CN113527494A

CN113527494A - Novel anti-tumor transformation receptor T cell

Info

Publication number: CN113527494A
Application number: CN202110827476.2A
Authority: CN
Inventors: 卢铀; 薛建新; 仝瑞占
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2020-11-26
Filing date: 2021-07-21
Publication date: 2021-10-22
Anticipated expiration: 2041-07-21
Also published as: CN113527494B

Abstract

The invention discloses a novel anti-tumor transformation receptor T cell, and belongs to the field of tumor immunotherapy. The novel anti-tumor transformation receptor T cell of the invention has the core of expressing an artificial recombinant receptor protein which is sequentially composed of the following segments: (1) a cell membrane targeting signal peptide, (2) an anti-PD-L1 single-chain antibody segment, (3) a CD28 transmembrane and intracellular segment; the sequence of the part (2) is shown in any one of SEQ ID NO. 5-8. The anti-tumor transformation receptor T cell can convert a PD-L1 inhibition signal on the surface of a cancer cell into an activation signal, activate the T cell and play a role in immunotherapy.

Description

Novel anti-tumor transformation receptor T cell

Technical Field

The invention belongs to the field of tumor immunotherapy, and particularly relates to a novel anti-tumor transformation receptor T cell.

Background

In recent years, research on the application of Adoptive Cellular Immunotherapy (ACI) in the treatment of hematological tumors and solid tumors has attracted general attention. In particular CAR-T (cardiac antigen receptor T cell), has been approved for the treatment of Acute Lymphoblastic Leukemia (ALL) and relapsed or refractory large B-cell lymphoma, for example, CD 19-targeted CAR-T has been approved for clinical treatment of hematological malignancies. However, CAR-T technology does not achieve the same therapeutic effect on solid tumors as hematological tumors because solid tumors have a more complex immunosuppressive microenvironment, including hypoxic, low nutritional metabolic environments, pathways mediating immunosuppression such as PD-L1/PD-1, and the like.

At present, the immune checkpoint inhibitor medicine taking PD-1 and PD-L1 as targets is greatly developed in clinic, and the killing capacity of T cells to tumor cells is restored by blocking the immune checkpoint pathway of PD-1 and PD-L1. However, the clinical efficacy of the antibody drug targeting PD-1 and PD-L1 is greatly limited because of the limited number of T cells of patients with advanced tumors and the lack of co-stimulation signals when the T cells kill the tumor cells, which leads to the death of the T cells due to exhaustion when the T cells kill the tumor cells.

The research on a product which can enhance the proliferation and the amplification capacity of T cells and is suitable for treating solid tumors is of great significance.

Disclosure of Invention

The invention aims to solve the problems that: provides a novel anti-tumor transformation receptor T cell (PDL1/CD 28T cell) and a preparation method and application thereof. The T cells of the conversion receptor can transmit an activation signal (from CD28 molecules downstream of the conversion receptor) instead of an inhibition signal by binding with PDL1, so that the immunosuppression of the PDL1 channel on the T cells is reversed, and the killing capacity and the proliferation activity of the T cells are further enhanced.

The technical scheme of the invention is as follows:

a PD-L1 antibody, the amino acid sequence of which is shown in any one of SEQ ID NO. 5-8.

A gene fragment, which codes protein with an amino acid sequence shown as any one of SEQ ID NO. 5-8.

The gene fragment has a sequence shown in any one of SEQ ID NO. 1-4.

An anti-tumor transition receptor, which comprises the following 4 parts in sequence:

(1) a cell membrane targeting signal peptide segment;

(2) anti-PD-L1 single-chain antibody segment;

(3) CD28 transmembrane segment;

(4) CD28 intracellular segment;

the sequence of the part (2) is shown in any one of SEQ ID NO. 5-8.

The sequence of the part (2) is shown as SEQ ID NO. 5.

The anti-tumor switch receptor is as described in the above, and the sequence of the part (1) is shown in SEQ ID NO. 15;

and/or the sequence of the part (3) is shown as SEQ ID NO. 17;

and/or the sequence of the part (4) is shown as SEQ ID NO. 18.

The anti-tumor transition receptor is as the aforementioned, and a connecting peptide segment is also arranged between the part (2) and the part (3), and preferably, the sequence of the connecting peptide segment is shown as SEQ ID NO. 16.

The sequence of the anti-tumor transformation receptor is shown as SEQ ID NO. 14.

A gene fragment encoding the aforementioned anti-tumor transition receptor.

A recombinant virus carrying the aforementioned gene fragment.

As with the recombinant viruses previously described, the viruses are lentiviruses.

A recombinant T cell carrying the aforementioned gene fragment and/or the aforementioned anti-tumor switch receptor.

The application of the PD-L1 antibody, the anti-tumor transformation receptor, the recombinant virus or the T cell in preparing the medicine for treating tumors.

The use as described above, the tumor is a solid tumor; preferably, the solid tumor is lung cancer, melanoma, bladder cancer, kidney cancer, liver cancer, nasopharyngeal cancer, esophageal cancer, cervical cancer, breast cancer or gastric cancer.

