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

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 of cells expressing anti-CD22 chimeric antigen receptor and PD-L1 blocking protein Methods, expression vectors and applications

technical field

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

Background technique

In the field of tumor treatment, chimeric antigen receptor T cell therapy has attracted more and more attention. Chimeric antigen receptor T cells (CAR T cells) are T cells that have been genetically engineered to have chimeric antigen receptors for use in immunotherapy.

Chimeric antigen receptor (CAR) is an artificially constructed transmembrane molecule encoded by a fusion gene, which can specifically target T cells to antigens on the surface of cancer cells to eliminate target cancer cells. A CAR consists of an extracellular domain (eg, 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 signal transduction. After the extracellular domain is specifically bound to the antigen, the intracellular domain initiates the signals required for cell activation, thereby promoting the proliferation of T cells and the release of cytokines. The transmembrane domain connects the extracellular and intracellular domains.

First-generation CARs linked scFv to intracellular signaling domains, such as CD3ζ, to induce antigen-specific T cell activation. Subsequent CARs incorporate single (second generation) or multiple (third generation) domains of other co-stimulatory signals, such as CD28, 4-1BB, or OX40, to further enhance and maintain T cell effector functions.

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

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

Chimeric antigen receptor (CAR) T cell therapy has achieved great success in hematological malignancies, but not in solid tumors. Overcoming the immune resistance of solid tumors to CAR T cell therapy has become an urgent need. solved problem.

In view of this, the present invention is proposed.

Contents of the invention

The object of the present invention is to provide a preparation method, expression vector and application of cells expressing anti-CD22 chimeric antigen receptor and PD-L1 blocking protein to solve the above technical problems.

The present invention is achieved like this:

An expression vector, 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;

The PD-L1 blocking protein is PD-L1 lacking the PD-1 transmembrane region and the intracellular signal region, the amino acid sequence of the anti-CD22 chimeric antigen receptor is shown in SEQ ID NO.1, and the PD-L1 blocking protein is The amino acid sequence is shown in SEQ ID NO.2.

CD22 is another B cell-specific antigen with a similar tissue distribution to CD19. This glycoprotein is expressed on the surface of cells of the B-cell lineage. CD22 has been shown to be a promising target in lymphoma and B-cell leukemia. Studies have shown that CART targeting CD22 achieves excellent anti-leukemia efficacy. Therefore, CD22 has potential as a target for CAR-T cell therapy in all patients. It could also provide a second option for patients who have relapsed after receiving CD19-targeted CART therapy.

The inventor constructed the first nucleic acid encoding the anti-CD22 chimeric antigen receptor and the second nucleic acid encoding the PD-L1 blocking protein on the same vector, and simultaneously utilized the CD22 target and the PD-1 target, through the antibody targeting CD22 Or its fragments, the T cells of the present invention can efficiently and specifically target tumor cells expressing the antigen CD22.

PD-L1 blocking protein is only composed of the extracellular domain of PD-1, and PD-L1 blocking protein can compete with endogenously expressed PD-1 to bind to PD-L1 on target cells, thereby blocking PD-1/ PD-L1 signaling pathway. Thus, the problem of immune tolerance of solid tumors to CART cell therapy is alleviated.

The corresponding T cells can be obtained by transfecting the expression vector provided by the present invention, and the T cells can realize the co-expression of anti-CD22 chimeric antigen receptor and PD-L1 blocking protein. After the PD-L1 expressed on the tumor cells is combined with the PD-L1 blocking protein expressed on the surface of the above-mentioned T cells of the present invention, the combination of PD-L1 expressed on the tumor cells and the PD-1 on the surface of the above-mentioned T cells is limited . Thus, the PD-1/PD-L1 signaling pathway is reduced or even blocked. Ultimately, the action time of the above-mentioned T cells of the present invention is prolonged, and the tumor killing effect is improved.

The present invention provides the first nucleic acid encoding the anti-CD22 chimeric antigen receptor. In other embodiments, it is not limited to the CD22 target, and CD19, CD20, CD30, CD123, BCMA, Her2, GPC3 can also be selected according to the targeting requirements , Mesothelin and other targets.

In other embodiments, the CD22-specific binding domain (ie, the first nucleic acid encoding the anti-CD22 chimeric antigen receptor) can 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 application of the present invention, the nucleotide sequence of the above-mentioned first nucleic acid is shown in SEQ ID NO.3, and the nucleotide sequence of the second nucleic acid is shown in SEQ ID NO.4.

