CN111533810A

CN111533810A - Double-chimeric antigen receptor T cell containing bifunctional immune switch molecule and application thereof

Info

Publication number: CN111533810A
Application number: CN202010386100.8A
Authority: CN
Inventors: 徐寒梅; 杨培伟
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2020-05-09
Filing date: 2020-05-09
Publication date: 2020-08-14

Abstract

The invention discloses a double-chimeric antigen receptor T cell containing a bifunctional immune switch molecule and application thereof, belonging to the field of tumor immunotherapy. The bifunctional immune molecule comprises two parts, and is composed of polypeptide with PD-L1 blocking function and FITC molecule for regulating and controlling double chimeric antigen receptor T cells. In vitro and in vivo experiments prove that the constructed bifunctional switch molecule can accurately regulate and control the activation and target cytotoxicity of the dual chimeric antigen receptor T cells, and simultaneously can prolong the proliferation capacity of the dual chimeric antigen receptor T cells and enhance the anti-tumor activity of the dual chimeric antigen receptor T cells. This combined modulation approach can significantly improve the safety of CAR-T cell immunotherapy and tumor clearance in solid tumors.

Description

Double-chimeric antigen receptor T cell containing bifunctional immune switch molecule and application thereof

Technical Field

The invention belongs to the field of tumor immunotherapy, and particularly relates to a construction method and application of a bifunctional immune switch molecule for regulating and controlling the activity of a double-chimeric antigen receptor T cell.

Background:

immunotherapy based on Chimeric Antigen Receptor (CAR) modified T cells has proven to be a promising therapeutic approach for cancer, especially in B cell malignancies with unprecedented success. CARs confer T cells the ability to recognize and eliminate tumors through surface-displayed single-chain variable regions with intracellular co-stimulatory and activating domains. Although CAR-T cell therapy exhibits significant efficacy in B cell acute lymphocytic leukemia patients, its efficacy in treating solid tumors is not as satisfactory.

The applications of CAR-T cells are primarily limited by the solid tumor microenvironment as well as off-target effects. Cells and related ligands that exert immunosuppressive activity in the solid tumor microenvironment can cause infiltrating T cells to lose antitumor activity, thereby causing immune escape of the tumor. In breast cancer, for example, the tumor microenvironment contains a number of tumor-associated macrophages and regulatory T cells that impair the anti-tumor activity of T cells by contacting the T cells or by secreting suppressive cytokines, rendering the patient ineffective in its anti-tumor immune response. In addition, activated T cells secrete large amounts of the effector cytokine IFN γ, resulting in elevated expression of PD-L1 in breast tumors. And the binding of PD-L1 and a T cell surface inhibitory receptor PD-1 can inhibit the anti-tumor activity of the T cell. Currently, checkpoint blockade therapies, particularly against PD-1/PD-L1, which block the T cell inhibitory signaling pathway through antibodies, have been shown to produce clinical responses in a range of tumor treatments. Whereas autocrine PD-1/PD-L1 antibodies by CAR-T cells, and PD-1-knockout CAR-T cells mediated by CRISPR, have shown positive results in preclinical or clinical studies by combining CAR-T cell therapy with PD-1/PD-L1 antibodies.

For off-target effects, CAR-T cells are clinically severely toxic when used in conjunction with solid tumor therapy, since solid tumor-associated antigens are often expressed in a range of normal tissues as well. For example, a breast cancer patient produces lethal inflammatory cytokine release after reinfusion of HER-2 targeted CAR-T cells, because HER-2 is also expressed on lung epidermal cells. There are currently a number of approaches to control CAR-T cell activity, including the introduction of suicide genes, the use of dual antigen receptors, and the control of CAR-T cell activity by exogenous switching molecules. Furthermore, our previous studies introduced a modulatory dual receptor CAR system, which is activeDependence on dual antigens and strict control by switching molecules^[1]. Specifically, T cells are co-regulated by a dual chimeric antigen receptor, a first chimeric antigen receptor (CD3 ζ intracellular signal domain) controlled by a functional switch molecule, and a second chimeric antigen receptor (4-1BB intracellular signal domain) controlled by the tumor associated antigen MSLN. The switch molecule is used as a bridge between effective target cells and controls the activation of the dual-receptor CAR-T cell and the clearance of the target cells.

Disclosure of Invention

Problems to be solved

Aiming at off-target toxicity and immunosuppression in the current CAR-T cell treatment process, the invention provides a bifunctional immune switch molecule which has the function of regulating and controlling the activity of double-receptor CAR-T cells on one hand, and the CAR-T cells can be activated only by simultaneously recognizing double antigens of MSLN and PD-L1 and the bifunctional switch molecule, so that the risk of off-target toxicity is reduced. On the other hand, the bifunctional switch molecule has PD-L1 blocking activity, so that the inhibition state of the CAR-T cell is relieved, and the antitumor activity is better exerted. In a word, the designed bifunctional immune switch molecule can simultaneously solve the off-target effect and the immune suppression state of the dual-receptor CAR-T cell, and the application of the CAR-T cell in solid tumors is expanded.

Technical scheme

In order to solve the problems, the technical scheme adopted by the invention is as follows:

a bifunctional immune switch molecule comprising a regulatory region of a bipartite chimeric antigen receptor and an immune checkpoint inhibitory region.

The bifunctional immune switch molecule is characterized in that: the immune checkpoint inhibitory region is a PD-L1 blocking polypeptide, and the sequence of the polypeptide is as follows: SEQ ID NO. 1.

The bifunctional immune switch molecule is characterized in that: the double chimeric antigen receptor regulating region is coupled FITC molecule.

The bifunctional immune switch molecule is characterized in that: the sequence is FITC-Acp-SNGLSQPV;

a bi-chimeric antigen receptor T cell comprising a bifunctional immune switch molecule, characterized in that: it contains the bifunctional immune switch molecule, a first chimeric antigen receptor and a second chimeric antigen receptor; wherein the first chimeric antigen receptor is composed of a CD8 alpha signal peptide, FITC scFv (SEQ ID NO.6), a CD8 alpha hinge region, a transmembrane region and a CD3 zeta intracellular signal domain which are connected in series, and the amino acid sequence of the first chimeric antigen receptor is as follows: SEQ ID No. 2; the second chimeric antigen receptor is composed of a CD8 alpha signal peptide, MSLNscFv (SEQ ID NO.5), a CD8 alpha hinge region and a transmembrane region, and a 4-1BB intracellular signal region in series, and the amino acid sequence of the second chimeric antigen receptor is as follows: SEQ ID No. 3;

the application of the double-chimeric antigen receptor T cell containing the bifunctional immune switch molecule in preparing a medicament for treating tumors.

