CN109694875B

CN109694875B - anti-CII chimeric antigen receptor coding gene, lentiviral plasmid, Treg immune cell and application thereof

Info

Publication number: CN109694875B
Application number: CN201811610740.1A
Authority: CN
Inventors: 刘明录; 王立新; 刘敏; 金海锋; 万磊; 冯建海; 卢永灿; 张传鹏; 强邦明
Original assignee: Shandong Xinrui Biotechnology Co ltd
Current assignee: Shanghai Xingrui Yida Biotechnology Co ltd
Priority date: 2018-12-27
Filing date: 2018-12-27
Publication date: 2022-04-29
Anticipated expiration: 2038-12-27
Also published as: CN109694875A

Abstract

The invention discloses an anti-CII chimeric antigen receptor coding gene, which comprises an Linker nucleic acid artificial sequence, a CII single-chain antibody nucleic acid artificial sequence, a CD8hinge region nucleic acid artificial sequence, a CD8 transmembrane region nucleic acid artificial sequence, a 2B4 co-stimulatory region nucleic acid artificial sequence, a CD3 zeta signaling region nucleic acid artificial sequence, a T2A self-cleavage region nucleic acid artificial sequence, a CTLA4 nucleic acid artificial sequence and a RQR8 molecular switch region nucleic acid artificial sequence. The invention also discloses a lentiviral plasmid with the CII-resistant chimeric antigen receptor coding gene, a Treg immune cell and application thereof. The invention enhances the immunosuppressive activity of CAR-Tregs cells on the one hand and ensures the safety of CAR-Treg cells on the other hand while ensuring the curative effect.

Description

anti-CII chimeric antigen receptor coding gene, lentiviral plasmid, Treg immune cell and application thereof

Technical Field

The invention relates to the field of biological genes, in particular to an anti-CII chimeric antigen receptor coding gene, a lentivirus plasmid, a Treg immune cell and application thereof.

Background

Rheumatoid Arthritis (RA) is a common autoimmune abnormal disease with high disability and influence on life quality worldwide, and the incidence rate is high, accounting for 2% -4% of the total population of Rheumatoid arthritis population according to statistics. RA is also considered to be a chronic syndrome, which is a disease in which the joints of hands and feet are swollen and damaged due to invasion of the joints of hands and feet by the immune system of the RA itself, and is manifested as nonspecific inflammation of peripheral joints. The diseased joint and its surrounding tissues now exhibit progressive destruction and cause dysfunction of the damaged joint, with autoreactive T lymphocytes playing an important role in their aberrant immune response.

Currently, the development of the disease is mainly controlled by drug therapy such as immunosuppressants and salicylates or surgical therapy such as synovium excision in the treatment of RA. Many studies and patents on RA have been reported, but many of them aim at treating RA by using new drugs, new dosage forms or RNA interference techniques, for example, patents such as CN200910112919.9, CN201380036874.6 and CN201380036874.6 invented new patches or new compositions, and no cell preparations for treating RA by using immune cells exist at present, so there is no preparation method thereof.

Therefore, the anti-CII chimeric antigen receptor coding gene, the lentivirus plasmid, the Treg immune cell and the application thereof are developed, so that the method has extremely high research value, and simultaneously has good economic benefit and industrial application potential.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides an anti-CII chimeric antigen receptor coding gene, a lentivirus plasmid, a Treg immune cell and application thereof.

In order to achieve the purpose, the technical scheme of the invention is as follows:

in a first aspect, the present invention provides an anti-CII chimeric antigen receptor encoding gene, wherein the encoding gene at least comprises a CII binding region, a CTLA4 functional region and a RQR8 molecular switch.

In the present invention, as a preferred embodiment, the anti-CII chimeric antigen receptor-encoding gene comprises Linker nucleic acid artificial sequence, CII single-chain antibody nucleic acid artificial sequence, CD8hinge region nucleic acid artificial sequence, CD8 transmembrane region nucleic acid artificial sequence, 2B4 costimulatory region nucleic acid artificial sequence, CD3 zeta signaling region nucleic acid artificial sequence, T2A self-cleavage region nucleic acid artificial sequence, CTLA4 nucleic acid artificial sequence, and RQR8 molecular switch region nucleic acid artificial sequence.

In the invention, as a preferred technical scheme, the anti-CII chimeric antigen receptor coding gene is shown in SEQ ID NO. 1.

In the invention, as a preferable technical scheme, the anti-CII chimeric antigen receptor comprises a Leader nucleic acid artificial sequence shown in SEQ ID NO.2 which is connected in sequence,

the CII binding region nucleic acid artificial sequence as shown in SEQ ID NO.3,

the CD8hinge region nucleic acid artificial sequence as shown in SEQ ID NO.4,

the CD8 transmembrane region nucleic acid artificial sequence as described in SEQ ID NO.5,

2B4 costimulatory region nucleic acid artificial sequence as set forth in SEQ ID NO.6,

the intracellular domain nucleic acid artificial sequence of CD3 ζ as set forth in SEQ ID NO.7,

the T2A nucleic acid artificial sequence as shown in SEQ ID NO.8,

the nucleic acid artificial sequence of CTLA4 as shown in SEQ ID NO.9,

the T2A nucleic acid artificial sequence as shown in SEQ ID NO.8,

the RQR8 nucleic acid artificial sequence as shown in SEQ ID NO. 10.

