga agagcctctc cctgtctccg 360
ggtaaa 366
<210> 7
<211> 366
<212> DNA
<213> unknown (unknown)
<400> 7
gagcccaaat cttgtgacaa aacttcaaca tgcccaccgt gcccagggca gccccgagaa 60
ccacaggtgt acaccctgcc cccatcccgg gatgagctga ccaagaacca ggtcagcctg 120
acctgcctgg tcaaaggctt ctatcccagc gacatcgccg tggagtggga gagcaatggg 180
cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgtcgg ctccttcttc 240
ctctacagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt cttctcatgc 300
tccgtgatgc atgaggctct gcacaaccac tacacgcaga agagcctctc cctgtctccg 360
ggtaaa 366
<210> 8
<211> 1725
<212> DNA
<213> unknown (unknown)
<400> 8
tcaccagaat tcatggagtt tgggctgagc tgggttttcc tcgttgctct tttaagaggt 60
gtccagtgtg acatccagat gacacagact acatcctccc tgtctgcctc tctgggagac 120
agagtcacca tcagttgcag ggcaagtcag gacattagta aatatttaaa ttggtatcag 180
cagaaaccag atggaactgt taaactcctg atctaccata catcaagatt acactcagga 240
gtcccatcaa ggttcagtgg cagtgggtct ggaacagatt attctctcac cattagcaac 300
ctggagcaag aagatattgc cacttacttt tgccaacagg gtaatacgct tccgtacacg 360
ttcggagggg ggactaagtt ggaaataaca ggctccacct ctggatccgg caagcccgga 420
tctggcgagg gatccaccaa gggcgaggtg aaactgcagg agtcaggacc tggcctggtg 480
gcgccctcac agagcctgtc cgtcacatgc actgtctcag gggtctcatt acccgactat 540
ggtgtaagct ggattcgcca gcctccacga aagggtctgg agtggctggg agtaatatgg 600
ggtagtgaaa ccacatacta taattcagct ctcaaatcca gactgaccat catcaaggac 660
aactccaaga gccaagtttt cttaaaaatg aacagtctgc aaactgatga cacagccatt 720
tactactgtg ccaaacatta ttactacggt ggtagctatg ctatggacta ctggggtcaa 780
ggaacctcag tcaccgtctc ctcagcggcc gagcccaaat cttgtgacaa aactcacaca 840
tgcccaccgt gcccagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 900
gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 960
gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1020
cccgtgctgg actccgtcgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 1080
aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1140
tacacgcaga agagcctctc cctgtctccg ggtaaaatct acatctgggc gcccttggcc 1200
gggacttgtg gggtccttct cctgtcactg gttatcaccc tttactgcag gagtaagagg 1260
agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1320
aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cagagtgaag 1380
ttcagcagga gcgcagacgc ccccgcgtac cagcagggcc agaaccagct ctataacgag 1440
ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 1500
gagatggggg gaaagccgca gagaaggaag aaccctcagg aaggcctgta caatgaactg 1560
cagaaagata agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg 1620
ggcaaggggc acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac 1680
gcccttcaca tgcaggccct gccccctcgc taagtcgacc aaagg 1725
<210> 9
<211> 1725
<212> DNA
<213> unknown (unknown)
<400> 9
tcaccagaat tcatggagtt tgggctgagc tgggttttcc tcgttgctct tttaagaggt 60
gtccagtgtg acatccagat gacacagact acatcctccc tgtctgcctc tctgggagac 120
agagtcacca tcagttgcag ggcaagtcag gacattagta aatatttaaa ttggtatcag 180
cagaaaccag atggaactgt taaactcctg atctaccata catcaagatt acactcagga 240
gtcccatcaa ggttcagtgg cagtgggtct ggaacagatt attctctcac cattagcaac 300
ctggagcaag aagatattgc cacttacttt tgccaacagg gtaatacgct tccgtacacg 360
ttcggagggg ggactaagtt ggaaataaca ggctccacct ctggatccgg caagcccgga 420
tctggcgagg gatccaccaa gggcgaggtg aaactgcagg agtcaggacc tggcctggtg 480