The application is characterized in that: the tumor comprises ovarian cancer, lung cancer, esophageal cancer, pancreatic cancer, gastric cancer, colon cancer, breast cancer, liver cancer, melanoma, head and neck cancer, cervical cancer and osteosarcoma.

Further, a method of preparing a T cell capable of expressing a chimeric antigen receptor by transfection, characterized in that: connecting the double chimeric antigen receptor gene to an over-expression vector, and transferring a target gene into primary T lymphocytes by packaging lentiviruses to ensure that the T cells express the double chimeric antigen receptor;

furthermore, the application of the double-chimeric antigen receptor T cell containing the bifunctional immune switch molecule in the field of preparing tumor medicaments for over-expressing tumor antigens MSLN and PD-L1 is provided.

The application is characterized in that the double-chimeric antigen receptor T cell containing the bifunctional immune switch molecule can treat cancers by various administration modes, including subcutaneous or intravenous injection, intramuscular injection and intratumoral injection.

A tumor cell therapeutic drug combination is characterized by comprising a bifunctional switch molecule or a double-chimeric antigen receptor T cell and pharmaceutically acceptable auxiliary materials.

The action mechanism is as follows:

as shown schematically in fig. 3, a T cell (sdCAR-T cell) comprising a regulatable dual chimeric antigen receptor (i.e., sdCAR) according to the invention comprises a dual chimeric antigen receptor and a bifunctional immune switch molecule (i.e., DT301), the sdCAR comprising a first and a second chimeric antigen receptor; the first chimeric antigen receptor mainly comprises an extracellular FITC scFv recognition region, an intracellular CD3 zeta signal domain and the like, and mainly recognizes the FITC part in DT 301; the second chimeric antigen receptor mainly comprises an extracellular MSLNscFv recognition region and an intracellular 4-1BB signal domain, and mainly recognizes MSLN on the surface of a tumor cell. Therefore, the sdCAR-T cells can be fully activated only after simultaneously recognizing FITC (FITC), namely MSLN (MSLN double antigen), of DT301, and the probability of off-target effect is effectively reduced. In addition, DT301 is composed of two parts, PD-L1 blocking polypeptide and coupled FITC molecule are respectively a molecule which plays a role of a switch, namely CAR-T cell initiation is realized by adding DT301, specifically, on one hand, after DT301 is added exogenously, the DT301 blocks polypeptide from being combined with PD-L1 on the surface of tumor cells through PD-L1, so that the coupled FITC molecule is anchored on the surface of the tumor cells, and further the activation of sdCAR-T cells can be regulated and controlled through FITC molecule and MSLN molecule expressed by the tumor cells, therefore, the sdCAR-T cells can be fully activated only after DT301 is added, and the controllability of the sdCAR-T cells is effectively increased. In a specific anti-tumor process, only when the tumor cells express MSLN and PD-L1 at the same time, DT301 is combined with PD-L1 after DT301 is added, then the sdCAR-T cells are activated by identifying MSLN and FITC antigens, thereby exerting anti-tumor effect, but DT301 is not added, or the tumor cells do not express MSLN and PD-L1 antigens at the same time, the sdCAR-T cells cannot be activated, and the results are shown in FIGS. 5-8, therefore, the safety is obviously improved. However, after the sdCAR-T cells were stimulated in vitro for a long period by tumor cells expressing both MSLN and PD-L1 and DT301 was added, the proliferative activity was significantly stronger than MSLN CAR-T cells, indicating that sdCAR-T cells showed better proliferative activity in combination with DT301 during long-term antitumor procedures, as shown in fig. 9. In vivo antitumor experiments also show that the sdCAR-T cells have stronger tumor cell clearing capacity and better in vivo proliferation activity by combining with DT301, and show better antitumor activity, while the sdCAR-T cells cannot play a role in clearing tumors by themselves for the group without adding DT301, as shown in FIGS. 10-12, the results show that the antitumor activity of the sdCAR-T cells in vivo depends on the existence of DT301, and when the sdCAR-T cells are combined with DT301, the antitumor activity of the sdCAR-T cells is better than that of MSCAR-T cells. In conclusion, by artificially adding DT301, multiple effects of reducing off-target toxicity, enhancing controllability of the sdCAR-T cells and relieving immunosuppression of the sdCAR-T cells can be simultaneously realized.

Advantageous effects

Compared with the prior art, the invention has the beneficial effects that:

1. the invention designs a novel bifunctional immune switch molecule DT301, the sequence of which is FITC-Acp-SNGLSQPV, the molecule is a molecule with relatively small molecular weight, and the molecule can play a role in starting and regulating through DT301, thereby reducing off-target toxicity, increasing controllability, removing multiple functions such as immunosuppression and the like, and the invention has the advantages of convenience, rapidness and high clinical value. Particularly, the PD-L1 blocking polypeptide is coupled with the FITC molecule, so that the activity of the double-receptor CAR-T cell reported previously can be accurately regulated^[1]. DT301 regulates the activation of sdCAR-T cells in conjunction with the tumor associated antigen MSLN after binding to PD-L1 on the surface of tumor cells. Compared with the previous report, by using high-expression PD-L1 and MSLN in the solid tumor, through DT301 as a bridge, the sdCAR-T cells are better limited at the solid tumor part, and the off-target effect is further reduced;

2. after the DT301 is added exogenously, the binding with PD-L1 on the surface of the tumor cell can not only control the activation of the sdCAR-T cell, but also block a PD-1/PD-L1 signal channel, thereby reducing the immunosuppressive activity of the sdCAR-T cell;

3. according to the application, due to the exogenous addition of DT301, the activation and cytotoxicity of the sdCAR-T cells are limited to the solid tumor part, and the immunosuppression state of the sdCAR-T cells can be relieved, so that the application range of the double-receptor CAR-T cells can be further expanded.