In a second aspect, the present invention provides a lentiviral plasmid with anti-CII chimeric antigen receptor, wherein the whole expression frame is synthesized according to the sequence of fusion gene fragment Leader-scFv (CII) -CD8-2B4-CD3 ζ -T2A-CTLA4-T2A-RQR8, and inserted between AsiSI and NotI cleavage sites of lentiviral plasmid pLent-C-GFP, so as to obtain plasmid pLent-CII-CTLA 4.

More specifically, the plasmid purification steps are: the pLent-CII-CTLA4 plasmid was extracted using an endotoxin-free plasmid extraction and purification kit (available from Solambio).

In a third aspect, the invention provides a Treg immune cell having a gene encoding an anti-CII chimeric antigen receptor.

In the present invention, as a preferred technical scheme, the Treg immune cells are obtained by a preparation method comprising:

transfecting a cell line 293T with pLent-CII-CTLA4 plasmid, then infecting Treg immune cells with the recombinant lentivirus, culturing the infected cells in a 37 ℃ and 5% CO2 incubator for 8 hours, collecting the cells, adding the virus solution again, culturing the cells in a 1000g and 32 ℃ centrifuge again for 90 minutes at 37 ℃ and in a 5% CO2 incubator, and repeating multiple infection in the way to improve the infection efficiency of the CIK cells; 2ml of culture supernatant was aspirated off, 2ml of fresh medium was added, and the expansion culture was continued for 17 days until the cells were expanded to a sufficient amount.

In a fourth aspect, the invention provides the use of Treg cells having a gene encoding an anti-CII chimeric antigen receptor for autoimmune diseases, particularly rheumatoid arthritis.

After the technical scheme is adopted, the invention has the beneficial effects that:

the invention innovatively provides CAR-Tregs cells for treating rheumatoid arthritis, and the CII is innovatively used as a target for treating autoimmune diseases. Type ii Collagen (CII) is mainly produced by chondrocytes, and is present in many tissues such as bones, joints, tendons, and the like. Anti-type II collagen antibodies, often found in RA inflammatory sites, play a role in the disease process, and the CII-targeting antibodies can treat inflammatory diseases, inhibit angiogenesis, and treat cancer and autoimmune diseases. CD4+ CD25+ Foxp3+ Regulatory T cells (Tregs) are a subset of T cells that control autoimmune reactivity in vivo, and are closely related to the development of autoimmune diseases. Tregs can suppress immune responses in a variety of ways, including the secretion of the suppressor cytokines IL-10, TGF-. beta.and the like. Tregs play a major role in the maintenance of peripheral immune tolerance and the prevention of autoimmune diseases, mainly by exerting an inhibitory immunoregulatory function. The invention constructs a CAR with anti-CII to target the treatment of autoimmune diseases, in particular to rheumatoid arthritis, and the designed coding gene at least comprises a CII binding region, a CTLA4 functional region and a RQR8 molecular switch, and also comprises a T2A nucleic acid artificial sequence positioned among the CII binding region, the CTLA4 functional region and the RQR8 molecular switch. Thus, after the anti-CII safe chimeric antigen receptor is transferred into an immune cell and translated into protein, T2A can make the receptor split into three parts by self-cutting, wherein the first part is a CAR structure of a CII antigen binding region, the second part is a CTLA4 functional region, and the third part is an RQR8 molecular switch action region. The invention enhances the immunosuppressive activity of CAR-Tregs cells on the one hand and ensures the safety of CAR-Treg cells on the other hand while ensuring the curative effect.

According to the invention, CII is used as a target for treating autoimmune diseases, and anti-type II collagen CAR is transfected into Tregs cells in peripheral blood of a patient collected and cultured in advance through a lentiviral vector to obtain the Tregs cells containing scFv (CII). The scFv (CII) can be combined with CII at the arthritis lesion, so that Tregs cells are gathered at the arthritis lesion, the immunosuppressive function of the Tregs cells is better exerted, and the effect of treating rheumatoid arthritis is further achieved.

The CAR structure of the invention is added with functional region elements of CTLA4, CTLA4 and 2B4 share a B7 molecular ligand, and CTLA-4 is combined with B7 molecules to induce T cell anergy and participate in the negative regulation of immune response. The addition of this element further enhances the immunosuppressive activity of CAR-Tregs cells.

According to the invention, the RQR8 molecular switch element is added into the CAR structure, the molecular switch has small molecular weight and consists of two antigen epitopes, rituximab, QBEND10 mab and the like can be clinically applied to directly induce apoptosis of CAR-Tregs through ADCC effect and CDC effect of antibodies, the clinical use safety of CAR-Tregs cells is ensured, and adverse reactions such as cytokine storm can be effectively stopped.

In conclusion, the invention enhances the immunosuppressive activity of the CAR-Tregs cells on the one hand and ensures the safety of the CAR-Treg cells on the other hand while ensuring the curative effect.