In a preferred embodiment of the application of the present invention, the above-mentioned expression vector is a lentiviral expression vector;

Preferably, the lentiviral expression vector is pLVX-IRES-Zsgreen lentiviral expression vector. In other embodiments, the lentiviral expression vector can also be adaptively selected according to needs.

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

Preferably, the transmembrane domain is selected from the transmembrane domain of a T cell receptor subunit, CD4, CD8 or CD28.

The purpose of setting the transmembrane domain is to express and interact on the surface of cells to guide the cellular response of immune cells to predetermined target cells.

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

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

The extracellular domain includes a hinge; the intracellular domain includes a co-stimulatory signal molecule and an intracellular signal transduction domain;

Preferably, the hinge is from CD8α.

The hinge serves to connect the transmembrane domain to the extracellular CD22-specific binding domain. In other embodiments the hinge may also be selected from FcRIIIa receptors.

The co-stimulatory signal molecule is selected from the co-stimulatory signal molecule of human 4-1BB, CD27, PD1, ICOS, OX40 or B7-H3; the intracellular signal transduction domain is the human CD3ζ signal transduction domain.

The intracellular signal transduction domain is responsible for the intracellular signal transduction after the extracellular CD22-specific binding domain binds to the target, causing immune cell activation and immune response. In other embodiments, the intracellular signal transduction domain can also be selected from CD3γ, CD5 or CD3ε.

Costimulatory signaling molecules refer to associated binding partners that specifically bind costimulatory ligands on T cells to mediate costimulatory responses of cells. In other embodiments, the co-stimulatory signal molecule can also be CD27, PD1, ICOS, OX40 or B7-H3.

The expression vector provided by the present invention consists of the first nucleic acid encoding anti-CD22 chimeric antigen receptor (CD22 SCFV), the transmembrane domain of CD8 (CD8 TM), 41-BB, CD3ζ, the second nucleic acid and the CD28 gene in sequence. become.

A method for preparing cells expressing anti-CD22 chimeric antigen receptor and PD-L1 blocking protein, the preparation method comprising: transfecting the above expression vector into host cells; preferably, the host cells are T cells.

In a preferred embodiment of the application of the present invention, the above-mentioned T cells are human peripheral blood mononuclear cells.

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

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

The inventors found that co-cultivating the anti-human PD-L1 antibody with the CAR T cells prepared by the cell preparation method provided by the present invention can rescue the expression of cytokines IL2 and TNFα, and promote the killing of tumor cells by T cells.

It should be noted that the monoclonal antibodies that can actually promote T cells to kill tumor cells are not limited to anti-human PD-L1 antibodies, and in other embodiments, adaptive adjustments can be made as needed.

The expression vector or the cells prepared by the above method can be used in the preparation of drugs for treating tumors.

In a preferred embodiment of the application of the present invention, the above-mentioned tumor is a PD-L1-positive or CD22-positive tumor;

Preferably, the tumor is any one of the following tumors: lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia and multiple myeloma.

In other embodiments, it can also be applied to the treatment of other solid tumors and hematological malignancies.

The present invention has the following beneficial effects:

The present invention constructs an expression vector, which can realize the co-expression of anti-CD22 chimeric antigen receptor and PD-L1 blocking protein, using CD22 target and PD-1 target, through the antibody or its fragment targeting CD22 , the T cells of the present invention can efficiently and specifically target tumor cells expressing the antigen CD22; the PD-L1 blocking protein can compete with endogenously expressed PD-1 to bind to PD-L1 on target cells, thereby blocking PD The -1/PD-L1 signaling pathway prolongs the action time of the above-mentioned T cells of the present invention and improves the tumor killing effect, thus alleviating the problem of immune tolerance of solid tumors to CAR T cell therapy. The cells prepared by the method for preparing cells expressing anti-CD22 chimeric antigen receptor and PD-L1 blocking protein provided by the present invention can be widely used in the preparation of solid tumor therapeutic drugs.

Description of drawings

In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and thus It should be regarded as a limitation on the scope, and those skilled in the art can also obtain other related drawings based on these drawings without creative work.