gcgccctcac agagcctgtc cgtcacatgc actgtctcag gggtctcatt acccgactat 540
ggtgtaagct ggattcgcca gcctccacga aagggtctgg agtggctggg agtaatatgg 600
ggtagtgaaa ccacatacta taattcagct ctcaaatcca gactgaccat catcaaggac 660
aactccaaga gccaagtttt cttaaaaatg aacagtctgc aaactgatga cacagccatt 720
tactactgtg ccaaacatta ttactacggt ggtagctatg ctatggacta ctggggtcaa 780
ggaacctcag tcaccgtctc ctcagcggcc gagcccaaat cttgtgacaa aacttacaca 840
tgcccaccgt gcccagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 900
gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 960
gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1020
cccgtgctgg actccgtcgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 1080
aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1140
tacacgcaga agagcctctc cctgtctccg ggtaaaatct acatctgggc gcccttggcc 1200
gggacttgtg gggtccttct cctgtcactg gttatcaccc tttactgcag gagtaagagg 1260
agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1320
aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cagagtgaag 1380
ttcagcagga gcgcagacgc ccccgcgtac cagcagggcc agaaccagct ctataacgag 1440
ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 1500
gagatggggg gaaagccgca gagaaggaag aaccctcagg aaggcctgta caatgaactg 1560
cagaaagata agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg 1620
ggcaaggggc acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac 1680
gcccttcaca tgcaggccct gccccctcgc taagtcgacc aaagg 1725
<210> 10
<211> 1725
<212> DNA
<213> unknown (unknown)
<400> 10
tcaccagaat tcatggagtt tgggctgagc tgggttttcc tcgttgctct tttaagaggt 60
gtccagtgtg acatccagat gacacagact acatcctccc tgtctgcctc tctgggagac 120
agagtcacca tcagttgcag ggcaagtcag gacattagta aatatttaaa ttggtatcag 180
cagaaaccag atggaactgt taaactcctg atctaccata catcaagatt acactcagga 240
gtcccatcaa ggttcagtgg cagtgggtct ggaacagatt attctctcac cattagcaac 300
ctggagcaag aagatattgc cacttacttt tgccaacagg gtaatacgct tccgtacacg 360
ttcggagggg ggactaagtt ggaaataaca ggctccacct ctggatccgg caagcccgga 420
tctggcgagg gatccaccaa gggcgaggtg aaactgcagg agtcaggacc tggcctggtg 480
gcgccctcac agagcctgtc cgtcacatgc actgtctcag gggtctcatt acccgactat 540
ggtgtaagct ggattcgcca gcctccacga aagggtctgg agtggctggg agtaatatgg 600
ggtagtgaaa ccacatacta taattcagct ctcaaatcca gactgaccat catcaaggac 660
aactccaaga gccaagtttt cttaaaaatg aacagtctgc aaactgatga cacagccatt 720
tactactgtg ccaaacatta ttactacggt ggtagctatg ctatggacta ctggggtcaa 780
ggaacctcag tcaccgtctc ctcagcggcc gagcccaaat cttgtgacaa aacttcaaca 840
tgcccaccgt gcccagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 900
gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 960
gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1020
cccgtgctgg actccgtcgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 1080
aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1140
tacacgcaga agagcctctc cctgtctccg ggtaaaatct acatctgggc gcccttggcc 1200
gggacttgtg gggtccttct cctgtcactg gttatcaccc tttactgcag gagtaagagg 1260
agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1320
aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cagagtgaag 1380
ttcagcagga gcgcagacgc ccccgcgtac cagcagggcc agaaccagct ctataacgag 1440
ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 1500
gagatggggg gaaagccgca gagaaggaag aaccctcagg aaggcctgta caatgaactg 1560