Drawings

FIG. 1 is a design diagram of a fusion gene fragment of the chimeric antigen receptor Leader-scFv (CII) -CD8-2B4-CD3 zeta-T2A-CTLA 4-T2A-RQR 8.

FIG. 2 is a graph showing the efficiency of expressing CAR from pLent-CII-CTLA 4-specific Treg cells of the present invention.

FIG. 3 shows the therapeutic effect of pLent-CII-CTLA4 specific Treg cells on arthritis mice.

Detailed Description

The technical solution of the present invention will be described in detail with reference to specific examples. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.

Example 1

An anti-CII chimeric antigen receptor coding gene, which comprises an artificial Linker nucleic acid sequence, an artificial CII single-chain antibody nucleic acid sequence, an artificial CD8hinge region nucleic acid sequence, an artificial CD8 transmembrane region nucleic acid sequence, an artificial 2B4 costimulatory region nucleic acid sequence, an artificial CD3 zeta signaling region nucleic acid sequence, an artificial T2A self-splicing region nucleic acid sequence, an artificial CTLA4 nucleic acid sequence and an artificial RQR8 molecular switch region nucleic acid sequence.

As shown in FIG. 1, the anti-CII chimeric antigen receptor encoding gene of the present example comprises a Leader nucleic acid artificial sequence SEQ ID NO.2, a CII binding region nucleic acid artificial sequence (SEQ ID NO.3), a CD8Hinge region nucleic acid artificial sequence (SEQ ID NO.4), a CD8 transmembrane region nucleic acid artificial sequence (SEQ ID NO.5), a 2B4 nucleic acid artificial sequence (SEQ ID NO.6), a CD3 zeta intracellular region nucleic acid artificial sequence (SEQ ID NO.7), a T2A nucleic acid artificial sequence (SEQ ID NO.8), a CTLA4 nucleic acid artificial sequence (SEQ ID NO.9), a T2A nucleic acid artificial sequence (SEQ ID NO.8) and a RQR8 nucleic acid artificial sequence (SEQ ID NO.10) which are sequentially linked.

Example 2

Plasmid preparation example of anti-CII chimeric antigen receptor encoding gene.

The preparation method of the plasmid for anti-CII chimeric antigen receptor coding gene comprises the following steps:

(1) according to the order of Leader-scFv (CII) -CD8-2B4-CD3 ζ -T2A-CTLA4-T2A-RQR8, the entire expression cassette was synthesized by Competition Biotechnology engineering (Shanghai) Co., Ltd and inserted between AsiSI and NotI cleavage sites of lentiviral plasmid pLent-C-GFP to obtain plasmid pLent-CII-CTLA 4. Finally, E.coli Top10 containing lentivirus plasmid pLent-CII-CTLA4 with correct sequencing is obtained. Extracting lentiviral plasmid pLent-CII-CTLA4 by using an endotoxin-free plasmid extraction and purification kit, and placing the plasmid in a refrigerator at the temperature of-20 ℃ for later use after the concentration of the plasmid is determined.

(2) An endotoxin-free plasmid extraction and purification kit (purchased from Solambio company) is adopted to extract pLent-CII-CTLA4 plasmid, and the specific steps are as follows: (1) 10ul of E.coli Top10 bacterial solution containing the lentiviral plasmid pLent-CII-CTLA4 was put in 5ml of E.coli Top10 bacterial solution containing AMP⁺In the resistant LB medium, the cells were cultured overnight for 14-18h on a 250rpm shaker. (2) 2ml of the amplified bacterial solution was centrifuged at 12000rpm for 1min at room temperature. (3) The supernatant was removed, 200. mu.l of solution P1 (containing RNase A) was added, and the cells were shaken by a vortex shaker until they were completely suspended. (4) Adding 200 mul of solution P2, and gently inverting the centrifuge tube 6-8 times to fully crack the thalli. (5) Adding 200 μ l of solution P3, immediately and gently turning upside down for 6-8 times, mixing well until white flocculent precipitate appears, centrifuging at 12000rpm for 10min at room temperature, transferring the supernatant to another clean centrifugal tube by a pipette carefully, and not sucking out the precipitate as much as possible. (6) Adding an endotoxin scavenger precooled on ice with the volume of 1/5 supernatant, shaking and mixing uniformly, wherein the solution becomes turbid, and carrying out ice bath for 2min until the solution becomes clear. (7) The solution was cooled in an ice bath at 37 ℃ for 5min and became cloudy with occasional shaking. Centrifuging at 12000rpm for 5min, separating the solution into two phases, the upper water phase containing plasmid DNA and the lower oil phase containing endotoxin. (8) The upper aqueous phase containing plasmid DNA was transferred to a new tube, the lower oil phase was discarded, care was taken not to suck the oil phase, and steps (6) to (8) were repeated three times. (9) Adding 600 μ l of solution P4, mixing, adding into adsorption column, standing at room temperature for 2min, centrifuging at 12000rpm for 1min, removing waste liquid from the collection tube, and replacing the adsorption column in the collection tube. (10) Adding 600 μ l of rinsing solution (checking whether absolute ethanol is added before use) into adsorption column, centrifuging at 12000rpm for 1min, discarding waste liquid, and placing adsorption column back into collection tube; this step was repeated once. (11) Centrifuging at 12000rpm for 2min, placing the adsorption column in an open room or 50 deg.C incubatorFor several minutes, the residual rinse was removed. (12) The absorption column is put into a clean centrifuge tube, 50-200 μ l of eluent preheated by water bath at 65 ℃ is dripped into the center of the adsorption membrane, the mixture is kept stand for 2min at room temperature and centrifuged for 1min at 12000rpm, and plasmid DNA is collected. (13) The concentration of the extracted lentiviral plasmid pLent-CII-CTLA4 is 326 ng/ul and is finally obtained by measuring the concentration of the nanodrop nucleic acid, and the lentiviral plasmid is placed in a refrigerator at the temperature of 20 ℃ below zero for standby.