The invention has the beneficial effects that:

the anti-tumor transformation receptor shortens the length of a PD-L1 antibody, can convert an inhibition signal of PD-L1 to a T cell into an activation signal, enhances the activity and the proliferation capacity of the T cell, and enables the T cell to have the capacity of treating solid tumors.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 is a schematic structural diagram of recombinant lentiviral vector pHBLV-EF 1A-alpha PD-L1-CD 28-T2A-Mcherry.

FIG. 2 is a graph showing the results of detection of lentivirus-transfected 293T cells, in which a is an inverted microscope image and b is a fluorescence microscope image.

FIG. 3 is a graph showing the results of detection of lentivirus-infected T cells.

FIG. 4 shows the expression of CD69 after 24 hours of the action of α PDL1/CD28T cells, unloaded T cells, control T cells and CD3 antibody + PD-L1 protein.

FIG. 5 shows the expression of 4-1BB after 24 hours of the interaction of α PDL1/CD28T cells, unloaded T cells, control T cells and CD3 antibody + PD-L1 protein.

FIG. 6 shows the secretion of IL2, IFN gamma and TNF alpha in the supernatant of alpha PDL1/CD28T cells, unloaded T cells and control T cells after 24 hours of action under different conditions.

FIG. 7 shows apoptosis of α PDL1/CD28T cells, T cells with no load, and control T cells after 48 hours of stimulation under different conditions.

FIG. 8 shows memory phenotype of α PDL1/CD28T cells, unloaded T cells, and control T cells after 48 hours of stimulation under different conditions.

FIG. 9 shows the proliferation of cell proliferation dye-labeled α PDL1/CD28T cells, T-cell null cells, and control T cells at various time points after the action of CD3 antibody + PD-L1 protein.

FIG. 10 shows the secretion of CD107a by α PDL1/CD28T cells, unloaded T cells, and control T cells after stimulation under different conditions.

FIG. 11 shows the results of the binding of each group of T cells to PDL 1.

FIG. 12 shows the expression results of the activation index of each group of T cells after conditioned stimulation.

FIG. 13 is the results of memory phenotype of various groups of T cells after conditioned stimulation.

FIG. 14 shows the results of CD107a secretion from various groups of T cells after conditioned stimulation.

Detailed Description

The raw materials and equipment used in the embodiment of the present invention are known products and obtained by purchasing commercially available products.

Example 1 preparation of anti-tumor transformation receptor T cells

First, Lentiviral construction

1. Construction of lentiviral vector plasmids

1.1 design of PD-L1 antibody Gene sequences

4 in total, which are respectively:

the first PD-L1 antibody gene sequence (SEQ ID NO. 1):

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGTAGCCTCTGGAAGTATCAGCAGTTTCGACGCAGTGCTCTGGTACCGCCGGGCTCCAGGGAAGCAGCGCGATTGGGTCGCAACTTCTTTTACCGCCGGTCACACAATCTATGAAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAGGAACACGGTGTATCTGCAAATGAACAGCCTGAAAACTGAGGACACAGGCGACTATTATTGTAATGCGAGGCGACTAGGTGCGCACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAAGAC

second PD-L1 antibody gene sequence (SEQ ID NO. 2):

GAGGTGCAGCTGGTGGAGTCCGGAGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGAGCTGCGTGGCTAGCGGCTCTCATCTTAGCTTCGACGCCGTGCTGTGGTACAGGCAGGCTCCTGGCAAGCAGCGGGATTGGGTGGCCACCAGCTTCACAGCTGGCTATACTATCTACGAGGACTCTGTGAAGGGCAGGTTCACCATCTCCCGCGATAACGCTAAGAATACAGTGTATCTGCAGATGAACTCTCTGCGCGCCGAGGACACAGCCGTGTACTATTGTAATGCCAGGCGGCTGGGCGCTCATTATTGGGGCCAGGGCACCCTGGTGACAGTGTCTTCC

the third PD-L1 antibody gene sequence (SEQ ID NO. 3):

GAGGTGCAGCTGGTGGAGTCCGGAGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGAGCTGCGTGGCTAGCGGCTCTGTTTCCAGCTTCGACGCCGTGCTGTGGTACAGGCAGGCTCCTGGCAAGCAGCGGGATTGGGTGGCCACCAGCTTCACAGCTGGTTATACCATCTACGAGGACTCTGTGAAGGGCAGGTTCACCATCTCCCGCGATAACGCTAAGAATACAGTGTATCTGCAGATGAACTCTCTGCGCGCCGAGGACACAGCCGTGTACTATTGTAATGCCAGGCGGCTGGGCGCTCATTATTGGGGCCAGGGCACCCTGGTGACAGTGTCTTCC

fourth PD-L1 antibody gene sequence (SEQ ID No. 4):

GAGGTGCAGCTGGTGGAGTCCGGAGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGAGCTGCGTGGCTAGCGGCTCTGATGTGAGCTTCGACGCCGTGCTGTGGTACAGGCAGGCTCCTGGCAAGCAGCGGGATTGGGTGGCCACCAGCTTCACAGCTGGCTGGGAGATCTACGAGGACTCTGTGAAGGGCAGGTTCACCATCTCCCGCGATAACGCTAAGAATACAGTGTATCTGCAGATGAACTCTCTGCGCGCCGAGGACACAGCCGTGTACTATTGTAATGCCAGGCGGCTGGGCGCTCATTATTGGGGCCAGGGCACCCTGGTGACAGTGTCTTCC

the corresponding amino acid sequence:

first PD-L1 antibody (SEQ ID NO. 5):

