CN111944850B

CN111944850B - Preparation method of cell for expressing anti-CD22 chimeric antigen receptor and PD-L1 blocking protein, expression vector and application

Info

Publication number: CN111944850B
Application number: CN202010891848.3A
Authority: CN
Inventors: 赵琦; 刘婕
Original assignee: University of Macau
Current assignee: University of Macau
Priority date: 2020-08-28
Filing date: 2020-08-28
Publication date: 2023-03-31
Anticipated expiration: 2040-08-28
Also published as: CN111944850A

Abstract

The invention discloses a preparation method, an expression vector and application of cells for expressing anti-CD22 chimeric antigen receptor and PD-L1 blocking protein, and relates to the technical field of medical biology. The expression vector is inserted with a first nucleic acid encoding an anti-CD22 chimeric antigen receptor and a second nucleic acid encoding a PD-L1 blocking protein. By using a CD22 target and a PD-L1 target and targeting an antibody or a fragment thereof of CD22, the T cell of the invention can efficiently and specifically target tumor cells expressing the antigen CD 22; the PD-L1 blocking protein can compete with the endogenously expressed PD-1 to bind to the PD-L1 on a target cell, so that a PD-1/PD-L1 signal channel is blocked, the action time of the T cell is prolonged, the tumor killing effect is improved, and the problem of the immune tolerance of solid tumors to CAR T cell therapy is relieved.

Description

Preparation method of cell for expressing anti-CD22 chimeric antigen receptor and PD-L1 blocking protein, expression vector and application

Technical Field

The invention relates to the technical field of medical biology, in particular to a preparation method, an expression vector and application of cells for expressing anti-CD22 chimeric antigen receptor and PD-L1 blocking protein.

Background

Chimeric antigen receptor T cell therapy is receiving increasing attention in the field of tumor therapy. Chimeric antigen receptor T cells (CAR T cells) are T cells genetically engineered to have a chimeric antigen receptor for immunotherapy.

Chimeric Antigen Receptor (CAR) is an artificially constructed transmembrane molecule encoded by a fusion gene that specifically targets T cells to antigens on the surface of cancer cells to eliminate the target cancer cells. CARs consist of an extracellular domain (e.g., a single chain antibody, scFv, of an antibody), a transmembrane domain, and an intracellular domain. The scFv of the extracellular domain is responsible for the recognition of specific antigens. The intracellular domain is responsible for the transduction of signals. When the extracellular domain specifically binds to an antigen, the intracellular domain initiates a signal required for cell activation, thereby promoting proliferation of T cells, release of cytokines, and the like. The transmembrane domain connects the extracellular domain and the intracellular domain.

The first generation CARs linked scfvs to an intracellular signaling domain, such as CD3 ζ, to induce antigen-specific T cell activation. Subsequent CARs incorporate single (second generation) or multiple (third generation) additional costimulatory signals, such as domains of CD28, 4-1BB or OX40, to further enhance and maintain effector function of T cells.

Although CAR T cell immunotherapy has the characteristics of high targeting, high killing activity, etc., due to the presence of immune checkpoint molecules, such as programmed death receptor-1 (PD-1), programmed cell death-ligand 1 (PD-L1), etc., tumor molecules can inactivate CAR T cells, thereby evading killing of CAR T cells.

PD-1 is an important immunosuppressive molecule. PD-1 is expressed primarily in T cells, activated T cells, monocytes and natural killer T cells (Keir et al). The structure of PD-1 includes an extracellular domain, a transmembrane domain, and an intracellular tail. PD-1 belongs to a synergistic inhibitory receptor and has 2 ligands, PD-L1 and programmed cell death-ligand 2 (PD-L2), respectively. PD-L1 is expressed in different malignancies, such as lung cancer, esophageal cancer, ovarian cancer, bladder cancer, malignant melanoma and glioma. In vivo, binding of PD-1 to PD-L1 or PD-L2 down regulates antigen-stimulated lymphocyte proliferation and cytokine production, ultimately leading to lymphocyte "depletion" to induce immune tolerance. Tumor cells in solid tumors can up-regulate the expression of PD-L1, which in turn provides a signal to down-regulate activated T cells, ultimately shutting down the immune response and inducing immune tolerance.

Chimeric Antigen Receptor (CAR) T cell therapy has had great success in hematologic malignancies, but not in solid tumors, overcoming the immune tolerance of solid tumors to CAR T cell therapy has become an urgent problem.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a preparation method, an expression vector and application of a cell for expressing an anti-CD22 chimeric antigen receptor and a PD-L1 blocking protein so as to solve the technical problems.

The invention is realized by the following steps:

an expression vector into which a first nucleic acid encoding an anti-CD22 chimeric antigen receptor and a second nucleic acid encoding a PD-L1 blocking protein are inserted;

the PD-L1 blocking protein is PD-L1 which lacks a PD-1 transmembrane region and an intracellular signal region, the amino acid sequence of the anti-CD22 chimeric antigen receptor is shown as SEQ ID NO.1, and the amino acid sequence of the PD-L1 blocking protein is shown as SEQ ID NO. 2.

CD22 is another antigen specific for B cells and has a tissue distribution similar to CD 19. The glycoprotein is expressed on the surface of cells of the B cell lineage. CD22 has proven to be a promising target for lymphomas and B-cell leukemias. Studies have demonstrated that CART targeting CD22 achieves superior anti-leukemia efficacy. Thus, CD22 has the potential to be a target for CAR-T cell therapy in all patients. It may also provide a second option for patients who received CART treatment targeting CD19 but who had unfortunately relapsed after recovery.

The inventor constructs a first nucleic acid encoding anti-CD22 chimeric antigen receptor and a second nucleic acid encoding PD-L1 blocking protein on the same carrier, and utilizes a CD22 target and a PD-1 target to effectively and specifically target tumor cells expressing the antigen CD22 by targeting an antibody or a fragment of the CD22.

The PD-L1 blocking protein only consists of the extracellular domain of PD-1, and the PD-L1 blocking protein can compete with the endogenously expressed PD-1 to bind to the PD-L1 on a target cell, thereby blocking a PD-1/PD-L1 signal channel. Thus, the problem of immune tolerance of solid tumors to CAR T cell therapy is alleviated.