cagaaagata agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg 1620
ggcaaggggc acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac 1680
gcccttcaca tgcaggccct gccccctcgc taagtcgacc aaagg 1725
<210> 11
<211> 1710
<212> DNA
<213> unknown (unknown)
<400> 11
tcaccagaat tcatggagtt tgggctgagc tgggttttcc tcgttgctct tttaagaggt 60
gtccagtgtg acatcgtcat gactcagtca cacgatttca tggcgacttc gctgggcgaa 120
cgcgctacca ttaactgtaa agcaagccaa gatgtaggga cagcggtcga ctggtaccaa 180
cagaagccag gccagccgcc caaactgctg atctattggg catcgacgcg acacacgggg 240
gtccctgaca gattttctgg aagtggctca gggacagatt tcacattgac aattacaaac 300
ctgcaatcgg aggacctcgc ggattacttc tgtcaacagt acaattcgta tcccctgacg 360
ttcgggcaag ggacaaagct cgacatcaag ggcggtggag ggtcaggtgg aggaggctcc 420
ggtgggggag ggagcggagg gggtggttcg gaggtgcagt tgcagcaatc aggcccggaa 480
cttaagaaac ccgggacctc agtaagaatc agctgtaaga caagcgggta cacgtttacc 540
gaatatacta tccattgggt gaagcaggcg cctggaaaat cgcttgaatg gatcgggaac 600
attaatccta ataacggggg aaccacgtac aaccagaagt ttgaggataa agccaccctt 660
actgtggaca aatccaaaaa ctctctgtat atggaattgc ggtccctgaa gaccgaggat 720
tcagccgtat actactgcgc ggcaggatgg aattttgatt attgggggca gggaacaaca 780
ttgacagtct cgagcgagcc caaatcttgt gacaaaacta ccacatgccc accgtgccca 840
gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 900
aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 960
tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1020
gtcggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1080
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1140
ctctccctgt ctccgggtaa aatctacatc tgggcgccct tggccgggac ttgtggggtc 1200
cttctcctgt cactggttat caccctttac tgcaggagta agaggagcag gctcctgcac 1260
agtgactaca tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc 1320
tatgccccac cacgcgactt cgcagcctat cgctccagag tgaagttcag caggagcgca 1380
gacgcccccg cgtaccagca gggccagaac cagctctata acgagctcaa tctaggacga 1440
agagaggagt acgatgtttt ggacaagaga cgtggccggg accctgagat ggggggaaag 1500
ccgcagagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1560
gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1620
ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1680
gccctgcccc ctcgctaagg atcccaaagg 1710
<210> 12
<211> 1710
<212> DNA
<213> unknown (unknown)
<400> 12
tcaccagaat tcatggagtt tgggctgagc tgggttttcc tcgttgctct tttaagaggt 60
gtccagtgtg acatcgtcat gactcagtca cacgatttca tggcgacttc gctgggcgaa 120
cgcgctacca ttaactgtaa agcaagccaa gatgtaggga cagcggtcga ctggtaccaa 180
cagaagccag gccagccgcc caaactgctg atctattggg catcgacgcg acacacgggg 240
gtccctgaca gattttctgg aagtggctca gggacagatt tcacattgac aattacaaac 300
ctgcaatcgg aggacctcgc ggattacttc tgtcaacagt acaattcgta tcccctgacg 360
ttcgggcaag ggacaaagct cgacatcaag ggcggtggag ggtcaggtgg aggaggctcc 420
ggtgggggag ggagcggagg gggtggttcg gaggtgcagt tgcagcaatc aggcccggaa 480
cttaagaaac ccgggacctc agtaagaatc agctgtaaga caagcgggta cacgtttacc 540
gaatatacta tccattgggt gaagcaggcg cctggaaaat cgcttgaatg gatcgggaac 600
attaatccta ataacggggg aaccacgtac aaccagaagt ttgaggataa agccaccctt 660
actgtggaca aatccaaaaa ctctctgtat atggaattgc ggtccctgaa gaccgaggat 720
tcagccgtat actactgcgc ggcaggatgg aattttgatt attgggggca gggaacaaca 780