QVQLVESGGGLVQAGGSLRLSCVASGSISSFDAVLWYRRAPGKQRDWVATSFTAGHTIYEDSVKGRFTISRDNARNTVYLQMNSLKTEDTGDYYCNARRLGAHYWGQGTQVTVSSEPKTPKPQD

second PD-L1 antibody (SEQ ID NO. 6):

EVQLVESGGGLVQPGGSLRLSCVASGSHLSFDAVLWYRQAPGKQRDWVATSFTAGYTIYEDSVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCNARRLGAHYWGQGTLVTVSS

the third PD-L1 antibody (SEQ ID NO. 7):

EVQLVESGGGLVQPGGSLRLSCVASGSVSSFDAVLWYRQAPGKQRDWVATSFTAGYTIYEDSVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCNARRLGAHYWGQGTLVTVSS

fourth PD-L1 antibody (SEQ ID No. 8):

EVQLVESGGGLVQPGGSLRLSCVASGSDVSFDAVLWYRQAPGKQRDWVATSFTAGWEIYEDSVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYYCNARRLGAHYWGQGTLVTVSS

1.2 Synthesis of fragment of the alpha PD-L1-CD28 Gene

Through a whole gene synthesis method, DNA sequences are synthesized, wherein the PD-L1 antibody gene sequences are respectively connected with a signal peptide, a CD28 hinge segment, a CD28 transmembrane segment and a CD28 intracellular segment, and the sequence is the signal peptide-PD-L1 antibody-CD 28 hinge segment-CD 28 transmembrane segment-CD 28 intracellular segment.

Taking the alpha PD-L1-CD28 corresponding to the first PD-L1 antibody gene as an example, the DNA sequence (SEQ ID NO.9) is:

note: the lower case non-italic part (SEQ ID NO.10) is a signal peptide segment; the upper case bold part is anti-PD-L1 segment sequence; the lower case italic part (SEQ ID NO.11) is the CD28 hinge (hinge) segment, which plays a role in ligation; the underlined part (SEQ ID NO.12) is a CD28 transmembrane segment; the upper italic part (SEQ ID NO.13) is the CD28 intracellular segment.

The protein sequence (SEQ ID NO.14) coded by the first alpha PD-L1-CD28 gene segment is as follows:

note: the lower case non-italic part (SEQ ID NO.15) is a signal peptide segment; the upper case bold part is anti-PD-L1 segment sequence; the lower case italic part (SEQ ID NO.16) is the CD28 hinge (hinge) segment, which serves for ligation; the underlined part (SEQ ID NO.17) is a CD28 transmembrane segment; the upper italic part (SEQ ID NO.18) is the CD28 intracellular segment.

The obtained alpha PD-L1-CD28 gene fragment is cloned to pHBLV-EF1A-MCS-T2A-Mcherry lentiviral vector, and as shown in figure 1, recombinant pHBLV-EF 1A-alpha PD-L1-CD28-T2A-Mcherry lentiviral vector is constructed.

Respectively taking 1 piece of 100 mu L competent cells (purchased from Beijing Ongzhike Biotechnology Co., Ltd.), adding 1 mu g of target plasmid pHBLV-EF 1A-alpha PD-L1-CD 28-T2A-Mchery into the competent cells, respectively adding 1 mu g of lentivirus packaging helper plasmids pSPAX2 and PMD2G into 100 mu L of the competent cells, respectively, fully mixing, respectively placing on ice for incubation for 30 minutes, immediately heating in a 42 ℃ water bath for 45 seconds, and then placing in an ice bath for 2 minutes. Then, LB medium containing 50. mu.g/mL ampicillin solution (the objective plasmid pHBLV-EF 1A-alpha PD-L1-CD 28-T2A-Mchery and the packaging plasmids pSPAX2 and PMD2G are ampicillin resistant) is respectively added, and after being fully mixed, the mixture is placed in a shaker at 37 ℃ and is subjected to constant temperature shaking culture at 220rpm/min for 12-16 hours.

The concentrations and purities of the target Plasmid and the packaged Plasmid were determined by QIAGEN Plasmid Plus Midi Kit and Nanodrop ultramicro spectrophotometer, respectively, and then stored in a refrigerator at-20 ℃.

2. Packaging of lentiviruses

Preparation of 293T cells: 293T cells in logarithmic growth phase were seeded in 6-well plates with cell density adjusted to 5X 10⁵Cells/well, cultured in 5% CO2 incubator at 37 ℃ until the cell density reaches 70-80% are ready for transfection.

The lentivirus packaging is carried out by using Lipofilter 3.0 (purchased from Shanghai Biotechnology, Inc., with the product number of HB-LF3-1000), and the specific steps are as follows:

adding 4 mu g of plasmid (0.8 mu g of pMD2.G +1.6 mu g of psPAX +1.6 mu g of target plasmid) into 250 mu L of DMEM medium, and gently blowing and uniformly mixing by using a gun; adding 6 μ L LLIPOFITER 3.0 into 250 μ L DMEM, blowing with a gun, mixing, and standing at room temperature for 5 min;

mixing the two solutions, and lightly blowing and uniformly mixing the two solutions by using a gun;

incubation for 20min at room temperature;

fourthly, uniformly adding the mixed solution into one hole of a 6-hole plate, and uniformly mixing;

culturing in a cell culture box for 6-12h, and replacing a fresh culture medium;

sixthly, observing the expression condition of red fluorescent protein of 293T cells by a fluorescence microscope after 24h of transfection (as shown in figure 2), and harvesting the virus after 48h of transfection.