Corresponding T cells can be obtained by transfecting the expression vector provided by the invention, and the T cells can realize the co-expression of the anti-CD22 chimeric antigen receptor and the PD-L1 blocking protein. Binding of PD-L1 expressed on tumor cells to PD-L1 on the surface of the above-mentioned T cells is restricted after binding of PD-L1 expressed on tumor cells to PD-L1 on the surface of the above-mentioned T cells of the present invention. Thus, the PD-1/PD-L1 signaling pathway is reduced or even blocked. Finally, the action time of the T cells is prolonged, and the tumor killing effect is improved.

The present invention provides a first nucleic acid encoding an anti-CD22 chimeric antigen receptor, and in other embodiments, is not limited to a CD22 target, and other targets such as CD19, CD20, CD30, CD123, BCMA, her2, GPC3, mesothelin, and the like, may be selected according to targeting requirements.

In other embodiments, the binding domain specific for CD22 (i.e., the first nucleic acid encoding the anti-CD22 chimeric antigen receptor) may also be a polypeptide sequence having at least 90% identity to a single-chain variable fragment (scFv). A binding domain specific for CD22 can bind to CD22.

In a preferred embodiment of the present invention, the nucleotide sequence of the first nucleic acid is shown as SEQ ID NO.3, and the nucleotide sequence of the second nucleic acid is shown as SEQ ID NO. 4.

In a preferred embodiment of the present invention, the expression vector is a lentiviral expression vector;

preferably, the lentiviral expression vector is a pLVX-IRES-Zsgreen lentiviral expression vector. In other embodiments, lentiviral expression vectors may also be adaptively selected as desired.

In a preferred embodiment of the present invention, the transmembrane domain is linked to the second nucleic acid in an expression vector;

preferably, the transmembrane domain is selected from the transmembrane domains of the T cell receptor subunit, CD4, CD8 or CD 28.

The transmembrane domain is designed to: express capacity on the cell surface and interact to direct the cellular response of immune cells to the intended target cells.

In other embodiments, the transmembrane domain may be derived from natural or synthetic sources. The transmembrane domain may be derived from any membrane-bound or transmembrane protein. For example, the transmembrane polypeptide may be a subunit of a T cell receptor such as α, β or δ. Synthetic transmembrane domains may contain predominantly hydrophobic residues such as leucine and valine.

In a preferred embodiment of the present invention, the expression vector further comprises: an extracellular domain and an intracellular domain.

The extracellular domain comprises a hinge; the intracellular domain comprises a costimulatory signaling molecule and an intracellular signaling domain;

preferably, the hinge is from CD8 α.

The hinge serves to link the transmembrane domain to an extracellular binding domain specific for CD22. In other embodiments the hinge may also be selected from the FcRIII α receptor.

The costimulatory signal molecule is selected from the group consisting of human 4-1BB costimulatory signal molecule, CD27, PD1, ICOS, OX40 or B7-H3; the intracellular signaling domain is a human CD3 ζ signaling domain.

The intracellular signaling domain is responsible for intracellular signaling upon binding of the extracellular binding domain specific for CD22 to the target, resulting in immune cell activation and immune response. In other embodiments, the intracellular signaling domain may also be selected from CD3 γ, CD5, or CD3 epsilon.

Costimulatory signaling molecules refer to cognate binding partners on T cells that specifically bind to costimulatory ligands, thereby mediating a costimulatory response by the cell. In other embodiments, the costimulatory signal molecule can also be CD27, PD1, ICOS, OX40, or B7-H3.

The expression vector provided by the invention is formed by connecting a first nucleic acid (CD 22 SCFV) for coding an anti-CD22 chimeric antigen receptor, a transmembrane domain (CD 8 TM) of CD8, 41-BB, CD3 zeta, a second nucleic acid and a CD28 gene in series in sequence.

A method for preparing a cell expressing an anti-CD22 chimeric antigen receptor and a PD-L1 blocker protein, the method comprising: transfecting host cells with the expression vector; preferably, the host cell is a T cell.

In a preferred embodiment of the present invention, the T cell is a human peripheral blood mononuclear cell.

A pharmaceutical composition, which comprises the expression vector or the cell prepared by the cell preparation method, and pharmaceutically acceptable excipient.

The pharmaceutical composition further comprises a soluble monoclonal antibody; preferably, the monoclonal antibody is an anti-human PD-L1 antibody.

The inventor finds that the co-culture of the anti-human PD-L1 antibody and the CAR T cell prepared by the cell preparation method can save the expression of cytokines IL2 and TNF alpha and promote the T cell to kill the tumor cell.

It should be noted that the monoclonal antibody that actually promotes the killing of tumor cells by T cells is not limited to the anti-human PD-L1 antibody, and in other embodiments, can be adaptively adjusted as needed.

The expression vector or the cell prepared by the method can be applied to preparing the medicine for treating the tumor.

In a preferred embodiment of the present invention, the tumor is a tumor positive for PD-L1 or CD 22;

preferably, the tumor is any one of the following tumors: lymphoma, hodgkin lymphoma, non-hodgkin lymphoma, leukemia, and multiple myeloma.

In other embodiments, it may be applied to the treatment of other solid tumors and hematological malignancies as well.

The invention has the following beneficial effects:

the invention constructs an expression vector which can realize the co-expression of anti-CD22 chimeric antigen receptor and PD-L1 blocking protein, and the T cell can efficiently and specifically target the tumor cell expressing the antigen CD22 by using the CD22 target and the PD-1 target and through targeting the CD22 antibody or the fragment thereof; the PD-L1 blocking protein can compete with the endogenously expressed PD-1 to bind to the PD-L1 on the target cell, so that a PD-1/PD-L1 signal channel is blocked, the action time of the T cell is prolonged, the tumor killing effect is improved, and the problem of the immune tolerance of the solid tumor to CAR T cell therapy is relieved. The cell prepared by the preparation method of the cell expressing the CD 22-resistant chimeric antigen receptor and the PD-L1 blocking protein can be widely used for preparing solid tumor treatment medicines.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and those skilled in the art can also obtain other related drawings based on the drawings without inventive efforts.