Description of the drawings:

FIG. 1 shows the flow assay results of DT301 binding to target cells (PANC-1 and MDA-MB-321);

FIG. 2 shows the expression of MSLN and PD-L1 on the surface of target cells (PANC-1 and MDA-MB-321);

FIG. 3 is a schematic representation of sdCAR-T cells in combination with DT301 for anti-tumor;

FIG. 4 is a blank control, MSLN CAR-T cell and sdCAR-T cell transfection positive results;

FIG. 5 shows the level of IL-2 secretion following DT 301-mediated sdCAR-T cell activation;

FIG. 6 shows the level of IFN- γ secretion following DT 301-mediated activation of sdCAR-T cells;

FIG. 7 is the DT 301-mediated expression level of CD69 following sdCAR-T cell activation;

FIG. 8 shows the result of DT 301-mediated long-term proliferation ability of sdCAR-T cells;

FIG. 9 shows the results of DT 301-mediated monitoring of the in vivo tumoricidal ability of sdCAR-T cells;

FIG. 10 is the tumor mass results after DT 301-mediated killing of sdCAR-T cells;

FIG. 11 shows the result of DT 301-mediated in vivo proliferation of sdCAR-T cells;

FIG. 12 is a graph showing the results of expression levels of multiple solid tumor heavy PD-L1;

FIG. 13 is a graph showing the results of expression levels of multiple solid tumor heavy MSLNs;

FIG. 14 is a schematic diagram of recombinant plasmid pLVX-CAR;

FIG. 15 is a plasmid map of MSLN overexpression vector.

Detailed Description

The terms: DT301, a bifunctional immune switch molecule; sdcAr-T cells, i.e., T cells that regulate dual chimeric antigen receptors; MSLN, mesothelin; MSLN CAR-T cells, and classical, secondary MSLN-targeted CAR-T cells; EGFP, enhanced green fluorescent protein; PANC-1 cells are human pancreatic cancer cell lines;

example 1 Synthesis of DT301 and target cell binding assay

The invention relates to a bifunctional immune switch molecule DT301, which has a sequence FITC-Acp-SNGLSQPV, wherein Acp represents β -aminocaproic acid, the SNGLSQPV sequence is from a PD-L1 inhibitory polypeptide (CN110669102A) disclosed previously, DT301 is Shanghai Jier polypeptide Co., Ltd, and the purity is more than 95%Two different target cells, MDA-MB-231 (PD-L1)⁺) And PANC-1 (PD-L1)^-) All purchased from ATCC. The specific scheme is as follows:

250nm,750nm and 1.5 mu M DT301 are respectively incubated with the target cells for 30min in a dark place, washed for 3 times by PBS, and then the binding capacity of DT301 on the target cells is monitored by an FITC channel of a flow cytometer. The flow assay results are detailed in FIG. 1, the binding rate of DT301 in MDA-MB-231 cells is 250 nm-76.4%, 750 nm-91.2% and 1.5. mu.M-96.8%, while DT301 is not bound at any concentration in PANC-1 cells.

The results show that:

MDA-MB-231 cells are human breast cancer cells, flow detection results show that only PD-L1 is expressed and MSLN is not expressed, PANC-1 cells are human pancreatic cancer cells, and flow results show that neither PD-L1 nor MSLN is expressed. DT301 was combined with MDA-MB-231 cells (PD-L1)⁺MSLN^-) Or PANC-1 cells (PD-L1)^-MSLN^-) The co-incubation results showed that DT301 only binds to MDA-MB-231 cells and not to PANC-1 cells, indicating its binding specificity for PD-L1.

Example 2 construction of sdcAr overexpression plasmid

1. Synthesis of Gene fragments

The sdcAR sequence is provided with a CD8 alpha signal peptide, a FITC scFv, a CD8 alpha hinge region, a CD8 alpha transmembrane region, a CD3 zeta intracellular signal region, an IRES, a CD8 alpha signal peptide, a MSLNscFv, a CD8 alpha hinge region, a CD8 alpha transmembrane region, a 4-1BB intracellular signal region, a P2A and an EGFP in sequence from 5 'to 3', the optimized complete sdcAR gene sequence is subjected to whole-gene synthesis, the nucleotide sequence is SEQ ID NO.4, the optimized complete sdcAR gene sequence is cloned into pLVX-Puro through specific enzyme cutting sites Xho I and Xba I, and finally a recombinant plasmid pLVX-Puro is constructed (see figure 14), the introduced EGFP fluorescent tag is used for detecting and tracking the constructed CAR-T cells, and the schematic diagram of the sdcAR-T cells is shown in figure 3.

2. Construction and sequencing of recombinant plasmids

The recombinant plasmid pLVX-CAR is sent to a company for sequencing, and the sequencing result is compared with the sequence of the sdCAR gene to be synthesized, so that the result proves that the obtained synthetic sequence is correct.

Example 3 target cells (MSLN)⁺PD-L1⁺，MSLN⁺PD-L1^-，MSLN^-PD-L1⁺，MSLN^-PD-L1^-) Construction of

Construction of MSLN overexpression vectors

Since the invention requires verification of the antigen dependence of sdCAR-T cells on PD-L1 and MSLN, four target cells, MSLN respectively, are required⁺PD-L1⁺，MSLN⁺PD-L1^-，MSLN^-PD-L1⁺，MSLN^-PD-L1^-. To obtain four target cells, MSLN was obtained by first screening^-PD-L1⁺And MSLN^-PD-L1^-And then by artificially over-expressing MSLN, four target cells satisfying the conditions can be obtained. Reference is made to the CDS region sequence of human mesothelin MSLN in the Ensembl database (ENSG 00000102854). The MSLN gene fragment was inserted into the lentiviral over-expression vector pLVX through the restriction sites Xho I and Xba I to form a pLVX-MSLN recombinant expression plasmid, as shown in FIG. 15. The recombinant plasmid is sent to a company for sequencing, and the sequencing result is compared with the pLVX-MSLN gene sequence to be synthesized, so that the result proves that the obtained synthetic sequence is correct.

2. Preparation of Lentiviral particles

1) A15 cm dish was prepared, complete medium (DMEM high-glucose) containing 10% Fetal Bovine Serum (FBS) was added, and 5 × 10 was inoculated⁶293T cells, incubated at 37 ℃ with 5% CO₂And (5) an incubator for overnight culture.

2) The constructed over-expression vector pLVX-MSLN (concentration about 100. mu.M) and lentiviral packaging plasmids (Lenti-GOI, pGP, pVSVG) (purchased from Addgene) were removed from the refrigerator, thawed at room temperature, and blown up and down using a pipette gun to mix well.

3) Phosphate Buffered Saline (PBS) was removed and warmed to room temperature. Adding 2mL of PBS into one hole of a 6-hole plate, respectively adding 10 mu g of Lenti-GOI, 6 mu g of pGP and 5 mu g of pVSVG, blowing and beating up and down by a liquid transfer gun, fully and uniformly mixing, adding 18 mu L of pLVX-MSLN, immediately blowing and beating up and down by a liquid transfer gun, uniformly mixing, and standing at room temperature for 10 min.