3. Preparation of lentivirus concentrate

The virus was filtered using a 0.22 μm (Millipore) frit. The viruses were concentrated using the PEG Virus Precipitation Kit (BioVision, USA, Cat. No. K904-50/200). To the harvested virus, PEG Solution (5X) (20ml of filtered virus supernatant +5ml of PEG) was added by volume, mixed well, and left to stand at 4 ℃ overnight. The harvested virus supernatant was centrifuged (4 ℃, 4000rpm/15 min) to precipitate the virus. The supernatant was decanted, dissolved in a Virus Re-suspension Solution, and stored at-80 ℃.

Second, lentivirus infection of T cells

T cell culture Medium

ImmunoCult^TMXF T Cell Expansion Medium (STEMCELL, cat # 10981) supplemented with 10ng/ml interleukin 2(STEMCELL, cat # 78036).

2. Method of producing a composite material

Inoculating T cells into 24-well cell culture plate at a concentration of 1 × 10⁶Cell/well, per cell: the proportion of the magnetic beads is 1: 3 Dynabeads Human T-Activator CD3/CD28(Gibco Corp., cat # 11131D) was added to activate T cells. After 24 hours by adding magnetic beads, the T cells were transferred to a well plate coated with RetroNectin (Recombinant Human fibrin fragment), a lentivirus concentrate (multiplicity of infection: 30) was added, and a susceptibility-promoting agent HitransG A (Shanghai Jikai Gene medicine science and technology) was addedManufactured by gmbh, product number genechem REVG004), mixed well, and cultured in an incubator at 37 ℃. Expanded T cells, designated α PDL1/CD28T cells, were harvested after passage of T cells every 2 days.

The beneficial effects of the alpha PDL1/CD28T cells of the invention are further illustrated in the form of experimental examples, and because the results of the four PDL1 antibody sequences are similar, the genes of the PD-L1 antibodies adopted in experimental examples 1-8 are the genes shown in SEQ ID NO. 4.

Experimental example 1 detection of infection efficiency of α PDL1/CD28T cells

1. Cell preparation

Alpha PDL1/CD28T cells, unloaded T cells (the alpha PD-L1-CD28 gene segment is omitted on the basis of example 1, and lentivirus empty vectors are transferred), and control T cells.

2. Antibody detection

Separately, biotinylated human PD-L1 protein was added to each cell suspension to a final concentration of 1 μ g/ml, incubated on ice for 30 minutes in the absence of light, washed 1 time with washing buffer, centrifuged at 300g for 5 minutes, the supernatant removed, and 50 μ L of 1: streptavidin APC conjugate (Thermo Fisher Scientific, cat. No. SA1005) diluted with 200PBS was mixed well and incubated for 30 minutes on ice in the absence of light. Wash 1 time with wash buffer, centrifuge at 300g for 5 minutes, remove the supernatant, 200 u L PBS buffer heavy suspension cells. And respectively carrying out detection analysis on the samples by using a flow cytometer.

3. Results

The results are shown in FIG. 3. The detection result shows that the alpha PDL1/CD28T cell can recognize PD-L1 at a ratio of 71.7 percent, while the idle T cell and the control T cell can hardly recognize PD-L1; the alpha PDL1/CD28T cells expressed RFP in 75.5% and the unloaded T cells in 89% and the control T cells did not express RFP.

4. Conclusion

The alpha PDL1/CD28T cell can better recognize PD-L1.

Experimental example 2 phenotypic analysis of activation status of α PDL1/CD28T cells

1. Method of producing a composite material

A96-well plate was prepared, and 60. mu.l of a coating solution was added to each well, wherein the coating solution had a composition of 1. mu.g/ml of CD3 antibody (Miltenyi, Inc., cat. No. 170-076-309) and/or 25. mu.g/ml of PD-L1 protein (Sino Biological, cat. No. 10084-H05H), and was allowed to stand overnight at 4 ℃.

The lentivirus-infected T cells (i.e., α PDL1/CD28T cells) obtained in example 1, unloaded T cells, and control T cells were individually seeded into each well at a cell count of 2X 10⁵Culturing in 37 deg.C incubator.

After 24h, the cells were collected from each well, washed 1 time with wash buffer, centrifuged at 300g for 5 minutes, the supernatant removed, and the cells resuspended in 100. mu.L of wash buffer to give a cell suspension.

1) Detection of CD 69: to each sample of the cell suspension, 5 μ L of a mouse anti-human CD69 monoclonal antibody labeled with PE-Cy7 (purchased from BD Biosciences, usa, product No. 557745) was added, and after incubation for 30 minutes in the absence of light on ice, the cell suspension was washed twice with a washing buffer, centrifuged at 300g for 5 minutes, the supernatant was removed, and then 200 μ L of PBS buffer was added to resuspend the cells, and each sample was detected by a flow cytometer.