Figure 1 shows a design scheme diagram of the vectors used to construct the CAR PDR T cells of the invention;

FIGS. 2A-2C show the results of 293T cells transfected with the vectors CD22ABPDR 4 ZS, CD22ABPDR 8 ZS and CD22 ABPDR28 ZS expressing CAR, PDR and ZsGreen1, respectively;

FIG. 2D shows the results of Western blot experiments of PDR expressed by 293T cells transfected with the vectors CD22ABPDR 4 ZS, CD22ABPDR 8 ZS and CD22 ABPDR28 ZS, respectively;

FIG. 3 shows a diagram of another design scheme for constructing a vector for use in CAR PDR T cells of the invention;

FIG. 4A shows the results of Q-PCR identification of the expression of the relevant proteins in human peripheral blood lymphocytes transfected with the vectors ZS, PDR, CD22AB and CD22 ABPDR;

FIG. 4B is a graph showing the results of stepwise expression of PD-1;

FIG. 5 shows that the tumor cell lines K562-PD-L1, raji-PD-L1, daudi-PD-L1 and BV173-PD-L1 used in the present invention stably highly express PD-L1;

FIG. 6A shows the ratio of CD4 and CD8 in human peripheral blood lymphocytes transfected with vectors ZS, PDR, CD22AB and CD22ABPDR after selection of CD 3T cells;

FIG. 6B shows the results of FACS detection of the activation marker CD27 expressed in human peripheral blood lymphocytes transfected with the vectors ZS, PDR, CD22AB and CD22 ABPDR;

FIG. 6C shows the results of FACS detection of the activation marker CD28 expressed in human peripheral blood lymphocytes transfected with the vectors ZS, PDR, CD22AB and CD22 ABPDR;

FIG. 6D shows the results of FACS detection of the activation marker CD69 expressed in human peripheral blood lymphocytes transfected with the vectors ZS, PDR, CD22AB and CD22 ABPDR;

FIG. 7A shows FACS detection of CD45RA and CD62L expression after 24 hours co-culture of human peripheral blood lymphocytes transfected with the vectors ZS, PDR, CD22AB and CD22ABPDR with the tumor cell line BV 173-PD-L1;

FIG. 7B shows the different proportions of CD45RA + CD 62L-cell types after 24 hours of co-culture of human peripheral blood lymphocytes transfected with the vectors ZS, PDR, CD22AB and CD22ABPDR with the tumor cell line BV 173-PD-L1;

FIG. 8 shows the lysis of tumor cell lines K562, raji, daudi and BV173 by human peripheral blood lymphocytes transfected with the vectors ZS, PDR, CD22AB and CD22 ABPDR;

FIG. 9A shows the effect of an anti-PD-1 antibody (anti-PD-1) on the expression of IL2 and TNF α by human peripheral blood lymphocytes transfected with the vector CD22ABPDR against the tumor cell line BV173 PD-L1;

FIG. 9B shows the effect of anti-PD-L1 antibody (anti-PD-L1) against the tumor cell line BV173 PD-L1 on the expression of IL2 and TNF α by human peripheral blood lymphocytes transfected with the vector CD22ABPDR.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

The invention aims at overcoming the immune check point inhibition of CAR T cell therapy, and designs a T cell aiming at a CD22 antigen, wherein the T cell co-expresses an anti-CD22 chimeric antigen receptor and a PD-L1 blocking protein, and the PD-L1 blocking protein is a dominant negative receptor of PD-1. The T cells are abbreviated CAR PDR T cells. In addition, tumor killing effects of anti-PD-L1 antibodies used in combination with the CAR PDR T cells of the present application were also investigated.

Example 1

This example constructs a series of anti-CD22 vectors to co-express CAR and PD-L1 blocking protein. The anti-CD22 CAR constructs used in the present invention are second generation CARs, all of which comprise a CD8 a leader sequence, an anti-CD22 specific scFv, a CD8 a hinge, and a TM (transmembrane domain). The intracellular domain comprises a 4-1BB costimulatory domain and a CD3 zeta signaling domain, referred to as CD22AB.

To identify the efficiency of immune checkpoint molecules in CAR T cells, we constructed vectors co-expressing CAR and PD-1 extracellular domains with different transmembrane domains (CD 4, CD8 and CD 28). Each vector was named 22ABPDR 4, 22ABPDR 8, and 22ABPDR28, respectively. The construction of the inserted gene and plasmid is shown in FIG. 1.

The vector comprises a first nucleic acid encoding a chimeric antigen receptor consisting of a single chain antibody of an Anti-CD22 antibody (Anti-CD 22), a CD8 transmembrane domain (TM), 41-BB, and CD3 zeta, and a nucleic acid encoding a PD-1 dominant negative receptor (PDR) consisting of a PD-1 extracellular domain and three different TMs. Vectors are constructed by means conventional in the art.

The CD22AB scFv was amplified by primers CD22AB F and CD22AB R in Table 1, the CAR signal transduction region was amplified by signal F and signal R, and then the CD22AB scFv and signal transduction region were ligated into one complete CAR gene by Overlap PCR.

The PD-L1 blocking protein gene and the transmembrane region of CD4/CD8/CD28 are amplified by PCR, and P2A is introduced to the 5' end of the transmembrane region of CD4/CD8/CD28 in a PCR amplification mode. Then, the transmembrane region and the CAR gene are connected through an Overlap PCR, and the DNA fragment is inserted into a lentiviral vector pLVX-EF1 alpha-IRES-ZsGreen 1 through enzyme cutting sites at two ends.

The PCR system was 50ul and included 0.5U Q5 DNA polymerase, 2mM MgCl ₂ 0.2mM dNTP mix, 20ng plasmid template, 0.25. Mu.M forward primer and 0.25. Mu.M reverse primer. The PCR reaction program is: pre-denaturation at 98 ℃ for 30s, denaturation for 10s,annealing at 58 ℃ for 20s, extension at 72 ℃ for 30 cycles, and final extension at 72 ℃ for 5min. The PCR products were electrophoresed in 1% agarose and the PCR fragments were recovered using an Omega gel recovery kit.