ttgacagtct cgagcgagcc caaatcttgt gacaaaactt caacatgccc accgtgccca 840
gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 900
aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 960
tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1020
gtcggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1080
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1140
ctctccctgt ctccgggtaa aatctacatc tgggcgccct tggccgggac ttgtggggtc 1200
cttctcctgt cactggttat caccctttac tgcaggagta agaggagcag gctcctgcac 1260
agtgactaca tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc 1320
tatgccccac cacgcgactt cgcagcctat cgctccagag tgaagttcag caggagcgca 1380
gacgcccccg cgtaccagca gggccagaac cagctctata acgagctcaa tctaggacga 1440
agagaggagt acgatgtttt ggacaagaga cgtggccggg accctgagat ggggggaaag 1500
ccgcagagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1560
gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1620
ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1680
gccctgcccc ctcgctaagg atcccaaagg 1710
<210> 13
<211> 1710
<212> DNA
<213> unknown (unknown)
<400> 13
tcaccagaat tcatggagtt tgggctgagc tgggttttcc tcgttgctct tttaagaggt 60
gtccagtgtg acatcgtcat gactcagtca cacgatttca tggcgacttc gctgggcgaa 120
cgcgctacca ttaactgtaa agcaagccaa gatgtaggga cagcggtcga ctggtaccaa 180
cagaagccag gccagccgcc caaactgctg atctattggg catcgacgcg acacacgggg 240
gtccctgaca gattttctgg aagtggctca gggacagatt tcacattgac aattacaaac 300
ctgcaatcgg aggacctcgc ggattacttc tgtcaacagt acaattcgta tcccctgacg 360
ttcgggcaag ggacaaagct cgacatcaag ggcggtggag ggtcaggtgg aggaggctcc 420
ggtgggggag ggagcggagg gggtggttcg gaggtgcagt tgcagcaatc aggcccggaa 480
cttaagaaac ccgggacctc agtaagaatc agctgtaaga caagcgggta cacgtttacc 540
gaatatacta tccattgggt gaagcaggcg cctggaaaat cgcttgaatg gatcgggaac 600
attaatccta ataacggggg aaccacgtac aaccagaagt ttgaggataa agccaccctt 660
actgtggaca aatccaaaaa ctctctgtat atggaattgc ggtccctgaa gaccgaggat 720
tcagccgtat actactgcgc ggcaggatgg aattttgatt attgggggca gggaacaaca 780
ttgacagtct cgagcgagcc caaatcttgt gacaaaactt caacatgccc accgtgccca 840
gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 900
aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 960
tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1020
gtcggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1080
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1140
ctctccctgt ctccgggtaa aatctacatc tgggcgccct tggccgggac ttgtggggtc 1200
cttctcctgt cactggttat caccctttac tgcaggagta agaggagcag gctcctgcac 1260
agtgactaca tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc 1320
tatgccccac cacgcgactt cgcagcctat cgctccagag tgaagttcag caggagcgca 1380
gacgcccccg cgtaccagca gggccagaac cagctctata acgagctcaa tctaggacga 1440
agagaggagt acgatgtttt ggacaagaga cgtggccggg accctgagat ggggggaaag 1500
ccgcagagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1560
gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1620
ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1680
gccctgcccc ctcgctaagg atcccaaagg 1710

Claims (10)

1. An antioxidant chimeric antigen receptor comprising an extracellular region including a spacer region having a Core Hinge region amino acid sequence of Core Hinge-Y1: CDKTYTCPPCP (SEQ ID NO: 3) or Core Hinge-Y2: CDKTSTCPPCP (SEQ ID NO: 4).