2) Detection of 4-1 BB: to each cell suspension sample, 5. mu.L of an APC-labeled mouse anti-human 4-1BB monoclonal antibody (purchased from BD Biosciences, USA, product No. 550890) was added, and after incubation for 30 minutes in the absence of light on ice, the cell suspension was washed twice with a washing buffer, centrifuged at 300g for 5 minutes, the supernatant was removed, and then 400. mu.L of a PBS buffer was added to resuspend the cells, and each sample was detected by a flow cytometer.

2. Results

The results of CD69 and 4-1BB detection are shown in FIGS. 4 and 5. It can be seen that the expression of CD69 and 4-1BB of T cells in each group is improved after the stimulation of the CD3 antibody; however, after the action of CD3 antibody and PD-L1 protein, the expression of alpha PDL1/CD28T cells CD69 and 4-1BB is further increased, but the expression of unloaded T cells and control T cells CD69 and 4-1BB is reduced.

3. Conclusion

The CD3 antibody can induce T cell activation, CD69, 4-1BB are markers of T cell activation, and the PD-L1 protein can bind to PD-1 on the surface of a T cell and inhibit the T cell activation.

The results of the experimental example show that the alpha PDL1/CD28T cells can convert inhibitory signals of PD-L1 protein into activating signals, and the killing capacity of the T cells to PD-L1 positive tumors is improved.

Experimental example 3 detection of cytokines secreted by α PDL1/CD28T cells

1. Method of producing a composite material

The culture supernatant after 24 hours of culture was subjected to ELISA kit (Thermo Fisher Scientific Co., Ltd., cat Nos. 77-7025, 88-7316) to detect the contents of IL2, IFN γ and TNF α in the culture supernatant.

2. Results

The cytokine detection results are shown in fig. 6, and after the CD3 antibody + PD-L1 protein acts, IL2, IFN γ and TNF α in the culture supernatant of α PDL1/CD28T cells are significantly increased compared to those of unloaded T cells and control T cells.

3. Conclusion

IL2, IFN gamma and TNF alpha are factors which can enhance NK cell activity and are generated after T cells are activated, and under the action of a CD3 antibody and PD-L1, alpha PDL1/CD28T cells secrete cytokines to be improved, so that the anti-tumor treatment is facilitated.

Experimental example 4 detection of apoptotic phenotype of α PDL1/CD28T cells

1. Method of producing a composite material

The lentivirus-infected T cells (i.e., α PDL1/CD28T cells) obtained in example 1, unloaded T cells, and control T cells were individually seeded into each well at a cell count of 2X 10⁵Is arranged atCulturing at 37 deg.C in incubator.

After 48h, the cells were collected from each well, washed 1 time with wash buffer, centrifuged at 300g for 5 minutes, the supernatant removed, and the cells resuspended in 100. mu.L of wash buffer to give a cell suspension.

Flow-type staining is carried out by adopting an Annexin V/PI apoptosis kit, and detection and analysis are respectively carried out on each sample by using a flow cytometer.

2. Results

The results are shown in fig. 7, and the ratio of early apoptosis and late apoptosis of alpha PDL1/CD28T cells is reduced compared with those of unloaded T cells and control T cells after the CD3 antibody + PD-L1 protein acts.

3. Conclusion

The alpha PDL1/CD28T cells have long service life and can play an anti-tumor role for a long time compared with common T cells.

Experimental example 5 detection of memory phenotype of α PDL1/CD28T cells

1. Method of producing a composite material

To each cell suspension sample, a PE-labeled mouse anti-human CD62L monoclonal antibody (purchased from BD Biosciences, USA, product No. 555544) 20. mu. L, PerCP-Cy5.5-labeled mouse anti-human CD45RO monoclonal antibody (purchased from BD Biosciences, USA, product No. 560607) was added. After incubation for 30 min on ice in the dark, the cells were washed twice with wash buffer, centrifuged at 300g for 5min, the supernatant was removed, and then 200. mu.L of PBS buffer was added to resuspend the cells. And (4) detecting and analyzing each sample by using a flow cytometer.

2. Results

As shown in FIG. 8, after the CD3 antibody + PD-L1 protein acts, the memory phenotype of alpha PDL1/CD28T cells is obviously changed,

t cells are obviously decreased, and T cells of effector memory and central memory are obviously increased.

3. Conclusion

Under the action of CD3 antibody and PD-L1 protein, alpha PDL1/CD28T cells can be converted into memory phenotype T cells more, the immune function can be exerted more rapidly, and the anti-cancer effect is stronger.

Experimental example 6 detection of proliferation phenotype of α PDL1/CD28T cells

1. Method of producing a composite material

The lentivirus-infected T cells (i.e., α PDL1/CD28T cells) obtained in example 1, unloaded T cells, and control T cells were individually seeded into each well, and each group of T cells was labeled with eBioscience Cell promotion dye (Thermo Fisher Scientific Co., Ltd., cat. No. 65-0840), seeded into the coated plate, and cultured in an incubator at 37 ℃.

Cells were harvested at 4, 8 days after plating, washed 1 time with wash buffer, centrifuged at 300g for 5 minutes, the supernatant removed, and the cells resuspended in 200. mu.L PBS buffer. And (4) detecting and analyzing each sample by using a flow cytometer.

2. Results

As shown in FIG. 9, the α PDL1/CD28T cell proliferation dye peak was shifted to the left by the action of CD3 antibody + PD-L1 protein.