Table 1 primer sequence table.

CAR F and CAR R amplify CD22AB CAR sequences, PD-1 EM F and PD-1 EM R amplify PD-L1 blocking protein, P2A F for insertion of P2A to the 5' terminal PD-1 extracellular sequence.

The nucleotide sequence of P2A is:

ggcgccaccaacttctccctgctgaagcaggccggcgacgtggaggagaaccccggcccc. The amino acid sequence of P2A is: GATNFSLLKQAGDVEENPGP.

CD4/8/28 TM M R is used for constructing different transmembrane sequences to the 3 'end of a PD-1 extracellular sequence, the vector does not contain a ZsGreen reporter gene, CD4 TM B R, CD8 TM NR and CD28 TM B R are used for constructing different transmembrane sequences to the 3' end of a PD-L1 blocking protein sequence, and the vector contains a ZsGreen reporter gene. The transmembrane region sequences are respectively:

the nucleotide sequence of CD4 TM is:

gccctgattgtgctggggggcgtcgccggcctcctgcttttcattgggctaggcatcttcttc, amino acid sequence of CD4 TM: ALIVLGGVAGLLLFIGLGIFF;

the nucleotide sequence of CD8 TM is:

atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc

amino acid sequence of CD8 TM: IYIWAPLAGTCGVLLLSLVITLYC;

the nucleotide sequence of CD28 TM is:

ttttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtg

the amino acid sequence of CD28 TM is:

FWVLVVVGGVLACYSLLVTVAFIIFWV。

5'LTR in FIG. 1 represents a 5' long terminal repeat; LS denotes a preamble sequence; anti-22 represents the coding sequence of a single-chain antibody of Anti-CD22 antibody-22 AB; CD8 TM represents the coding sequence for the CD8 transmembrane domain; 41-BB and CD3 zeta represent the coding sequences of costimulatory signaling molecule 41-BB and intracellular signaling domain CD3 zeta, respectively; 2A represents the coding sequence of the Thoseaasgna virus 2A peptide; PD-1 EM + CD4 TM, PD-1 EM + CD8 TM and PD-1 EM + CD28 TM represent the coding sequences of the combination of the extracellular domain of PD-1 with the transmembrane domains CD4, CD8 and CD28, respectively; IRES denotes the coding sequence of the internal ribosome entry site; zsGreen1 represents the coding sequence of a fluorescent protein.

Three plasmid vectors, plasmid CD22ABPDR 4 ZS, are respectively constructed, wherein the extracellular domain of PD-1 is combined with the CD4 transmembrane domain; plasmid CD22ABPDR 8 ZS wherein the extracellular domain of PD-1 is combined with the CD8 transmembrane domain; and plasmid CD22 ABPDR28 ZS, wherein the extracellular domain of PD-1 is combined with the CD28 transmembrane domain.

The three plasmid vectors described above, CD22ABPDR 4 ZS, CD22ABPDR 8 ZS and CD22 ABPDR28 ZS, were transfected into 293T cells, respectively.

Subsequently, transfected 293T cells were cultured, harvested, and subjected to FACS detection.

The FACS assay comprises the following steps:

CD22 expression on tumor cell lines was stained with FITC anti-human CD22 antibody. CAR expression in transduced T cells was measured using flow cytometry with CD22-Fc protein and anti-Phycoerythrin (PE) conjugated anti-human IgG. PDR expression in transduced T cells was detected with mouse anti-human PD-1 and APC conjugated goat anti-mouse IgG. For T cell typing, CAR T cells or mock T cells were stained with APC-conjugated anti-human CD3, PE-conjugated anti-human CD4, PE/CY 7-conjugated anti-human CD 8. For T cell activation, CAR T cells or control T cells were stained with APC-conjugated anti-human CD8 and PE-conjugated CD27, PE-conjugated CD28, PE-conjugated CD 69. For T cell subtypes, CAR T cells or mock T cells were stained with APC-conjugated anti-human CD3, PE-conjugated anti-human CD45, PE/CY 7-conjugated CD 62L. For cytotoxic function of T cells, CAR T or mock T cells were stained with APC-conjugated anti-human CD8 and PE-conjugated anti-human granzyme B. All staining was at 1X 10 ⁶ Individual cells were processed in 100. Mu.l FACS buffer (PBS +3% FBS) protected from light at 4 ℃ for one hour for the primary antibody and 30 minutes for the secondary antibody. Cytokines were detected using mixed magnetic beads coated with PE conjugated anti-human IL2, IL4, IL6, IL10, TNF and IFN γ antibodies of different fluorescence intensities. All samples were filtered through a 70. Mu.l cell filter, except for commercial magnetic beads, prior to loading into the flow cytometer. Using BD Accuri ^TM Flow cytometric analysis was performed on C6 or Beckman Cytoflex S.

293T cells transfected with the above three vectors, CD22ABPDR 4 ZS, CD22ABPDR 8 ZS and CD22 ABPDR28 ZS, were tested for expression of CAR, PDR and ZsGreen1, respectively, using Fluorescence Activated Cell Sorting (FACS).

FIGS. 2A-2C show the results of FACS detection of cultured 293T cells after transfection, respectively, and FIG. 2A shows the expression of CAR from 293T cells transfected with the vectors CD22ABPDR 4 ZS, CD22ABPDR 8 ZS and CD22 ABPDR28 ZS, respectively. FACS counts of CAR expressed by 293T cells transfected with the vector CD22ABPDR 4 ZS (referred to as CD22AB CAR) expressed a value of 90.6; FACS counts of CAR expressed by 293T cells transfected with the vector CD22ABPDR 8 ZS (referred to as CD22AB CAR) expressed a value of 72.5; the FACS count of CAR expressed by 293T cells transfected with the vector CD22 ABPDR28 ZS (referred to as CD22AB CAR) represented a value of 87.5.

Shown in figure 2B are the PDR expression of 293T cells transfected with vectors 22ABPDR 4 ZS, 22ABPDR 8 ZS, and 22ABPDR28 ZS, respectively. FACS counts for PDR expressed from 293T cells transfected with the vector CD22ABPDR 4 ZS, indicated a value of 89.6; FACS counts for PDR expressed from 293T cells transfected with the vector CD22ABPDR 8 ZS indicated a value of 58.5; the FACS count of PDR expressed from 293T cells transfected with the vector CD22 ABPDR28 ZS represented a value of 96.9.