4) The above-described complex of the overexpression plasmid pLVX-MSLN and the various packaging plasmids was added dropwise to 293T cells cultured overnight, the dish was gently shaken, and mixed well. The dishes were placed in a 5% CO2 incubator at 37 ℃.

5) After 6-8h of incubation (preferably 8h in this example), the medium containing the transfection reagent is removed and replaced with fresh complete medium.

6) After 48-72h of continuous culture (48 h is preferred in this example), the virus-containing culture supernatant was collected from the dishes.

7) The supernatant of the culture medium obtained above was filtered through a 0.45 μm filter membrane, transferred to a centrifuge tube, and centrifuged at 50000g and 4 ℃ for 2 hours at high speed.

8) After centrifugation, the liquid in the centrifuge tubes was carefully aspirated in a biosafety cabinet, the pellet was resuspended by adding 500. mu.L LPBS buffer, and the virus was stored at-80 ℃.

9) Titer of the resulting lentivirus particles the titer of the MSLN overexpressing virions was 1.62 × 10⁸TU/mL。

3. Lentiviral infection of tumor cells

1) Recovering the cell strain of the low generation from liquid nitrogen, carrying out conventional culture and passage for 5 times, and adjusting the cell state to a logarithmic growth phase.

2) Prepare a new 6 well plate, according to 5 × 10⁵The cells were seeded at a density of one well, 2 wells were co-seeded, and complete medium was added to 2 mL/well. Placing the well plate at 37 deg.C and 5% CO₂And (5) culturing in an incubator overnight.

3) One tube of the over-expressed lentivirus was taken out of the-80 ℃ freezer and placed in a37 ℃ water bath for rapid thawing. The cells were removed from the incubator, fresh complete medium was replaced, polybrene was added to the medium in one well to a final concentration of 6ug/mL, then 100uL lentivirus was added, gently pipetted and mixed well, the well plate was placed in a centrifuge and centrifuged at 800g for 1 hour. After centrifugation, the well plate was placed at 37 ℃ in 5% CO₂The cultivation was continued in the incubator for 24 hours.

4) The well plate was removed from the incubator, the medium containing the viral supernatant in the well plate was removed, fresh complete medium was added and incubation continued for 2 days.

5) The well plate was removed from the incubator, and then the culture medium was removed from 2 wells of the well plate, and a culture medium containing 1ug/ml puromycin was added.

6) And continuously culturing for 5 days by taking the cells of the control wells which are not infected by the virus as a control until the cells of the control wells die completely, wherein the cells which survive in the wells infected by the virus are the successfully constructed stable cell strain.

7) The expression of the target gene in the stable cell strain is verified by adopting a flow cytometer or WB.

8) Obtaining target cells (MSLN)⁺PD-L1⁺，MSLN⁺PD-L1^-，MSLN^-PD-L1⁺，MSLN^-PD-L1^-)。

Example 4 preparation of SdCAR-T cells

Preparation of sdCAR Lentiviral particles

1) A15 cm dish was prepared, complete medium (DMEM high-glucose) containing 10% Fetal Bovine Serum (FBS) was added, and inoculated with 5 × 10⁶293T cells, incubated at 37 ℃ with 5% CO₂And (5) an incubator for overnight culture.

2) The over-expression vector pLVX-CAR (concentration about 100. mu.M) constructed in example 2 and lentiviral packaging plasmids (Lenti-GOI, pGP, pVSVG) (purchased from Addgene) were removed from the refrigerator, thawed at room temperature, and then pipetted up and down to mix well.

3) Phosphate Buffered Saline (PBS) was removed and warmed to room temperature. And (3) adding 2mL of PBS into one hole of a 6-hole plate, respectively adding 10 mu g of Lenti-GOI, 6 mu g of pGP and 5 mu g of pVSVG, blowing and beating the mixture up and down by a liquid transfer gun to be fully and uniformly mixed, adding 18 mu L of pLVX-CAR, immediately blowing and beating the mixture up and down by a liquid transfer machine to be uniformly mixed, and standing the mixture at room temperature for 10 min.

4) The above-described complex of the over-expression plasmid pLVX-CAR and various packaging plasmids was added dropwise to 293T cells cultured overnight, the dish was gently shaken, and mixed well. Placing the culture dish at 37 ℃ and 5% CO₂An incubator.

9) Titer of the resulting lentivirus particles the titer of the virus particles overexpressing sdcAR was 1.18 × 10⁸TU/mL。

2. Peripheral blood mononuclear cell harvesting

Peripheral Blood Mononuclear Cells (PBMC) include lymphocytes and monocytes, which have different volume, morphology and density from other cells, and the Cell density of erythrocytes and granulocytes is about 1.092g/mL, while the density of lymphocytes and monocytes is about 1.070 g/mL. The lymphocyte separating medium Ficoll reagent is prepared by mixing 60% of Ficoll and 34% of diatrizoate according to a ratio of 2:1, and has the characteristic of specific gravity of about 1.077 +/-0.001. The lymphocytes are separated from other blood cells by centrifugation, and are distributed according to a density gradient. The specific implementation method comprises the following steps: uniformly mixing 1mL of fresh anticoagulation blood with a PBS buffer solution according to a ratio of 1:1, carefully adding 1mL of Ficoll separating medium, centrifuging at room temperature of 2000rpm for 20min, dividing cells in a centrifuge tube into four layers from bottom to top, and collecting a second milky white thin film layer on the centrifuge tube as a PBMC layer; and (3) fully and uniformly mixing the PBMC with 5mL of PBS buffer solution, centrifuging at 1000rpm for 10min, and repeatedly washing twice to obtain the PBMC with higher purity.

The PBMC obtained by separation is 5 × 10 per ml⁵The cells were seeded at a density of 15cm in RPMI1640 containing 10% FBS and the cytokine IL-2 was added to a final concentration of 50U/mL, and the plates were placed at 37 ℃ and 5% CO₂The cells were cultured in an incubator for 48 hours, and the state of the cells was observed under a microscope after the culture was completed. Cell shape after PBMC culture was observedThe state is obviously differentiated, one part is suspension cells which are grown in an aggregation mode and are round and transparent, and the other part is cells which are attached to the wall and grow in a fusiform mode. The cells growing in suspension should be lymphocytes and the cells growing adherently should be monocytes, as analyzed.