3. Conclusion

After the action of CD3 antibody and PD-L1 protein, the proliferation capacity of alpha PDL1/CD28T cells is improved.

Experimental example 7 functional assay for secretion of CD107a from α PDL1/CD28T cells

1. Method of producing a composite material

The lentivirus-infected T cells (i.e., alpha PDL1/CD28T cells) obtained in example 1, unloaded T cells and control T cells were respectively inoculated into each well, 5. mu.l of CD107a-APC antibody was added to each well, mixed well, centrifuged at 500rpm for 3min, and incubated at 37 ℃ in an incubator.

After 1 hour, GolgiStop (purchased from BD Biosciences, USA, product code 554724) was diluted at a ratio of 1:150 in a serum-free medium, and added to a 96-well plate at 10. mu.l/well, mixed well and cultured in an incubator at 37 ℃.

After 2.5 hours, single cell suspensions were prepared by aspirating cells from 96-well plates, stained with antibodies to CD3-BV510, CD8-FITC, 2. mu.l/well, incubated on ice for 30 minutes in the dark, washed twice with wash buffer, centrifuged at 300g for 5 minutes, the supernatant removed, and the cells resuspended in 200. mu.L PBS. And (4) detecting and analyzing each sample by using a flow cytometer.

2. Results

As shown in fig. 10, compared to the unloaded T cells and the control T cells, the CD107a secretion of the α PDL1/CD28T cells stimulated by the CD3 antibody + PDL1 protein was significantly increased, while the CD107a secretion of the unloaded T cells and the control T cells was inhibited after the action of PDL 1.

3. Conclusion

Alpha PDL1/CD28T cells have a stronger capacity to secrete CD107 a.

Experimental example 8 functional experiments comparing alpha PDL1/CD28T cells with other T cells which convert receptors

1. Method of producing a composite material

The structure of the conversion receptor of the T cell is PD1-CD 28. Alpha PDL1/CD28T cells, PD1/CD 28T cells and unloaded T cells were prepared contemporaneously by means of lentivirus infection, and in experimental examples 1, 2, 5 and 7, functional comparisons were made between alpha PDL1/CD28T cells and PD1/CD 28T cells.

2. Results

The results of the binding assay of each group of T cells to PDL1 are shown in fig. 11. Under the condition that the infection efficiency of alpha PDL1/CD28T cells and PD1/CD 28T cells is equivalent (the expression of red fluorescent labels is about 70 percent), the binding capacity of the alpha PDL1/CD28T cells to PDL1 is obviously higher than that of the PD1-CD28-T cells.

The expression results of the activation index of each group of T cells after conditioned stimulation are shown in fig. 12. After the alpha CD3 acts for 24 hours, the expression of T cell activation indexes of each group is obviously increased; with the increase of PDL1 dosage, the expression of the no-load T cell activation index is obviously reduced, while the expression of the alpha PDL1/CD28T cell and the expression of the alpha PDL1/CD28T cell activation index are further increased, which indicates the conversion effect on PDL1 inhibition signals. Under the same condition of stimulation of alpha CD3+ PDL1, the activation index of alpha PDL1/CD28T cells is higher than that of PD1-CD28-T cells.

The results of the memory phenotype of each group of T cells after conditioned stimulation are shown in FIG. 13. After 24h of action of alpha CD3, each group of T cells

The proportion is obviously reduced, and the proportion of central memory cells is increased; idle T cells with increasing PDL1 dose

Increased ratio, decreased ratio of central memory T cells, and alpha PDL1/CD28T cells and PD1/CD 28T cells

The proportion continuously decreases and the proportion of central memory T cells continuously increases. Alpha PDL1/CD28T cells under the same conditions of alpha CD3+ PDL1 stimulation

The ratio is lower than that of PD1/CD 28T cells, and the central memory ratio is higher than that of PD1/CD 28T cells.

The results of CD107a secretion from each group of T cells following conditional stimulation are shown in figure 14. 4h after the action of the alpha CD3, the secretion of CD107a by the T cells in each group is obviously increased; with increasing dose of PDL1, unloaded T cells 107a decreased significantly, while α PDL1/CD28T cells and PD1/CD 28T cells CD107a secretion was further increased, suggesting its role in converting PDL1 inhibitory signals. Under the same condition of stimulation of alpha CD3+ PDL1, the secretion of CD107a of alpha PDL1/CD28T cells is higher than that of PD1/CD 28T cells.

In conclusion, after the alpha PDL1/CD28T cell is acted by a CD3 antibody and a PD-L1 protein, an inhibition signal of the PD-L1 protein is converted into an activation signal, the secretion of T cell factors is promoted, the apoptosis of the T cell is reduced, the memory phenotype T cell is increased, and the proliferation capacity of the T cell is improved. Compared with common T cells, the alpha PDL1/CD28T cells have stronger anti-tumor capacity and the capacity of treating solid tumors.