The expression of ZsGreen1 by 293T cells transfected with the vectors CD22ABPDR 4 ZS, CD22ABPDR 8 ZS and CD22 ABPDR28 ZS is shown in figure 2C. FACS counts for ZsGreen1 expressed from 293T cells transfected with vector CD22ABPDR 4 ZS indicated a value of 88.0; FACS counts for ZsGreen1 expressed from 293T cells transfected with the vector CD22ABPDR 8 ZS indicated a value of 90.2; the FACS count of ZsGreen1 expressed from 293T cells transfected with vector CD22 ABPDR28 ZS indicated a value of 84.9.

By western blot experiments (fig. 2D), we can see that CD22 ABPDR28 can be highly expressed in 293T cells, which suggests that CD22 ABPDR28 can be applied to later human PBMC cells since 293T is a human cell. Thus, CD28 TM may be effective in facilitating the combined expression provided by the present invention. We used this vector abbreviated CD22 ABPDR28 to complete the rest of the experiments.

Western blotting procedure:

1ug of the above plasmid was transfected into 293T cells in 6-well plates, after 48 hours, the cell layer was washed twice with PBS, 100ul of RIPA per well was added, incubated on ice for 30min, and the lysate was transferred to a 1.5ml centrifuge tube and centrifuged at 12000rpm for 15min. The supernatant was transferred to a new 1.5ml EP tube and stored at-20 ℃ until use.

The protein is measured by BCA assay, which comprises absorbing 5ul protein solution, diluting to 25ul with PBS, adding 200ul BCA working solution, incubating at 37 deg.C for 30min, measuring absorbance at OD562, and calculating protein concentration according to absorbance by standard curve.

Then, 35ug of total protein was aspirated, a volume of SDS loadingbuffer was added, samples were centrifuged at 12000rpm for 5min after heating at 95 ℃ and all samples were loaded into SDSpage wells, electrophoresed at 80V for 30min, then protein samples were separated by electrophoresis at 120V for 45min, and then the samples on the gel were transferred to PVDF membrane under the condition of half-dry transfer to 20v 30min. After the electrotransformation, the membrane is placed in 5% skimmed milk powder and sealed for 1h at room temperature. Incubation with murine anti-human PD-1 antibody (1 500) and murine anti-human β -Actin (1 5000) antibody overnight at 4 ℃, after three PBST washes, incubation for 1h with HRP-anti-mouse Fc (1 5000) at room temperature. PBST was washed three times, developed with Super Signal West Pico chemistry primer Substrate and photographed with BioRad ChemiDoc MP Imaging System.

Example 2

This example constructs T cells co-expressing CAR and PDR with CD28, respectively. The construction of the inserted gene and plasmid is shown in FIG. 3. Four plasmid vectors ZS, PDR, CD22AB and CD22ABPDR were constructed by means of routine techniques in the art. Wherein the vector ZS is a control vector containing nucleic acids encoding IRES and ZsGreen 1; the vector PDR is a control vector containing nucleic acids encoding PD-1 EM and CD28 TM; vector CD22AB is a control vector containing nucleic acids encoding Anti-CD22, CD8 TM, 41-BB, CD3 ζ, IRES, and ZsGreen 1; the carrier CD22ABPDR is identical to the carrier CD22 ABPDR28 ZS in example 1.

The four plasmid vectors are transfected into human peripheral blood lymphocytes to construct CAR T cells.

Transfected human peripheral blood lymphocytes were cultured, harvested, and then subjected to FACS detection and Q-PCR identification.

The preparation method of PBMC cells comprises the following steps: 30-50ml of residual blood in the buffer Coat is transferred into a new 50ml centrifuge tube, diluted to 200ml by PBS, mixed evenly, transferred into 28ml diluted blood sample to the centrifuge tube, added with 21ml Ficoll gently, set the centrifuge speed to increase 3 and decrease 0,400g is centrifuged for 30min. The buffy coat was transferred to a new 50ml centrifuge tube and washed twice with 10 fold PBS and centrifuged at 100g 10min. The PBMCs were then resuspended in 10% DMSO-containing FBS and diluted after counting to a cell concentration of 2X 107/ml and each frozen in a liquid nitrogen tank for use.

CAR T cells were constructed as follows: after recovery of PBMCs, the cells were cultured in IL 2-containing RIPM +10% FBS + P.S. + Glutamine medium, and Dynabeads were added at a rate of 1:1. After 24h, the virus was added to PBMCs containing 5ug/ml polybrene at MOI = 5-10, the cell concentration was controlled at about 2 × 106, the virus was centrifuged at 570g at 32 ℃ for 1h, the virus was repeatedly infected once at 48h, the virus was changed to IL 2-containing RIPM +10 FBS P.S. + Glutamine medium without polybrene after 72h, the beads were removed after one week, and half of the medium was changed every other day until the required amount was reached.

PBMC separation: PBMCs used in the present invention are from healthy donors. PBMC were isolated from the intermediate white layer using Ficoll-Paque PLUS according to the manufacturer's instructions. And then the purified PBMC is obtained through the lysis of the erythrocytes and the removal of the NK cells.

The results of the Q-PCR identification are shown in FIG. 4A, where it can be seen that PDR is highly expressed in human PBMC cells transfected with plasmid CD22ABPDR and plasmid PDR.

After further testing for changes in PD-1 expression, we can see that PD-1 expression is down-regulated day by day after activation, as shown in figure 4B. On day 12, no expression of PD-1 was detected, which means that PD-1 was not expressed when PBMCs were cultured in vitro after 12 days.

Example 3

This example demonstrates the effect of constructed CAR T cells.

(1) A cell line expressing PD-L1 was constructed.