Magnetic bead sorting of T cells

In this embodiment, the target T cells are mainly sorted by a magnetic bead sorting method, and the specific steps are as follows:

1) amplifying PBMC obtained from the previous step in vitro to 10⁶～10⁷；

2) Centrifuging: 300g, 10min, and completely discarding the supernatant;

3) once cell resuspension: according to each 10⁷Total cell number 80. mu.L of buffer was added to thoroughly resuspend the cells;

4) magnetic labeling: according to each 10⁷ Total cell number 20 μ L of magnetic beads of CD3 were added; fully and uniformly mixing, and then placing in a refrigerator at the temperature of 2-8 ℃ for standing for 15 min;

5) primary cleaning: according to each 10⁷Adding 1-2 mLbuffer into the total cell number, uniformly mixing, centrifuging for 10min at 300g, and completely removing supernatant;

6) and (3) secondarily resuspending the cells: 500 μ Lbuffer was added to resuspend the cells;

7) rinsing the column: adding a proper amount of buffer according to different sorting columns;

8) loading: adding the whole resuspended cells into a sorting column;

9) secondary cleaning: collecting unlabeled cells and washing with an appropriate buffer;

10) and (3) eluting the target cells: after the washing is finished, the sorting column is quickly taken out from the magnetic field and put into a proper collecting pipe, a proper amount of buffer is added, and finally, the cells are quickly pressed out by using a special injection piston.

Sorting results showed that CD3 was obtained by magnetic bead sorting⁺The positive rate of T cells was about 92.4%.

4. Lentiviral infection of T lymphocytes

1) Activated T cells were collected, centrifuged (1000rpm, 5min) and the supernatant discarded, and 1mL of fresh medium was taken to resuspend the cells. Using a magnetic bead sorting rack, adsorbing magnetic beads on the wall of the centrifugal tube by using a magnetic field bar CD3/CD28, slowly sucking the cell suspension, sucking the cell suspension to another centrifugal tube, and repeating the process for 2 times.

2) Prepare a new 6 well plate, as per 3 × 10⁵The cells were seeded at a density of one well, 2 wells in total, and complete medium was added to 3 mL/well. Placing the well plate at 37 deg.C and 5% CO₂And (5) culturing in an incubator overnight.

3) One tube of the over-expressed lentivirus (viral particles of sdCAR) was removed from the-80 ℃ freezer and placed in a37 ℃ water bath for rapid thawing. The cells were removed from the incubator, fresh complete medium was replaced, polybrene was added to the medium in one well to a final concentration of 6ug/mL, then 50uL of lentivirus was added, gently pipetted and mixed well, the well plate was placed in a centrifuge and centrifuged at 800g for 1 hour. After centrifugation, the well plate was placed at 37 ℃ in 5% CO₂The cultivation was continued in the incubator for 24 hours.

4) The well plate was removed from the incubator, the medium containing the viral supernatant in the well plate was removed, fresh complete medium was added, incubation was continued for 2 days and GFP expression was observed.

The transfection results are shown in FIG. 4, and show that the cells were transfected successfully, and the transfection efficiency is about 43%, i.e., the sdCAR-T cells are obtained.

Example 5 DT 301-mediated in vitro activation assay of sdCAR-T cells

This example details the study of DT 301-mediated in vitro activation assay of sdCAR-T cells. The indicators tested were the number of the T cell surface activating molecule CD69 and the levels of cytokines IL-2 and IFN γ produced during activation. In this experiment, the target cells selected were the target cells obtained in example 3. Totally, 5 groups are designed (each group is provided with three complex holes), and the specific scheme is as follows:

the first group: the effector cell is sdCAR-T cell, and the target cell is MDA-MB-231 cell (MSLN)^-PD-L1⁺) Adding DT 301;

and the second group: the effector cell is sdCAR-T cell, and the target cell is MDA-MB-231-MSLN cell (MSLN)⁺PD-L1⁺) DT301 is not added;

and (3) grouping three: the effector cell is sdCAR-T cell, and the target cell is PANC-1-MSLN cell (MSLN)⁺PD-L1^-) Adding DT 301;

and (4) grouping four: the effector cell is sdCAR-T cell, and the target cell is MDA-MB-231-MSLN cell (MSLN)⁺PD-L1⁺) Adding DT 301;

and group five: the effector cell is MSLN-CAR-T cell, and the target cell is MDA-MB-231-MSLN cell (MSLN)⁺PD-L1⁺) DT301 is not added.

The implementation steps are as follows:

1) respectively recovering effector cells and target cells, selecting an RPMI1640 culture medium to culture for 24h, and then performing recovery on effector cells and target cells according to an effective-to-target ratio of 2:1, and adding DT301 into the corresponding group of holes.

2) After each addition of acetyl methyl Gibberellic Acid (Gibberellic Acid AcetoxyMethyl Ester) for overnight culture (8h), 800g was centrifuged for 5min, and the cells and supernatant were collected separately.

3) And respectively detecting the contents of IL-2 and IFN gamma in the collected supernatant by adopting an ELISA method.

4) The cells obtained were collected and directly labeled with flow antibody CD69, and the number of activated T cells was flow-detected.

The level of cytokine IL-2 in cell supernatants is shown in detail in FIG. 5, and the level of cytokine IFN γ in cell supernatants is shown in detail in FIG. 6. The results of the flow assay for CAR-T cell surface activator CD69 are shown in FIG. 7.

Discussion of the results: this experiment was conducted by measuring the activation of sdCAR-T cells in the absence or presence of DT301 between sdCAR-T cells and different target cells to investigate the dependence of sdCAR-T cell activation on DT 301. The experimental result shows that the sdCAR-T cell can be activated only under the conditions that the target cell simultaneously expresses MSLN and PD-L1 and DT301 is added, and the characteristics of dual-antigen dependence and DT301 regulation of the sdCAR-T cell are illustrated.

Example 6 DT301 in vitro proliferation assay for enhanced Bichimeric antigen receptor T cells

This example details the study of the CAR-T cell proliferation assay in vitro. The indicator tested was the change in the number of T cells over time after long-term stimulation. In this experiment, the target cells selected were constructed MDA-MB-231-MSLN cells. The sdCAR-T cells and the target cells were incubated for 21 days. The experimental groups were designed as:

the first group: the effector cell is sdCAR-T cell, and the target cell is MDA-MB-231-MSLN cell (MSLN)⁺PD-L1⁺) Adding DT 301;

and the second group: the effector cell is MSLN-CAR-T cell, and the target cell is MDA-MB-231-MSLN cell (MSLN)⁺PD-L1⁺) DT301 is not added.