SEQUENCE LISTING

<110> Sichuan university Hospital in western China

<120> a novel anti-tumor transition receptor T cell

<130> GYKH1352-2021P0113537CCZ

<150> 2020113667589

<151> 2020-11-26

<160> 18

<170> PatentIn version 3.5

<210> 1

<211> 372

<212> DNA

<213> Artificial sequence

<400> 1

caggtgcagc tggtggagtc tgggggaggc ttggtgcagg ctggggggtc tctgagactc 60

tcctgtgtag cctctggaag tatcagcagt ttcgacgcag tgctctggta ccgccgggct 120

ccagggaagc agcgcgattg ggtcgcaact tcttttaccg ccggtcacac aatctatgaa 180

gactccgtga agggccgatt caccatctcc agagacaacg ccaggaacac ggtgtatctg 240

caaatgaaca gcctgaaaac tgaggacaca ggcgactatt attgtaatgc gaggcgacta 300

ggtgcgcact actggggcca ggggacccag gtcaccgtct cctcagaacc caagacacca 360

aaaccacaag ac 372

<210> 2

<211> 345

<212> DNA

<213> Artificial sequence

<400> 2

gaggtgcagc tggtggagtc cggaggagga ctggtgcagc ctggcggctc cctgagactg 60

agctgcgtgg ctagcggctc tcatcttagc ttcgacgccg tgctgtggta caggcaggct 120

cctggcaagc agcgggattg ggtggccacc agcttcacag ctggctatac tatctacgag 180

gactctgtga agggcaggtt caccatctcc cgcgataacg ctaagaatac agtgtatctg 240

cagatgaact ctctgcgcgc cgaggacaca gccgtgtact attgtaatgc caggcggctg 300

ggcgctcatt attggggcca gggcaccctg gtgacagtgt cttcc 345

<210> 3

<211> 345

<212> DNA

<213> Artificial sequence

<400> 3

gaggtgcagc tggtggagtc cggaggagga ctggtgcagc ctggcggctc cctgagactg 60

agctgcgtgg ctagcggctc tgtttccagc ttcgacgccg tgctgtggta caggcaggct 120

cctggcaagc agcgggattg ggtggccacc agcttcacag ctggttatac catctacgag 180

gactctgtga agggcaggtt caccatctcc cgcgataacg ctaagaatac agtgtatctg 240

cagatgaact ctctgcgcgc cgaggacaca gccgtgtact attgtaatgc caggcggctg 300

ggcgctcatt attggggcca gggcaccctg gtgacagtgt cttcc 345

<210> 4

<211> 345

<212> DNA

<213> Artificial sequence

<400> 4

gaggtgcagc tggtggagtc cggaggagga ctggtgcagc ctggcggctc cctgagactg 60

agctgcgtgg ctagcggctc tgatgtgagc ttcgacgccg tgctgtggta caggcaggct 120

cctggcaagc agcgggattg ggtggccacc agcttcacag ctggctggga gatctacgag 180

gactctgtga agggcaggtt caccatctcc cgcgataacg ctaagaatac agtgtatctg 240

cagatgaact ctctgcgcgc cgaggacaca gccgtgtact attgtaatgc caggcggctg 300

ggcgctcatt attggggcca gggcaccctg gtgacagtgt cttcc 345

<210> 5

<211> 124

<212> PRT

<213> Artificial sequence

<400> 5

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

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

20 25 30

Ala Val Leu Trp Tyr Arg Arg Ala Pro Gly Lys Gln Arg Asp Trp Val

35 40 45

Ala Thr Ser Phe Thr Ala Gly His Thr Ile Tyr Glu Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Gly Asp Tyr Tyr Cys Asn

85 90 95

Ala Arg Arg Leu Gly Ala His Tyr Trp Gly Gln Gly Thr Gln Val Thr

100 105 110

Val Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Asp

115 120

<210> 6

<211> 115

<212> PRT

<213> Artificial sequence

<400> 6

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ser His Leu Ser Phe Asp

20 25 30

Ala Val Leu Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Asp Trp Val

35 40 45

Ala Thr Ser Phe Thr Ala Gly Tyr Thr Ile Tyr Glu Asp Ser Val Lys

50 55 60

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

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Arg Arg Leu Gly Ala His Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 7

<211> 115

<212> PRT

<213> Artificial sequence

<400> 7

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

1 5 10 15

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

20 25 30

Ala Val Leu Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Asp Trp Val

35 40 45

Ala Thr Ser Phe Thr Ala Gly Tyr Thr Ile Tyr Glu Asp Ser Val Lys

50 55 60

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

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Arg Arg Leu Gly Ala His Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 8

<211> 115

<212> PRT

<213> Artificial sequence

<400> 8

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

1 5 10 15

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

20 25 30

Ala Val Leu Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Asp Trp Val

35 40 45

Ala Thr Ser Phe Thr Ala Gly Trp Glu Ile Tyr Glu Asp Ser Val Lys

50 55 60

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

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Arg Arg Leu Gly Ala His Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 9