To identify the function of PDR expressed on CAR T cells, tumor cells need to express PD-L1. Since tumor cell lines of Raji, K562, daudi and BV173 did not express endogenous PD-L1, the inventors constructed tumor cell lines of Raji, K562, daudi and BV173 Raji-PD-L1, K562-PD-L1, daudi-PD-L1 and BV-173-PD-L1 expressing PD-L1, respectively, using lentiviruses on the basis of tumor cell lines of Raji, K562, daudi and BV 173. By flow cytometry analysis, all the constructed cell lines were confirmed to stably and highly express PD-L1 (see FIG. 5).

(2) Detecting expression of key activation markers.

The ratios of CD4 and CD8 expressed on human PMBC transfected with the four plasmid vectors ZS, PDR, 971 and 971PDR in example 2 and other activated cell markers were analyzed using flow cytometry.

As can be seen in fig. 6A, the proportions of CD4 and CD8 expressed on human PMBC transfected with plasmid vectors 971 and 971PDR, respectively, were similar. As can be seen from FIG. 6B, although higher CD8 was shown in human PMBC transfected with plasmid vector 971 ⁺ There was no difference in expression of the T cell, but activated cell marker CD 27. However, fig. 6C and 6D show that human PMBC transfected by plasmid 971 had lower CD28 expression and higher CD69 expression than human PMBC transfected by the other three plasmids.

From these results, transfection of plasmid 971 into human PBMCs reduced CD28 expression but increased CD69 expression, while plasmid PDR restored CD28 expression and down-regulated CD69 expression. In conclusion, the introduction of the PDR plasmid did not substantially affect the activation function of human PBMCs.

(3) Effect of PDR on the differentiation function of CD22 CART cells.

PDR expressed in human peripheral blood lymphocytes transfected with vector 971PDR can effectively target PD-L1 expressing tumor cells, and PDR can compete with endogenously expressed PD-1 for binding to PD-L1 on target cells, thereby enhancing activation of CAR T cells. CAR T cells targeted to tumor cells can promote differentiation of T cells into different subsets of T cells.

Human PMBC transfected with the four plasmid vectors ZS, PDR, CD22AB and CD22ABPDR were co-cultured with the tumor cell line BV173-PD-L1 constructed in example 3. After 24 hours, cells were harvested and stained with antibody, and after CD8+ T cells were selected, effector cells CD62L-CD45RA + were analyzed, and it was found that the proportion of effector T cells of human PMBC transfected by plasmid vector CD22AB was significantly higher than that of human PMBC transfected by plasmid vector CD22ABPDR (see fig. 7B). In FIG. 7A, the expression of CD62L + CD45RA + is positive by NaiveT cells, and CD45RA + CD 62L-is effector cells, which shows the differentiation capability of the cells into effector cells, further indicating that PDR can effectively target tumor cells expressing PD-L1, and promote the differentiation of T cells.

This means that PDR expressed on plasmid CD22ABPDR transfected human PBMC cells can effectively target PD-L1 expressed on tumor cells. PDR expressed on plasmid CD22ABPDR transfected human PBMC cells can target PD-L1 on PD-L1 expressing tumor cells, contributing to targeting tumor cells by plasmid CD22ABPDR transfected human PBMC cells to increase the anti-tumor efficacy of human PBMC cells transfected by plasmid CD22ABPDR, as well as accelerating differentiation of human PBMC cells transfected by plasmid CD22ABPDR into terminal effector cells. This result indicates that CAR T cell-expressing PDRs can serve as a secondary target for PD-L1-expressing tumor cells. Thus, T cell activation can be enhanced by CD22ABPDR CAR T cells that target CAR and PD-1 simultaneously. Targeting CAR T cells to tumor cells can improve the anti-tumor efficacy of CD22ABPDR on the one hand, and can accelerate the differentiation of CD22ABPDR CAR T cells into end effector cells on the other hand.

(4) Co-expression of PDR with CAR enhances tumor killing of CAR T cells.

CAR T cells can be directly targeted to antigen-expressing tumor cells by methods not limited to MHC complexes.

The tumor killing effect of human peripheral blood lymphocytes transfected by the vectors ZS, PDR, CD22AB and CD22ABPDR was further examined.

T cells are cultured in RPMI medium +10% fetal calf serum +100 units/ml +1% double antibody human interleukin-2, and tumor cells are cultured in RPMI medium +10% fetal calf serum. Tumor killing effect of each of the autologous peripheral blood lymphocytes was analyzed by using calcein-AM. As can be seen from fig. 8, human peripheral blood lymphocytes transfected by the vector CD22ABPDR have enhanced anti-tumor efficiency against Raji, daudi and BV173 tumor cell lines highly or moderately expressing CD22 antigen compared to human peripheral blood lymphocytes transfected by the vector CD22AB, and K562 does not express CD22. This demonstrates that co-expression of PD-l PDR in CAR T cells enhances the tumor killing effect of CAR T cells.

Cytotoxicity assay methods:

lysis of tumor cells by CAR T cells was analyzed with calcein-AM (Invitrogen, C3100 MP). Target cells were incubated in 10 μm working solution of calcein-AM at a density of 1X 106 cells/ml for 30min at 37 ℃. The cells were then washed three times with complete medium to remove residual calcein-AM and then resuspended at a density of 1 × 105 cells/ml.

Next, the target cells are co-cultured with effector CAR T or mock T cells. Transfer 100. Mu.l per well to a 96-well plate as target. CAR T cells and target cells were added to 96-well plates in triplicate at dilutions from 20/1 to 2.5/1, with a total volume of 200 μ Ι per well. Spontaneous target tumor control wells and maximum release target control wells were set and 100 μ l of medium was added to each well to a total volume of 200 μ l. Then, a set of negative controls was set, and 200. Mu.l of the medium was added as a negative control. The plates were then placed at 37 ℃ and 5% CO ₂ In a humid atmosphere incubator.

5% CO at 37 ℃% ₂ After 3 hours of incubation, 2. Mu.l triton X-100 was added to the positive wells. 5% CO at 37 ℃ ₂ Incubation was continued for 1 hour, and then the plate was centrifuged at 1500rpm for 5 minutes. Next, 100. Mu.l of the cell lysis supernatant was transferred to a white opaque 96-well plate. Fluorescence was measured using a Perkinelmer Multimode Reader at 495/515. For use inThe percentage of tumor cell lysis was calculated by the following formula:

test samples: the number of fluorescein released by the killed tumor; spontaneous sampling: fluorescein naturally released by tumor cells; maximum value of sample: all fluorescein in tumor cells; maximum value of the culture medium: background in culture medium.