Example 3

The invention provides an embodiment of a Treg immune cell with an anti-CII chimeric antigen receptor encoding gene. The preparation method comprises the following steps: the pLent-CII-CTLA4 plasmid is subjected to lentivirus packaging firstly, and then Tregs immune cells are infected by the recombinant lentivirus.

(1) Lentivirus packaging and titer detection

A Lentiviral Packaging Kit is adopted, and the specific method comprises the following steps: the lentivirus packaging cell line 293T is inoculated in a 10cm culture dish containing DMEM and 10% FBS, cultured at 37 ℃ under the condition of 5% CO2, and prepared for transfection when the anchorage rate is 70% -80%. A sterile 1.5ml EP tube or 15ml centrifuge tube was used to prepare the reaction system as follows: serum-free DMEM: 4 ml; pLent-CII-CTLA4 plasmid: 10 mu g of the mixture; GM easy (TM) Lentiviral Mix: 10 μ l (10 μ g); HG Transgene Reagent: 60 μ l. After mixing, the mixture was left at room temperature for 20min, and then dropped into a dish containing 293T cells, and then cultured in a CO2 incubator. After transfection 24, the cell culture solution was carefully aspirated off and discarded in a waste liquid cup containing a disinfectant solution, and then 15ml of fresh culture medium containing 10% serum was added to continue the culture. After 48h of liquid change, the cell supernatant was aspirated into a 50ml centrifuge tube, centrifuged at 500g for 5min at 4 ℃, filtered through a 0.45 μm filter and transferred to a new centrifuge tube. The virus particles in the supernatant can then be directly assayed for titer. The titer of the 100. mu.l of the virus solution is determined by using a lentiviral vector (HIV P24) rapid detection card, and the titer of the recombinant lentivirus is 1.76X 10⁷TU/ml。

(2) Preparation of Tregs cells

Peripheral blood mononuclear cells were isolated from 60ml of peripheral blood using TBD sample density separation (from tertiary amines of tianjin) organisms. CD4+ CD25+ CD127 dim/-Regulatory T Cell Isola is usedt Kit II (purchased from Miltenyi) isolated Tregs cells. Taking the separated mononuclear cells every 10 th⁷Adding 10ul of Biotin-Antibody into each cell, mixing well, incubating at 2-8 deg.C for 5min, adding 20ul of Anti-Biotin Microbeads/10⁷Mixing the cells, incubating at 2-8 deg.C for 10min, adjusting the volume of the cells to 500 μ l, loading onto LD column, collecting cell suspension flowing down after column chromatography as CD4+ T cells (negative selection), washing with PBS, centrifuging, and adding PBS for 10 times⁷The cells were resuspended in 90. mu.l PBS, 10 times⁷Adding CD25MicroBeads 20ul into each cell, mixing uniformly, incubating for 15min at 2-8 ℃, adding 1ml PBS, washing and centrifuging, pouring out supernatant, suspending to 500 mu l, passing through MS column, removing separator, placing 1ml PBS on MS column, rapidly removing cells, collecting CD4+ CD25+ T cells (positive selection), adding Treg culture medium and continuing culturing. And (3) reserving a part of cells for flow cytometry detection, wherein the sorting rate of CD4+ CD25+ positive cells detected by a flow cytometer reaches over 86%.

(3) Lentiviral infected Tregs cell and expansion culture of infected Tregs cell

Tregs cells were infected with the recombinant lentivirus described above at MOI ═ 5. After the infected cells are cultured in a 5% CO2 culture box at 37 ℃ for 12 hours, the cells are collected, the supernatant is discarded, the same amount of virus solution, Polybrene (8 mu g/ml) and cell culture solution are added again, the cells are cultured in a 5% CO2 culture box at 37 ℃, the culture supernatant is discarded after 12 hours, fresh Treg culture medium is added again, the expansion culture is continued, and the cells are cultured for 17 days until the cells are expanded to a sufficient amount. Chimeric antigen receptor expression was detected by FC500 flow cytometer (purchased from BECKMAN corporation) FL1 channel (fig. 2). The positive rate of the recombinant lentivirus infected Tregs cells is 32.8 percent by taking uninfected Tregs cells as negative control.