<211> 768

<212> DNA

<213> Artificial sequence

<400> 9

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atcccacagg tgcagctggt ggagtctggg ggaggcttgg tgcaggctgg ggggtctctg 120

agactctcct gtgtagcctc tggaagtatc agcagtttcg acgcagtgct ctggtaccgc 180

cgggctccag ggaagcagcg cgattgggtc gcaacttctt ttaccgccgg tcacacaatc 240

tatgaagact ccgtgaaggg ccgattcacc atctccagag acaacgccag gaacacggtg 300

tatctgcaaa tgaacagcct gaaaactgag gacacaggcg actattattg taatgcgagg 360

cgactaggtg cgcactactg gggccagggg acccaggtca ccgtctcctc agaacccaag 420

acaccaaaac cacaagacgc ggccgcaatt gaagttatgt atcctcctcc ttacctagac 480

aatgagaaga gcaatggaac cattatccat gtgaaaggga aacacctttg tccaagtccc 540

ctatttcccg gaccttctaa gcccttttgg gtgctggtgg tggttggggg agtcctggct 600

tgctatagct tgctagtaac agtggccttt attattttct gggtgaggag taagaggagc 660

aggctcctgc acagtgacta catgaacatg actccccgcc gccccgggcc cacccgcaag 720

cattaccagc cctatgcccc accacgcgac ttcgcagcct atcgctcc 768

<210> 10

<211> 66

<212> DNA

<213> Artificial sequence

<400> 10

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atccca 66

<210> 11

<211> 126

<212> DNA

<213> Artificial sequence

<400> 11

gcggccgcaa ttgaagttat gtatcctcct ccttacctag acaatgagaa gagcaatgga 60

accattatcc atgtgaaagg gaaacacctt tgtccaagtc ccctatttcc cggaccttct 120

aagccc 126

<210> 12

<211> 81

<212> DNA

<213> Artificial sequence

<400> 12

ttttgggtgc tggtggtggt tgggggagtc ctggcttgct atagcttgct agtaacagtg 60

gcctttatta ttttctgggt g 81

<210> 13

<211> 123

<212> DNA

<213> Artificial sequence

<400> 13

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120

tcc 123

<210> 14

<211> 256

<212> PRT

<213> Artificial sequence

<400> 14

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly

20 25 30

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

35 40 45

Ser Ile Ser Ser Phe Asp Ala Val Leu Trp Tyr Arg Arg Ala Pro Gly

50 55 60

Lys Gln Arg Asp Trp Val Ala Thr Ser Phe Thr Ala Gly His Thr Ile

65 70 75 80

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

85 90 95

Arg Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr

100 105 110

Gly Asp Tyr Tyr Cys Asn Ala Arg Arg Leu Gly Ala His Tyr Trp Gly

115 120 125

Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Thr Pro Lys Pro

130 135 140

Gln Asp Ala Ala Ala Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp

145 150 155 160

Asn Glu Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu

165 170 175

Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu

180 185 190

Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val

195 200 205

Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His

210 215 220

Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys

225 230 235 240

His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser

245 250 255

<210> 15

<211> 22

<212> PRT

<213> Artificial sequence

<400> 15

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro

20

<210> 16

<211> 42

<212> PRT

<213> Artificial sequence

<400> 16

Ala Ala Ala Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu

1 5 10 15

Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro

20 25 30

Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro

35 40

<210> 17

<211> 27

<212> PRT

<213> Artificial sequence

<400> 17

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 18

<211> 41

<212> PRT

<213> Artificial sequence

<400> 18

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

Claims

1. A PD-L1 antibody, characterized in that: the amino acid sequence of the polypeptide is shown in any one of SEQ ID NO. 5-8.

2. A gene fragment, characterized in that: the coded amino acid sequence of the protein is shown as any one of SEQ ID NO. 5-8.

3. The gene segment of claim 2, wherein: the sequence of the gene fragment is shown in any one of SEQ ID NO. 1-4.

4. An anti-tumor switch receptor characterized by: the sequence of the polypeptide sequentially comprises the following 4 parts:

(1) a cell membrane targeting signal peptide segment;

(2) anti-PD-L1 single-chain antibody segment;

(3) CD28 transmembrane segment;

(4) CD28 intracellular segment;

the sequence of the part (2) is shown in any one of SEQ ID NO. 5-8.

5. The anti-tumor switch receptor of claim 4, wherein: the sequence of the part (2) is shown as SEQ ID NO. 5.

6. The anti-tumor switch receptor of claim 4, wherein: the sequence of the part (1) is shown as SEQ ID NO. 15;

and/or the sequence of the part (3) is shown as SEQ ID NO. 17;

and/or the sequence of the part (4) is shown as SEQ ID NO. 18.

7. The anti-tumor transforming receptor according to any one of claims 4 to 6, wherein: a connecting peptide segment is also arranged between the part (2) and the part (3), and the sequence of the connecting peptide segment is preferably shown as SEQ ID NO. 16.

8. The anti-tumor transforming receptor according to any one of claims 4 to 6, wherein: the sequence is shown as SEQ ID NO. 14.

9. A gene fragment encoding the anti-tumor transition receptor according to any one of claims 4 to 8.

10. A recombinant virus, characterized in that: the virus carries the gene segment of claim 9.

11. The recombinant virus of claim 10, wherein: the virus is a lentivirus.

12. A recombinant T cell, characterized in that: the T cell carries the gene segment of claim 9 and/or the anti-tumor transition receptor of any one of claims 4 to 8.

13. Use of the PD-L1 antibody of claim 1, the anti-tumor switch receptor of any one of claims 4-8, the recombinant virus of any one of claims 10-11, or the T cell of claim 12 in the manufacture of a medicament for the treatment of a tumor.

14. Use according to claim 13, characterized in that: the tumor is a solid tumor; preferably, the solid tumor is lung cancer, melanoma, bladder cancer, kidney cancer, liver cancer, nasopharyngeal cancer, esophageal cancer, cervical cancer, breast cancer or gastric cancer.