The method comprises the following specific implementation steps:

1) recovering the target cells MDA-MB-231-MSLN, selecting RPMI1640 culture medium to culture for 24h, and treating with mitomycin C for 30min to make the target cells lose the proliferation capacity.

2) Resuscitating effector cells, sdCAR-T cells or MSLN-CAR-T cells, at an effective to target ratio of 2:1, paving the board.

3) Fresh tumor cells were added every 7 days for stimulation.

4) The percentage of cells of CAR-T was measured by flow and the total number of cells was measured by a cytometer.

5) CAR-T cell numbers were calculated. The cell proliferation results are shown in detail in FIG. 8.

Discussion of the results: this experiment compares the antitumor activity of sdCAR-T cells with MSLN CAR-T cells by incubating them with MDA-MB-231-MSLN cells for long periods of time. The experimental results show that sdcAR-T cells have stronger anti-tumor activity than MSLN CAR-T cells when co-incubated with target cells for a long period of time.

Example 7 DT301 enhanced in vivo tumoricidal assay of Dual receptor CAR-T cells

Selecting female nude mice BALB/c of 6-8 weeks, injecting tumor cells into nude mice subcutaneously, and after the tumor cells are tumorigenic in vivo, injecting corresponding effector cells and switch molecules into tail veins of each group respectively. Four experimental groups were designed, each group consisting of 6 nude mice. The specific scheme is as follows:

the first group: a PBS control group;

and the second group: effector cells were sdcAr-T cells, DT300 was added (no FITC);

and (3) grouping three: the effector cells are sdCAR-T cells, and DT301 is added;

and (4) grouping four: the effector cells are second-generation CAR-T cells specific to MSLN, and DT301 is not added;

day 0 when tumor cells were inoculated and day 7 when 1 × 10 was injected⁷The mouse is subjected to in vivo imaging to continuously observe the size of the tumor in the body, and the in vivo load of the tumor characterized by in vivo imaging is shown in figure 8. Tumor weight see FIG. 10 and 50. mu.L of nude mouse blood taken at day 14, the number of CAR-T cells in peripheral blood was measured, and the experimental results are shown in FIG. 11. Through experimental verification and analysis, the sdCAR-T cell + DT301 has better capacity of inhibiting tumor growth compared with other three groups, the tumor fluorescence intensity is lower, and the survival time of the sdCAR-T cell in vivo is obviously prolonged.

Discussion of the results: in vivo experiment results show that the treatment of the sdCAR-T cells and DT301 can effectively mediate the elimination of the tumor of the mice, and the anti-tumor activity is stronger than that of the MSLN CAR-T cells; in addition, the tumor of the mice administered with the sdCAR-T cells alone was not inhibited, indicating that the antitumor activity of the sdCAR-T cells depends on the addition of DT301, reflecting the controllability of the sdCAR-T cells.

Example 8 use of double chimeric antigen receptor T cells in combination with DT301 in solid tumors

According to the in vitro and in vivo test results of the sdcAR-T cell combined DT301, in order to further verify the treatment effect and the applicable tumor type of the combined treatment strategy in the solid tumor, the selected solid tumor cells all highly express MSLN and PD-L1. Reference to related documents^[2-5]And corresponding clinical test data, the indications of the CAR-T cell combined DT301 treatment are mainly selected from pancreatic cancer AsPC-1, lung cancer A549, gastric cancer MG803, colon cancer SW480, breast cancer MCF7, liver cancer SMCC7721, melanoma A375, cervical cancer Hela, kidney cancer A498, prostate cancer PC-3, tongue squamous carcinoma Cal-27, bladder cancer BT-B, ovarian cancer OV90 and nasopharyngeal carcinoma 5-8F cells. First, the flow antibody is used to detect the level of carcinoembryonic antigen expressed on the surface of these solid tumor cells, and the detection results of these solid tumors are summarized in FIGS. 12 and 13. The result of the detectionIt was shown that all of these selected tumor cells highly expressed the response antigen. The above test results indicate that the sdCAR-T cell combination DT301 of the present invention is suitable for use in a variety of solid tumor types.

[1].Zhang E,Gu J,Xue J et al.Accurate control of dual-receptor-engineered T cell activity through a bifunctional anti-angiogenic peptide[J].J Hematol Oncol,2018,11:44

[2].Lv J,Zhao R,Wu D et al.Mesothelin is a target of chimeric antigenreceptor T cells for treating gastric cancer[J].J Hematol Oncol,2019,12:18

[3].Deng L,Ke X,He Z et al.A MSLN-targeted multifunctionalnanoimmunoliposome for MRI and targeting therapy in pancreatic cancer[J].IntJ Nanomedicine,2012,7:5053-5065

[4].Kendrick ZW,Firpo MA,Repko RC et al.Serum IGFBP2 and MSLN asdiagnostic and prognostic biomarkers for pancreatic cancer[J].HPB(Oxford),2014,16:670-676

[5].Sotoudeh M,Shirvani SI,Merat S et al.MSLN(Mesothelin),ANTXR1(TEM8),and MUC3A are the potent antigenic targets for CAR T cell therapy ofgastric adenocarcinoma[J].J Cell Biochem,2019,120:5010-5017

Sequence listing

<110> university of Chinese pharmacy

<120> double-chimeric antigen receptor T cell containing bifunctional immune switch molecule and application thereof

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<213> PD-L1 Block polypeptide (2 Ambystoma laterale x Ambystoma jeffersonianum)

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Ser Asn Gly Leu Ser Gln Pro Val

1 5

<210>2

<211>458

<212>PRT

<213> first chimeric antigen receptor (2 Ambystoma laterale x Ambystoma jeffersonanum)

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Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Ala Ser Asp Val Val Met Thr Gln Thr Pro Leu