(5) Effect of anti-PD-1 and anti-PD-L1 antibodies on tumor killing effect of PDR and CAR co-expressing T cells.

The method for measuring the secretion of the cytokine comprises the following steps:

cytokine release was detected using the BD Cytometric Bead Array (CBA) human Th1/Th2 cytokine kit II. In complete RPMI-1640, 1X 10 ⁶ Effector T cells and 1X 10 ⁵ The individual target cells were co-cultured. After 24 hours, the medium was collected and cytokines were measured.

Specifically, a vial of lyophilized standard beads was added to a 15ml tube along with 2ml of assay buffer to reconstitute the standard to the highest standard. The mixture was pipetted up and down several times. Then, 300. Mu.l of the highest standard solution was transferred to a 1:2 dilution tube containing 300. Mu.l of assay buffer and mixed well. Then, serial dilutions were performed continuously by transferring 300 μ Ι, from 1:2 to 1:4, and so on up to 1. A negative control tube was set up containing 300. Mu.l of assay buffer.

Prior to mixing, each cytokine capture bead suspension was vortexed vigorously and then 100 μ Ι of capture beads were added to a single tube. Vortexed again vigorously to mix the beads well and centrifuged at 200 Xg for 5min. Then, an equal volume of assay buffer was added to resuspend the mixed magnetic beads and 50 μ Ι of mixed magnetic beads were added to 10 new tubes. Then, 50. Mu.l of human cytokine standard dilution or negative control was added to all tubes containing 50. Mu.l of mixed magnetic beads and 50. Mu.l of PE detection reagent. The tubes were incubated and left at room temperature for three hours in the dark. After one wash with wash buffer, the magnetic beads were resuspended in 300 μ l wash buffer for flow cytometry analysis.

Data was analyzed using FlowJo. After selecting PE-conjugated anti-human IL2, IL4, IL6, IL10, TNF and IFN γ antibody coated magnetic beads of different fluorescence intensities at the circle, the average fluorescence intensity of PE was measured by software. And drawing a standard curve by taking MFI as a horizontal coordinate and the concentration of the cytokine as a vertical coordinate.

In complete RPMI-1640, from 1X 10 ⁵ The supernatants of the co-cultures of individual targeted tumor cells were sampled. The mixture of magnetic beads needs to be pre-treated to detect cytokines in FBS-containing media. After transferring and mixing the magnetic beads, the mixture was centrifuged at 200 × g for 5 minutes. The supernatant was discarded and the mixed magnetic beads were resuspended in an equal volume of serum enhancement buffer. The mixture was incubated at room temperature for 30 minutes, protected from light, and centrifuged at 200 Xg for 5 minutes. Then, an equal volume of assay buffer was added to resuspend the mixed magnetic beads.

Samples were divided into triplicates and centrifuged at 1500rpm for five minutes to discard cell debris, and 50 μ Ι of supernatant was transferred to a new tube. Then, 50. Mu.l of the mixed magnetic beads and 50. Mu.l of the PE detection reagent were added to all tubes and incubated for 3 hours at room temperature in the dark. After washing once with the wash buffer, the magnetic beads were resuspended in 300. Mu.l of wash buffer for flow cytometry analysis.

Data was analyzed using FlowJo. After sorting the magnetic beads coated with PE conjugated anti-human IL2, TNF α antibodies of different fluorescence intensities, the average fluorescence intensity of PE was calculated by the software. The concentration of each cytokine released into the supernatant was then calculated using a standard curve.

Human peripheral blood lymphocytes transfected with the vector CD22ABPDR were co-cultured with the tumor cell line BV173-PD-L1 at a ratio of 10.

The co-cultivation conditions include: CD22AB PDR or CD22ABCAR T cells were mixed with tumor cell line BV173 or BV173-PD-L1 at 10:1 in RPMI medium +10% fetal bovine serum +1% double antibody for 24h. The cell culture conditions were: 37 ℃ C. 5% CO ₂ An incubator.

One group of co-culture systems was supplemented with 50nM of anti-human PD-1 antibody. After 24 hours, supernatants were collected to analyze cytokine changes. The results show that addition of anti-PD-1 antibody failed to rescue IL2 and TNF α expression when co-cultured with BV173-PD-L1 (see, FIG. 9A).

In addition, 50nM of anti-human PD-L1 antibody was added to the other set of co-culture systems. After 24 hours, supernatants were collected to analyze cytokine changes. The results show that the addition of anti-PD-L1 antibody rescued the expression of IL2 and TNF α when co-cultured with BV173-PD-L1 (see, FIG. 9B).

This means that inhibition of cytokine IL2 and TNF α expression was induced by PD-L1 expressed on CAR T cells. Thus, the use of anti-PD-L1 antibodies may further promote tumor killing of T cells co-expressing PDR and CAR.

Taken together, by comparing the different functions between CAR and PDR + CAR T cells in co-culture with tumor cell lines with or without PD-L1 expression, many differences were found in cytotoxicity, cell type differentiation, cell marker expression and cytokines.

The results indicate that co-expressed PDRs in CAR T cells can significantly inhibit the secretion of IL2 and TNF α compared to CD22 CAR. Cytokine inhibition was independent of tumor cells with or without PD-L1 expression. When we analyzed PD-L1 expression on CAR T cells, we can see that PD-L1 can be upregulated on CAR T cells with or without PDR expression.