Example 4

The control effectiveness of the molecular switch on the specific Treg cells containing pLent-CII-CTLA4 is analyzed.

Specific Treg cells containing pLent-CII-CTLA4 were cultured at a density of 1X 10⁵Inoculating 100ul of the strain/ml into a 96-well plate, and culturing in an incubator at 37 ℃ and 5% CO2 for 24 h; 10nM rituximab (product of Roche) was added, 20. mu.L of CCK-8 (product of MCE) per well after 12h, followed byAfter further incubation for 2h, the cells were detected by an enzyme-linked immunosorbent assay, and OD was read at a wavelength of 450 nm. CAR-Trges cell control group without rituximab and blank control group without rituximab only added.

The death rate of Trges cells was ═ 100% x [1- (plus rituximab group OD value-blank control group OD value)/(not plus rituximab group OD value-blank control group OD value) ]. The death rate of the Trges cells is 59.27%, and the control rate of rituximab on the activity of the CAR-Trges cells is 98.13% of the death rate/positive rate of recombinant lentivirus infected CIK; the result shows that the activity of the specificity Treg cells containing pLent-CII-CTLA4 designed by the invention is controlled by rituximab, the clinical use safety of CAR-Tregs cells is ensured, and adverse reactions such as cytokine storm can be effectively stopped.

Example 5

Therapeutic effects of Treg immunocytes having an anti-CII chimeric antigen receptor-encoding gene on RA mouse models. The method specifically comprises the following steps: analyzing the pathological degree of each joint of the mouse model, and changing the content of pLent-CII-CTLA4 specific Treg cells at the joints and factors such as peripheral blood TNF-alpha, IFN-gamma and the like.

(1) The degree of each joint lesion of the mouse model is analyzed by the specific Treg cells containing pLent-CII-CTLA 4.

About 20g of 7-8 weeks old mice (purchased from Guangzhou university of traditional Chinese medicine) were bred in animal houses (room temperature 23 + -2 ℃, humidity 50% + -10%). Under aseptic conditions, 100. mu.l of collagen emulsion fully emulsified by intradermal injection of type II Collagen (CII) and incomplete fortune-respect adjuvant was taken from 3 spots on days 1 and 21 of the tail root. The arthritis performance such as red and swollen joints of the mice and the arthritis score are gradually increased and are obviously used as the standard for successful modeling.

The mice were randomly divided into 4 groups of 10 mice each and the injection treatment experiment was started. The experimental groups were respectively:

a. in the control group, the tail part of the patient is injected with normal saline with the same volume, and the patient is continuously observed for 14 d;

b. one treatment group, tail vein injection 2X 10⁶Carrying out the injection of the same dosage for the second time 2 days after the first injection of each cell/pLent empty vector Tregs cell, and continuously observing for 14 d;

c. two groups of treatment, tail intravenous injection 2X 10⁶Carrying out secondary injection with the same dosage 2 days after the primary injection of each pLent-CII-CTLA4 specific Tregs cell/pLent-CII-CTLA 4 specific Tregs cell, and continuously observing for 14 d;

d. three groups of treatment, tail intravenous injection 2X 10⁶Carrying out the second injection with the same dosage 2 days after the first injection of each pLent-CII specific Tregs cell/pLent-CII specific Tregs cell, and continuously observing for 14 d;

joint and extraarticular lesions were observed daily and scored for arthritis and recorded, and a comparative plot of arthritic lesions was made, as shown in figure 3. Wherein, the pathological change degree of each joint is classified according to 5 grades, 0: no red swelling; 1, red and swollen is limited to one toe; 2, more than one toe is red and swollen; red and swollen involving all toes and instep; 4: the whole paw and ankle joint are severely inflamed.

The treatment effect is summarized as follows: pLent-CII-CTLA4 specific Tregs cells > pLent-CII specific Tregs cells > general Tregs cells.

(2) Peripheral blood TNF-alpha and IFN-gamma factor change analysis.

Venous blood was collected from 15 gastrodiad-drunken mice after immunotherapy and TNF-. alpha.levels in serum were measured using a Human TNF-alpha Quantikine ELISA Kit (purchased from R & D Systems). Serum TNF-alpha levels of (52.66 +/-9.32), (41.95 +/-9.10), (28.35 +/-6.94) and (32.43 +/-7.82) ng/ml in the control group and the first to the third groups of experiments respectively, and P is less than 0.01. IFN-. gamma.levels in serum were determined using a Human IFN-gamma Quantikine ELISA Kit (available from R & D Systems). The specific operation steps are shown in the specification. The serum IFN-gamma levels of the control group and the treatment group to the three groups are respectively (134.53 + -19.26), (101.91 + -16.42), (68.58 + -7.98), (79.453 + -7.31) ng/ml, and P is less than 0.01. The result shows that the functional region element of CTLA4 is added into the CAR structure of the Tregs, so that the Tregs inhibit effector cells to generate IFN-gamma and TNF alpha, and the immunosuppressive activity of the CAR-Tregs cells is further enhanced.