20 25 30

Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser

35 40 45

Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu Arg Trp Tyr

50 55 60

Leu Gln Lys Pro Gly Gln Ser Pro Lys Val Leu Ile Tyr Lys Val Ser

65 70 75 80

Asn Arg Val Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly

85 90 95

Thr Asp Phe Thr Leu Lys Ile Asn Arg Val Glu Ala Glu Asp Leu Gly

100 105 110

Val Tyr Phe Cys Ser Gln Ser Thr His Val Pro Trp Thr Phe Gly Gly

115 120 125

Gly Thr Lys Leu Glu Ile Lys Ser Ser Ala Asp Asp Ala Lys Lys Asp

130 135 140

Ala Ala Lys Lys Asp Asp Ala Lys Lys Asp Asp Ala Lys Lys Asp Gly

145 150 155 160

Gly Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Gly

165 170 175

Ala Met Lys Leu Ser Cys Val Thr Ser Gly Phe Thr Phe Gly His Tyr

180 185 190

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

195 200 205

Ala Gln Phe Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

210 215 220

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

225 230 235 240

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Thr Gly Ile Tyr

245 250 255

Tyr Cys Thr Gly Ala Ser Tyr Gly Met Glu Tyr Leu Gly Gln Gly Thr

260 265 270

Ser Val Thr Val Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro

275 280 285

Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys

290 295 300

Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala

305 310 315 320

Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu

325 330 335

Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Val Lys Phe Ser Arg

340 345 350

Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn

355 360 365

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

370 375 380

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

385 390 395 400

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

405 410 415

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

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Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

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Ala Leu His Met Gln Ala Leu Pro Pro Arg

450 455

<210>3

<211>376

<212>PRT

<213> second chimeric antigen receptor (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400>3

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Pro

20 25 30

Glu Leu Glu Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser

35 40 45

Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His

50 55 60

Gly Lys Ser Leu Glu Trp Ile Gly Leu Ile Thr Pro Tyr Asn Gly Ala

6570 75 80

Ser Ser Tyr Asn Gln Lys Phe Arg Gly Lys Ala Thr Leu Thr Val Asp

85 90 95

Lys Ser Ser Ser Thr Ala Tyr Met Asp Leu Leu Ser Leu Thr Ser Glu

100 105 110

Asp Ser Ala Val Tyr Phe Cys Ala Arg Gly Gly Tyr Asp Gly Arg Gly

115 120 125

Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly Ser Asp Ile Glu

145 150 155 160

Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val

165 170 175

Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr

180 185 190

Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser

195 200 205

Lys Leu Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Ser Gly Ser Gly

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Asn Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu Asp Asp Ala

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Thr Tyr Tyr Cys Gln Gln Trp Ser Lys His Pro Leu Thr Tyr Gly Ala

245 250 255

Gly Thr Lys Leu Glu Ile Lys Ser Ser Thr Thr Thr Pro Ala Pro Arg

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Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg

275 280 285

Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly

290 295 300

Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr

305 310 315 320

Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg

325 330 335

Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro

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Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu

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Glu Glu Glu Gly Gly Cys Glu Leu

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<210>4

<211>3116

<212>DNA

<213>sdCAR (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400>4

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccgggatccc aggtacaact gcagcagtct gggcctgagc tggagaagcc tggcgcttca 120