After further using anti-PD-1 and anti-PD-L1 antibodies to block the binding of PD-L1 to its receptor to see if some effect was produced on the cytokine secretion of CAR T cells, we found that only anti-PD-L1 antibodies could block the binding of PD-L1 to PD-1 and CD80 simultaneously, indicating that PD-L1 expressed on CAR T cells could inhibit the function of CAR T cells.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Australian university

<120> preparation method of cell expressing anti-CD22 chimeric antigen receptor and PD-L1 blocking protein, expression vector and

applications of

<160> 4

<170> PatentIn version 3.5

<210> 1

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<213> Artificial sequence

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

20 25 30

Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser

35 40 45

Val Ser Ser Asn Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser

50 55 60

Arg Gly Leu Glu Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr

65 70 75 80

Asn Asp Tyr Ala Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp

85 90 95

Thr Ser Lys Asn Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu

100 105 110

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

115 120 125

Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

145 150 155 160

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

165 170 175

Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Trp Ser Tyr Leu

180 185 190

Asn Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Asn Leu Leu Ile Tyr

195 200 205

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

210 215 220

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu

225 230 235 240

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ile Pro Gln Thr

245 250 255

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser

370 375 380

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

385 390 395 400

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

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

420 425 430

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

435 440 445

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

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

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Leu His Met Gln Ala Leu Pro Pro Arg

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Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu

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Ala Val Leu Gln Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro

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Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val

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Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser

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Glu Ser Phe Val Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr

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Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp

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Cys Arg Phe Arg Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met

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Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly

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Ala Ile Ser Leu Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala

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Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro

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Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln Phe Trp Val Leu Val

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

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Phe Ile Ile Phe Trp Val

210

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atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

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acctgtgcca tcagcggcga tagcgtgtcc agcaatagcg ccgcctggaa ctggatcaga 180

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aacgactacg ccgtgtccgt gaagtcccgg atcaccatca accccgacac cagcaagaac 300

cagttctccc tgcagctgaa cagcgtgacc cccgaggata ccgccgtgta ctactgcgcc 360

agagaagtga ccggcgacct ggaagatgcc ttcgacatct ggggccaggg cacaatggtc 420

accgtgtcta gcggtggcgg tggctcgggc ggtggtgggt cgggtggcgg cggatctgac 480

atccagatga cacagagccc cagctccctg agcgccagcg tgggagacag agtgaccatc 540

acctgtcggg ccagccagac catctggtcc tacctgaact ggtatcagca gcggcctggc 600

aaggccccca acctgctgat ctatgccgcc agctcactgc agagcggcgt gcccagcaga 660

ttttccggca gaggcagcgg caccgacttc accctgacaa tcagttccct gcaggccgag 720

gacttcgcca cctactactg ccagcagagc tacagcatcc cccagacctt cggccagggg 780

accaagctgg aaatcaaaac cacgacgcca gcgccgcgac caccaacacc ggcgcccacc 840

atcgcgtcgc agcccctgtc cctgcgccca gaggcgtgcc ggccagcggc ggggggcgca 900

gtgcacacga gggggctgga cttcgcctgt gatatctaca tctgggcgcc cttggccggg 960

acttgtgggg tccttctcct gtcactggtt atcacccttt actgcaaacg gggcagaaag 1020

aaactcctgt atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa 1080

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ttcagcagga gcgcagacgc ccccgcgtac cagcagggcc agaaccagct ctataacgag 1200

ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 1260

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aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 1380

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<212> DNA

<213> Artificial sequence

<400> 4

ggcgccacca acttctccct gctgaagcag gccggcgacg tggaggagaa ccccggcccc 60

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 120

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 180

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 240

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 300

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 360

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 420

tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 480

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 540

aggccagccg gccagttcca attttgggtg ctggtggtgg ttggtggagt cctggcttgc 600

tatagcttgc tagtaacagt ggcctttatt attttctggg tg 642

Claims

1. A pharmaceutical composition comprising

-an expression vector or a cell,

-soluble monoclonal antibodies, and

-a pharmaceutically acceptable excipient which is capable of forming,

wherein the expression vector is inserted with a first nucleic acid encoding an anti-CD22 chimeric antigen receptor and a second nucleic acid encoding a PD-L1 blocker protein; the PD-L1 blocking protein is PD-L1 which lacks a PD-1 transmembrane region and an intracellular signal region, the amino acid sequence of the anti-CD22 chimeric antigen receptor is shown as SEQ ID NO.1, the amino acid sequence of the PD-L1 blocking protein is shown as SEQ ID NO.2,

the cells were prepared by the following steps: transfecting a host cell with said expression vector to obtain said cell, and

the soluble monoclonal antibody is an anti-human PD-L1 antibody.

2. The pharmaceutical composition of claim 1, wherein the nucleotide sequence of the first nucleic acid is shown as SEQ ID No.3, and the nucleotide sequence of the second nucleic acid is shown as SEQ ID No. 4.

3. The pharmaceutical composition of claim 2, wherein the expression vector is a lentiviral expression vector.

4. The pharmaceutical composition of claim 3, wherein the lentiviral expression vector is a pLVX-IRES-Zsgreen lentiviral expression vector.

5. The pharmaceutical composition of claim 3, wherein the first nucleic acid and the second nucleic acid are both concatemeric with a transmembrane domain on the expression vector.

6. The pharmaceutical composition of claim 5, wherein the transmembrane domain is selected from the group consisting of a T cell receptor subunit, a transmembrane domain of CD4, CD8 or CD 28.

7. The pharmaceutical composition of claim 6, wherein the T cell receptor subunit is an alpha subunit, a beta subunit, or a delta subunit.

8. The pharmaceutical composition of claim 5, further comprising on said expression vector: an extracellular domain and an intracellular domain.

9. The pharmaceutical composition of claim 8, wherein the extracellular domain comprises a hinge; the intracellular domain includes a costimulatory signaling molecule and an intracellular signaling domain.

10. The pharmaceutical composition of claim 9, wherein the hinge is from CD8 a;

the costimulatory signal molecule is selected from the group consisting of human 4-1BB costimulatory signal molecule, CD27, PD1, ICOS, OX40 or B7-H3; the intracellular signaling domain is a human CD3 zeta signaling domain, CD3 gamma, CD5, or CD3 epsilon.

11. The pharmaceutical composition of claim 5, wherein the host cell is a T cell.

12. Use of a pharmaceutical composition according to any one of claims 1-11 for the preparation of a medicament for the treatment of a tumor that is PD-L1 positive or CD22 positive; the tumor is any one of the following tumors: lymphoma, hodgkin's lymphoma, non-hodgkin's lymphoma, leukemia, and multiple myeloma.