(3) Content of pLent-CII-CTLA4 specific Tregs cells at joints

Collecting mouse joint cavity hydrops for 5ml after 15 days of immunotherapy by conventional joint puncture, diluting with appropriate amount of PBS, standing for 5min, removing bottom residue, and mixing suspension 20Centrifuging at 00rpm for 5min, discarding the supernatant and resuspending with 1ml PBS; putting the Ficoll separating medium into a centrifuge tube, and adding the synovial fluid precipitate suspension into the centrifuge tube along the tube wall at a position of 1cm from the separating medium by using a suction tube; centrifuging at 2000rpm for 20min, sucking the middle white lymphocyte layer with a pipette, and transferring to a new centrifuge tube; adding PBS with 2 times volume, mixing uniformly, centrifuging at 2000rpm for 5min, discarding supernatant, and washing repeatedly; resuspend monocytes with PBS to a cell concentration of approximately 1X 10⁶Per ml; the CD4+ CD25+ Foxp3+ Tregs cell content was measured by FC500 flow cytometry (purchased from BECKMAN). The percentage of synovial fluid Tregs cells in CD4+ cells in the treated group (14.86%) was significantly higher than the percentage of synovial fluid Tregs cells in CD4+ cells in the treated group (10.93%). The result shows that scFv (CII) contained in the pLent-CII-CTLA4 specific Tregs cell can be combined with CII at the arthritis lesion, so that the Tregs cell is gathered at the arthritis lesion, the immunosuppressive function of the Tregs cell is better exerted, and the effect of treating rheumatoid arthritis is further achieved.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the present invention, and they should be construed as being included in the following claims and description.

Sequence listing

<110> Shandong Xingyi Biotechnology Ltd

<120> anti-CII chimeric antigen receptor coding gene, lentiviral plasmid, Treg immune cell and application thereof

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ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt 2220

ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca 2280

ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa 2340

gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg 2400

cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg 2460

gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac 2520

taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag 2580

cagagatcca gtttggttag taccgggccc gctctagaca tgtccaatat gaccgccatg 2640

ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag 2700

cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc 2760

caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg 2820

gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca 2880

tcaagtgtat catatgccaa gtccgccccc tattgacgtc aatgacggta aatggcccgc 2940

ctggcattat gcccagtaca tgaccttacg ggactttcct acttggcagt acatctacgt 3000

attagtcatc gctattacca tggtgatgcg gttttggcag tacaccaatg ggcgtggata 3060

gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt 3120

ttggcaccaa aatcaacggg actttccaaa atgtcgtaat aaccccgccc cgttgacgca 3180

aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt tagtgaaccg 3240

tcagaatttt gtaatacgac tcactatagg gcggccggga ttcgtcgact ggatccggta 3300

ccgaggagat ctgccgccgc gatcgcassn ymatggccct gcctgtgaca gccctgctgc 3360

tgcctctggc tctgctgctg catgccgcta gacccgaggt gcagctgttg gagtctgggg 3420

gaggcttggt acagcctggg gggtccctga gactctcctg tgcagcctct ggattcacct 3480

ttagcagcta tgccatgagc tgggtccgcc aggctccagg gaaggggctg gagtgggtct 3540

cagctattag tggtagtggt ggtagcacat actacgcaga ctccgtgaag ggccggttca 3600

ccatctccag agacaattcc aagaacacgc tgtatctgca aatgaacagc ctgagagccg 3660

aggacacggc cgtatattac tgtgcgaaag caggctactc tctctttgac tactggggcc 3720

agggaaccct ggtcaccgtc tcctcaggag gaggaggaag tggaggagga ggatctggag 3780

gaggaggatc tgsgaaattg tgctgactca gtctccaggc accctgtctt tgtctccagg 3840

ggaaagagcc accctctcct gcagggccag tcagagtgtt agcagcagct acttagcctg 3900

gtaccagcag aaacctggcc aggctcccag gctcctcatc tatggtgctt ccagcagggc 3960

cactggcatc ccagacaggt tcagtggcag tgggtctggg acagacttca ctctcaccat 4020

cagcagactg gagcctgaag attttgcagt gtattactgt cagcaggatc aaggcatgcc 4080

tctgacgttc ggccaaggga ccaaggtgga aatcaaaacc acgacgccag cgccgcgacc 4140

accaacaccg gcgcccacca tcgcgtcgca gcccctgtcc ctgcgcccag aggcgtgccg 4200

gccagcggcg gggggcgcag tgcacacgag ggggctggac ttcgcctgtg atcdhngatc 4260

tacatctggg cgcccttggc cgggacttgt ggggtccttc tcctgtcact ggttatcacc 4320

ctttactgcc dtransmaaa cggggcagaa agaaactcct gtatatattc aaacaaccat 4380

ttatgagacc agtacaaact actcaagagg aagatggctg tagctgccga tttccagaag 4440

aagaagaagg aggatgtgaa ctgbbcstma gagtgaagtt cagcaggagc gcagacgccc 4500

ccgcgtacaa gcagggccag aaccagctct ataacgagct caatctagga cgaagagagg 4560

agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga aagccgagaa 4620

ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg gcggaggcct 4680

acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat ggcctttacc 4740

agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag gccctgcccc 4800

ctcgccdtag aaggccgagg gagcctgctg acatgtggcg atgtggagga aaacccagga 4860

ccataatggc ttgccttgga tttcagcggc acaaggctca gctgaacctg gctaccagga 4920

cctggccctg cactctcctg ttttttcttc tcttcatccc tgtcttctgc aaagcaatgc 4980

acgtggccca gcctgctgtg gtactggcca gcagccgagg catcgccagc tttgtgtgtg 5040

agtatgcatc tccaggcaaa gccactgagg tccgggtgac agtgcttcgg caggctgaca 5100

gccaggtgac tgaagtctgt gcggcaacct acatgatggg gaatgagttg accttcctag 5160

atgattccat ctgcacgggc acctccagtg gaaatcaagt gaacctcact atccaaggac 5220

tgagggccat ggacacggga ctctacatct gcaaggtgga gctcatgtac ccaccgccat 5280

actacctggg cataggcaac ggaacccaga tttatgtaat tgatccagaa ccgtgcccag 5340

attctgactt cctcctctgg atccttgcag cagttagttc ggggttgttt ttttatagct 5400

ttctcctcac agctgtttct ttgagcaaaa tgctaaagaa aagaagccct cttacaacag 5460

gggtctatgt gaaaatgccc ccaacagagc cagaatgtga aaagcaattt cagccttatt 5520

ttattcccat caatgaaggc cgagggagcc tgctgacatg tggcgatgtg gaggaaaacc 5580

caggaccata tgtccttact ctaacccctc tctctgttct ggaggtggag gatctgagtt 5640

acctacccag ggaacatttt caaatgtttc tacaaatgta tcccctgcta agcctacaac 5700

aactgcatgt ccttactcta acccctctct ctgttctgga ggtggaggat ctcctgctcc 5760

tcgtcctcct acccctgctc ctactatcgc gtcgcagccc ctgtccctgc gcccagaggc 5820

gtgccggcca gcggcgggtg gcgcagtgca cacgaggggt ctggacttcg cctgtgatat 5880

ctacatctgg gcgcccttgg ccgggacttg tggggtcctt ctcctgtcac tggttatcac 5940

cctttactgc aaccacagaa ataggagaag agtttgcaag tgtcctagac ctgttgttta 6000

gacgcgtmny mgcggccgcn tnymggccgc tcgagatggt gagcaagggc gaggagctgt 6060

tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca 6120

gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct 6180

gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg 6240

tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca 6300

tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga 6360

cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca 6420

tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc 6480

acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc 6540

gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca 6600

tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga 6660

gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg 6720

ggatcactct cggcatggac gagctgtaca agaataaggt ttatccgatc caccggatct 6780

agataagata aacggccggc cgcggtctgt acaagtagga ttcgtcgagg gacctaataa 6840

cttcgtatag catacattat acgaagttat acatgtttaa gggttccggt tccactaggt 6900

acaattcgat atcaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt 6960

gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc 7020

tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg 7080

gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac 7140

tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc 7200

cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc 7260

ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa 7320

atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc 7380

cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc 7440

ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg 7500

ggccgcctcc ccgcatcgat accgtcgacc tcgatcgaga cctagaaaaa catggagcaa 7560

tcacaagtag caatacagca gctaccaatg ctgattgtgc ctggctagaa gcacaagagg 7620

aggaggaggt gggttttcca gtcacacctc aggtaccttt aagaccaatg acttacaagg 7680

cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact 7740

cccaacgaag acaagatatc cttgatctgt ggatctacca cacacaaggc tacttccctg 7800

attggcagaa ctacacacca gggccaggga tcagatatcc actgaccttt ggatggtgct 7860

acaagctagt accagttgag caagagaagg tagaagaagc caatgaagga gagaacaccc 7920

gcttgttaca ccctgtgagc ctgcatggga tggatgaccc ggagagagaa gtattagagt 7980

ggaggtttga cagccgccta gcatttcatc acatggcccg agagctgcat ccggactgta 8040

ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc 8100

cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt 8160

tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta 8220

gcagcatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 8280

cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 8340

ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 8400

tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 8460

gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 8520

gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 8580

gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 8640

ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 8700

ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag 8760

ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 8820

gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 8880

ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 8940

tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 9000

ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca 9060

gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg 9120

tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac 9180

cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg 9240

ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc 9300

gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta 9360

caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac 9420

gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc 9480

ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac 9540

tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact 9600

caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa 9660

tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt 9720

cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca 9780

ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa 9840

aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac 9900

tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg 9960

gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggtcccgcgc acatttcccc 10020