gtgaagatat cctgcaaggc ttctggttac tcattcactg gctacaccat gaactgggtg 180

aagcagagcc atggaaagag ccttgagtgg attggactta ttactcctta caatggtgct 240

tctagctaca accagaagtt caggggcaag gccacattaa ctgtagacaa gtcatccagc 300

acagcctaca tggacctcct cagtctgaca tctgaagact ctgcagtcta tttctgtgca 360

agggggggtt acgacgggag gggttttgac tactggggcc aagggaccac ggtcaccgtc 420

tcctcaggtg gaggcggttc aggcggcggt ggctctagcg gtggtggatc ggacatcgag 480

ctcactcagt ctccagcaat catgtctgca tctccagggg agaaggtcac catgacctgc 540

agtgccagct caagtgtaag ttacatgcac tggtaccagc agaagtcagg cacctccccc 600

aaaagatgga tttatgacac atccaaactg gcttctggag tcccaggtcg cttcagtggc 660

agtgggtctg gaaactctta ctctctcaca atcagcagcg tggaggctga agatgatgca 720

acttattact gccagcagtg gagtaagcac cctctcacgt acggtgctgg gacaaagttg 780

gaaatcaaaa gcagcaccac taccccagca ccgaggccac ccaccccggc tcctaccatc 840

gcctcccagc ctctgtccct gcgtccggag gcatgtagac ccgcagctgg tggggccgtg 900

catacccggg gtcttgactt cgcctgcgat atctacattt gggcccctct ggctggtact 960

tgcggggtcc tgctgctttc actcgtgatc actctttact gtaagcgcgg tcggaagaag 1020

ctgctgtaca tctttaagca acccttcatg aggcctgtgc agactactca agaggaggac 1080

ggctgttcat gccggttccc agaggaggag gaaggcggct gcgaactgta acgcccctct 1140

ccctcccccc cccctaacgt tactggccga agccgcttgg aataaggccg gtgtgcgttt 1200

gtctatatgt tattttccac catattgccg tcttttggca atgtgagggc ccggaaacct 1260

ggccctgtct tcttgacgag cattcctagg ggtctttccc ctctcgccaa aggaatgcaa 1320

ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag acaaacaacg 1380

tctgtagcga ccctttgcag gcagcggaac cccccacctg gcgacaggtg cctctgcggc 1440

caaaagccac gtgtataaga tacacctgca aaggcggcac aaccccagtg ccacgttgtg 1500

agttggatag ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg 1560

aaggatgccc agaaggtacc ccattgtatg ggatctgatc tggggcctcg gtgcacatgc 1620

tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg 1680

gttttccttt gaaaaacacg atgataagct tgccacaacc cacaaggaga cgaccttcca 1740

tggccctccc tgtcaccgcc ctgctgcttc cgctggctct tctgctccac gccgctcggc 1800

ccgctagcga cgtcgttatg actcaaacac cactatcact tcctgttagt ctaggtgatc 1860

aagcctccat ctcttgcaga tctagtcaga gcctcgtaca cagtaatgga aacacctatt 1920

tacgttggta cctgcagaag ccaggccagt ctccaaaggt cctgatctac aaagtttcca 1980

accgagtttc tggggtccca gacaggttca gtggcagtgg atcagggaca gatttcacac 2040

tcaagatcaa cagagtggag gctgaggatc tgggagttta tttctgctct caaagtacac 2100

atgttccgtg gacgttcggt ggaggcacca agcttgaaat taagtcctct gctgatgatg 2160

ctaagaagga tgctgctaag aaggatgatg ctaagaaaga tgatgctaag aaagatggtg 2220

gcgtcaaact ggatgagact ggaggaggct tggtgcaacc tgggggggcc atgaaactct 2280

cctgtgttac ctctggattc acttttggtc actactggat gaactgggtc cgccagtctc 2340

cagagaaagg actggagtgg gtagcacaat ttagaaacaa accttataat tatgaaacat 2400

attattcaga ttctgtgaaa ggcagattca ccatctcaag agatgattcc aaaagtagtg 2460

tctatctgca aatgaacaac ttaagagttg aagacacggg tatctattac tgtacgggtg 2520

cttcctatgg tatggaatac ttgggtcaag gaacctcagt caccgtctcc accactaccc 2580

cagcaccgag gccacccacc ccggctccta ccatcgcctc ccagcctctg tccctgcgtc 2640

cggaggcatg tagacccgca gctggtgggg ccgtgcatac ccggggtctt gacttcgcct 2700

gcgatatcta catttgggcc cctctggctg gtacttgcgg ggtcctgctg ctttcactcg 2760

tgatcactct ttactgtcgc gtgaaattca gccgcagcgc agatgctcca gcctaccagc 2820

aggggcagaa ccagctctac aacgaactca atcttggtcg gagagaggag tacgacgtgc 2880

tggacaagcg gagaggacgg gacccagaaa tgggcgggaa gccgcgcaga aagaatcccc 2940

aagagggcct gtacaacgag ctccaaaagg ataagatggc agaagcctat agcgagattg 3000

gtatgaaagg ggaacgcaga agaggcaaag gccacgacgg actgtaccag ggactcagca 3060

ccgccaccaa ggacacctat gacgctcttc acatgcaggc cctgccgcct cggtaa 3116

<210>5

<211>244

<212>PRT

<213>MSLN scFv(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400>5

Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro

1 5 10 15

Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr

20 25 30

Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu

35 40 45

Trp Ile Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln

50 55 60

Lys Phe Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr

65 70 75 80

Ala Tyr Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr

85 90 95

Phe Cys Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Ser Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro

130 135 140

Ala Ile Met SerAla Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser

145 150 155 160

Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Gly

165 170 175

Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly

180 185 190

Val Pro Gly Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser Leu

195 200 205

Thr Ile Ser Ser Val Glu Ala Glu Asp Asp Ala Thr Tyr Tyr Cys Gln

210 215 220

Gln Trp Ser Lys His Pro Leu Thr Tyr Gly Ala Gly Thr Lys Leu Glu

225 230 235 240

Ile Lys Ser Ser

<210>6

<211>256

<212>PRT

<213>FITC scFv(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400>6

Ala Ser Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser

1 5 10 15

Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val

20 25 30

His Ser Asn Gly Asn Thr Tyr Leu Arg Trp Tyr Leu Gln Lys Pro Gly

35 40 45

Gln Ser Pro Lys Val Leu Ile Tyr Lys Val Ser Asn Arg Val Ser Gly

50 55 60

Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

65 70 75 80

Lys Ile Asn Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser

85 90 95

Gln Ser Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu

100 105 110

Ile Lys Ser Ser Ala Asp Asp Ala Lys Lys Asp Ala Ala Lys Lys Asp

115 120 125

Asp Ala Lys Lys Asp Asp Ala Lys Lys Asp Gly Gly Val Lys Leu Asp

130 135 140

Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Gly Ala Met Lys Leu Ser

145 150 155 160

Cys Val Thr Ser Gly Phe Thr Phe Gly His Tyr Trp Met Asn Trp Val

165 170 175

Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val Ala Gln Phe Arg Asn

180 185 190

Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp Ser Val Lys Gly Arg

195 200 205

Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser Val Tyr Leu Gln Met

210 215 220

Asn Asn Leu Arg Val Glu Asp Thr Gly Ile Tyr Tyr Cys Thr Gly Ala

225 230 235 240

Ser Tyr Gly Met Glu Tyr Leu Gly Gln Gly Thr Ser Val Thr Val Ser

245 250 255

Claims

1. A bifunctional immune switch molecule comprising a regulatory region of a bipartite chimeric antigen receptor and an immune checkpoint inhibitory region.

2. A bifunctional immune switch molecule according to claim 1 characterized in that: the immune checkpoint inhibitory region is a PD-L1 blocking polypeptide, and the sequence of the polypeptide is as follows: SEQ ID number 1.

3. A bifunctional immune switch molecule according to claim 1 characterized in that: the double chimeric antigen receptor regulating region is coupled FITC molecule.

4. A bifunctional immune switch molecule according to any of claims 1-3 characterized in that: the sequence is FITC-Acp-SNGLSQPV.

5. A dual chimeric antigen receptor comprising a first chimeric antigen receptor and a second chimeric antigen receptor; the first chimeric antigen receptor comprises a CD8 alpha signal peptide, a FITC scFv, a CD8 alpha hinge region and transmembrane region and a CD3 zeta intracellular signal region, the amino acid sequence of the FITC scFv is SEQ ID NO.6, the second chimeric antigen receptor comprises a CD8 alpha signal peptide, an MSLN scFv, a CD8 alpha hinge region and transmembrane region and a 4-1BB intracellular signal region, and the amino acid sequence of the MSLN scFv is SEQ ID NO. 5.

6. The bipartite antigen receptor of claim 5, wherein the bipartite antigen receptor is introduced into an immune cell by lentivirus; the immune cells include T cells, monocyte-macrophages, NK cells.

7. A bi-chimeric antigen receptor T cell comprising a bifunctional immune switch molecule, characterized in that: comprising the bifunctional immune switch molecule of any one of claims 1-4, a first chimeric antigen receptor and a second chimeric antigen receptor; wherein the first chimeric antigen receptor is composed of a CD8 alpha signal peptide, a FITC scFv, a CD8 alpha hinge region, a transmembrane region and a CD3 zeta intracellular signal region which are connected in series, and the amino acid sequence of the first chimeric antigen receptor is as follows: SEQ ID number 2; the second chimeric antigen receptor is formed by connecting a CD8 alpha signal peptide, MSLN scFv, a CD8 alpha hinge region and a transmembrane region in series, and a 4-1BB intracellular signal region in series, and the amino acid sequence of the second chimeric antigen receptor is as follows: SEQ ID number 3.

8. The use of the bi-chimeric antigen receptor T cell of claim 7 containing a bifunctional immune switch molecule in the preparation of a medicament for the treatment of tumors.

9. Use according to claim 8, characterized in that: the tumor comprises ovarian cancer, lung cancer, esophageal cancer, pancreatic cancer, gastric cancer, colon cancer, breast cancer, liver cancer, melanoma, head and neck cancer, cervical cancer and osteosarcoma.

10. A pharmaceutical composition for treating tumor cells, comprising the bi-chimeric antigen receptor T cell of claim 7 containing a bifunctional immune switch molecule, and a pharmaceutically acceptable excipient.