2. The chimeric antigen receptor according to claim 1, further comprising a transmembrane region and an intracellular region, wherein the extracellular region further comprises a single-chain antibody, and wherein the transmembrane region is connected to the intracellular region at both ends of the spacer region, and wherein the spacer region is a constant-chain fragment of immunoglobulin G.
3. The chimeric antigen receptor of claim 1, wherein the spacer has the gene sequence of IgG HCH3-Y1 cDNA (SEQ ID NO: 6) or IgG HCH3-Y2 cDNA (SEQ ID NO: 7).
4. An immune cell expressing the chimeric antigen receptor of any one of claims 1-3.
5. The immune cell of claim 4, wherein the immune cell comprises a CAR-T cell, a CAR-NK cell, a CAR-B cell, or a CAR-MP cell.
6. The immune cell of claim 4, wherein the immune cell is used to kill tumor cells.
7. The immune cell of claim 6, wherein the immune cell is used to kill solid tumor cells or hematologic tumor cells.
8. The immune cell of claim 4, wherein the method of preparing the immune cell comprises:
synthesizing cDNA of chimeric antigen receptor;
preparing a lentiviral plasmid from the cDNA;
preparing lentiviral particles from the lentiviral plasmid;
obtaining mononuclear cells and performing pre-culture;
transfecting the lentivirus particles into the pre-cultured mononuclear cells, and culturing to obtain immune cells.
9. The chimeric antigen receptor according to claim 8, wherein the lentiviral particle is prepared by a method comprising:
culture of 6X10 Using DMEM Medium containing 10% fetal bovine serum6A HEK293T cell;
when the cell degree of polymerization reaches about 80% on the next day, the cell is stabilized for 2-3 hours after the cell is changed into a culture solution without 10% FBS and antibiotics;
the lentiviral plasmid comprises a pCCL-CAR plasmid, and the pCCL-CAR plasmid with the final concentration of 6.9 mug/ml, the pMDLg-pRRE plasmid with the final concentration of 3.4 mug/ml, the pMD2.G plasmid with the final concentration of 2 mug/ml and the pRSV-Rev plasmid with the final concentration of 1.71 mug/ml are respectively added into a culture solution and are uniformly mixed to obtain a plasmid culture solution;
adding 35 mu g of polyethyleneimine with the concentration of 1 mu g/mu l into 1 ml of DMEM culture solution, and uniformly mixing to obtain a polyethyleneimine culture solution;
culturing polyethyleneimine, adding the polyethyleneimine into material culture, uniformly mixing, and standing at room temperature for 20 minutes to obtain a first transfection solution;
adding the first transfection solution into the stabilized HEK293T cells for transfection;
after 4 hours of transfection, the culture solution was changed to DMEM containing 10% fetal bovine serum;
after culturing for 48 hours, collecting cell culture solution, and filtering with a 0.22 μm filter membrane;
centrifuging the filtrate at 26000rpm and 4 deg.C for 2 hr, and collecting precipitate;
resuspend the pellet with XVivo15 whole medium to obtain lentiviral particles.
10. The chimeric antigen receptor according to claim 8, wherein the method for obtaining an immune cell comprises:
taking 50ml of peripheral blood, and performing density gradient centrifugation on Ficoll lymphocyte separation liquid to obtain mononuclear cells;
the mononuclear cell is expressed at 2x106Cell density of/ml in X-VIVO 15 whole culture medium, 37 deg.C, 5% CO2Culturing for 2 hours, and collecting suspension cells;
suspension cells at 1.2 × 106Inoculating the cells with the density of/ml into a culture bottle, adding CD3/CD28 magnetic beads according to the ratio of the cells to the magnetic beads being 1:3, and adding interleukin 2 with the final concentration of 100U/ml for culturing overnight;
adding the lentivirus particles for transfection;
after cell transfection, the cells were cultured for 5 days, and the magnetic beads were removed by a magnetic holder to obtain immune cells.
CN202011001854.3A 2020-09-22 2020-09-22 Antioxidant chimeric antigen receptor and immune cell Pending CN112111014A (en)

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