gaaaagtgcc acctgac 10037

<210> 2

<211> 57

<212> DNA

<213> Homo sapiens

<400> 2

atggagtttg ggctgagctg ggttttcctc gttgctcttt ttagaggtgt ccagtgt 57

<210> 3

<211> 720

<212> DNA

<213> Homo sapiens

<400> 3

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gaaagcaggc 300

tactctctct ttgactactg gggccaggga accctggtca ccgtctcctc aggaggagga 360

ggaagtggag gaggaggatc tggaggagga ggatctgaaa ttgtgctgac tcagtctcca 420

ggcaccctgt ctttgtctcc aggggaaaga gccaccctct cctgcagggc cagtcagagt 480

gttagcagca gctacttagc ctggtaccag cagaaacctg gccaggctcc caggctcctc 540

atctatggtg cttccagcag ggccactggc atcccagaca ggttcagtgg cagtgggtct 600

gggacagact tcactctcac catcagcaga ctggagcctg aagattttgc agtgtattac 660

tgtcagcagg atcaaggcat gcctctgacg ttcggccaag ggaccaaggt ggaaatcaaa 720

<210> 4

<211> 135

<212> DNA

<213> Homo sapiens

<400> 4

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210> 5

<211> 72

<212> DNA

<213> Homo sapiens

<400> 5

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gc 72

<210> 6

<211> 126

<212> DNA

<213> Homo sapiens

<400> 6

aagagaggcc ggaagaagct gctgtacatc ttcaagcagc ccttcatgcg gcccgtgcag 60

accacccagg aagaggacgg ctgcagctgt cggttccccg aggaagaaga aggcggctgc 120

gaactg 126

<210> 7

<211> 336

<212> DNA

<213> Homo sapiens

<400> 7

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 8

<211> 54

<212> DNA

<213> Homo sapiens

<400> 8

gaaggccgag ggagcctgct gacatgtggc gatgtggagg aaaacccagg acca 54

<210> 9

<211> 669

<212> DNA

<213> Homo sapiens

<400> 9

atggcttgcc ttggatttca gcggcacaag gctcagctga acctggctac caggacctgg 60

ccctgcactc tcctgttttt tcttctcttc atccctgtct tctgcaaagc aatgcacgtg 120

gcccagcctg ctgtggtact ggccagcagc cgaggcatcg ccagctttgt gtgtgagtat 180

gcatctccag gcaaagccac tgaggtccgg gtgacagtgc ttcggcaggc tgacagccag 240

gtgactgaag tctgtgcggc aacctacatg atggggaatg agttgacctt cctagatgat 300

tccatctgca cgggcacctc cagtggaaat caagtgaacc tcactatcca aggactgagg 360

gccatggaca cgggactcta catctgcaag gtggagctca tgtacccacc gccatactac 420

ctgggcatag gcaacggaac ccagatttat gtaattgatc cagaaccgtg cccagattct 480

gacttcctcc tctggatcct tgcagcagtt agttcggggt tgttttttta tagctttctc 540

ctcacagctg tttctttgag caaaatgcta aagaaaagaa gccctcttac aacaggggtc 600

tatgtgaaaa tgcccccaac agagccagaa tgtgaaaagc aatttcagcc ttattttatt 660

cccatcaat 669

<210> 10

<211> 408

<212> DNA

<213> Homo sapiens

<400> 10

tgtccttact ctaacccctc tctctgttct ggaggtggag gatctgagtt acctacccag 60

ggaacatttt caaatgtttc tacaaatgta tcccctgcta agcctacaac aactgcatgt 120

ccttactcta acccctctct ctgttctgga ggtggaggat ctcctgctcc tcgtcctcct 180

acccctgctc ctactatcgc gtcgcagccc ctgtccctgc gcccagaggc gtgccggcca 240

gcggcgggtg gcgcagtgca cacgaggggt ctggacttcg cctgtgatat ctacatctgg 300

gcgcccttgg ccgggacttg tggggtcctt ctcctgtcac tggttatcac cctttactgc 360

aaccacagaa ataggagaag agtttgcaag tgtcctagac ctgttgtt 408

Claims

1. An anti-CII chimeric antigen receptor coding gene, which is characterized in that: the anti-CII chimeric antigen receptor coding gene is obtained by sequentially connecting the following sequences in series:

a Leader nucleic acid artificial sequence shown as SEQ ID NO.2,

the CII binding region nucleic acid artificial sequence shown in SEQ ID NO.3,

the CD8hinge region nucleic acid artificial sequence shown in SEQ ID NO.4,

the CD8 transmembrane region nucleic acid artificial sequence shown in SEQ ID NO.5,

2B4 costimulatory region nucleic acid artificial sequence shown in SEQ ID NO.6,

the intracellular domain nucleic acid artificial sequence of CD3 ζ as shown in SEQ ID NO.7,

the T2A nucleic acid artificial sequence shown in SEQ ID NO.8,

CTLA4 nucleic acid artificial sequence shown in SEQ ID NO.9,

the T2A nucleic acid artificial sequence shown in SEQ ID NO.8,

RQR8 nucleic acid artificial sequence shown in SEQ ID NO. 10.

2. A lentiviral plasmid characterized by: comprising the anti-CII chimeric antigen receptor-encoding gene of claim 1.

A Treg immune cell characterized by: comprising the anti-CII chimeric antigen receptor-encoding gene of claim 1.

4. Use of the Treg immune cells of claim 3 in the manufacture of a medicament for the treatment of rheumatoid arthritis.