Figure 1 shows a schematic diagram of the design of the carrier used to construct the CAR PDR T cells of the present invention;

Figure 2A-Figure 2C show the results of expressing CAR, PDR and ZsGreen1 in 293 T cells transfected by vectors CD22ABPDR 4 ZS, CD22ABPDR 8 ZS and CD22ABPDR 28ZS, respectively;

Figure 2D shows the results of Western blot experiments of PDR expressed by 293 T cells transfected with vectors CD22ABPDR 4 ZS, CD22ABPDR 8 ZS and CD22ABPDR 28 ZS, respectively;

Figure 3 shows another design scheme diagram of the carrier used to construct the CAR PDR T cells of the present invention;

Figure 4A shows the results of Q-PCR identification of related protein expression in human peripheral blood lymphocytes transfected by vectors ZS, PDR, CD22AB and CD22ABPDR;

Figure 4B is a graph showing the results of progressive expression of PD-1;

Figure 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 and highly express PD-L1;

Figure 6A shows the ratios of CD4 and CD8 in human peripheral blood lymphocytes transfected with vectors ZS, PDR, CD22AB and CD22ABPDR after CD3 T cells were encircled;

Figure 6B shows the FACS detection results of the activation marker CD27 expressed in human peripheral blood lymphocytes transfected with vectors ZS, PDR, CD22AB and CD22ABPDR;

Figure 6C shows the FACS detection results of the activation marker CD28 expressed in human peripheral blood lymphocytes transfected with vectors ZS, PDR, CD22AB and CD22ABPDR;

Figure 6D shows the FACS detection results of the activation marker CD69 expressed in human peripheral blood lymphocytes transfected with vectors ZS, PDR, CD22AB and CD22ABPDR;

Figure 7A shows the FACS detection of CD45RA and CD62L expression after human peripheral blood lymphocytes transfected by vectors ZS, PDR, CD22AB and CD22ABPDR were co-cultured with tumor cell line BV173-PD-L1 for 24 hours;

Figure 7B shows the different proportions of CD45RA+CD62L- cell types after co-culture of human peripheral blood lymphocytes transfected with vectors ZS, PDR, CD22AB and CD22ABPDR with the tumor cell line BV173-PD-L1 for 24 hours;

Figure 8 shows the lysis of tumor cell lines K562, Raji, Daudi and BV173 by human peripheral blood lymphocytes transfected with vectors ZS, PDR, CD22AB and CD22ABPDR;

Figure 9A shows the effect of anti-PD-1 antibody (anti-PD-1) on the expression of IL2 and TNFα in human peripheral blood lymphocytes transfected by vector CD22ABPDR against the tumor cell line BV173 PD-L1;

Figure 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 TNFα in human peripheral blood lymphocytes transfected with the vector CD22ABPDR.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

The characteristics and performance of the present invention will be described in further detail below in conjunction with the examples.

In order to overcome the immune checkpoint inhibition of CAR T cell therapy, the present invention designs a T cell for CD22 antigen, which co-expresses anti-CD22 chimeric antigen receptor and PD-L1 blocking protein, and the PD-L1 blocking protein is Dominant negative receptor for PD-1. The T cells are referred to as CAR PDR T cells for short. In addition, the tumor killing effect of anti-PD-L1 antibody combined with the CAR PDR T cells of the present application was also studied.

Example 1

In this example, a series of anti-CD22 vectors were constructed 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 contain a CD8α leader sequence, an anti-CD22-specific scFv, a CD8α hinge, and a TM (transmembrane domain). The intracellular domain contains the 4-1BB co-stimulatory domain and the CD3ζ signaling domain, known 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 regions (CD4, CD8, and CD28). Each vector was designated 22ABPDR 4, 22ABPDR 8 and 22ABPDR28, respectively. The construction of the inserted gene and plasmid is shown in Figure 1.

The vector includes the first nucleic acid encoding a chimeric antigen receptor consisting of a single-chain antibody of an anti-CD22 antibody (Anti-CD22), a CD8 transmembrane domain (TM), 41-BB, and CD3ζ, and a chimeric antigen receptor composed of a PD-1 cell Nucleic acid of the PD-1 dominant-negative receptor (PDR) composed of the ectodomain and three different TMs. The vectors are constructed by conventional technical means in the art.

CD22AB scFv was amplified by CD22AB F and CD22AB R in primer Table 1, and the CAR signal transduction region was amplified by signal F and signal R, and then CD22AB scFv and signal transduction region were connected into a complete CAR gene by Overlap PCR.

The PD-L1 blocking protein gene and the transmembrane region of CD4/CD8/CD28 were amplified by PCR, and P2A was introduced into the 5' end of the transmembrane region of CD4/CD8/CD28 by PCR amplification. Then, the transmembrane region and the CAR gene were connected by Overlap PCR, and inserted into the lentiviral vector pLVX-EF1α-IRES-ZsGreen1 at the restriction sites at both ends.

The PCR system is 50ul, including 0.5U Q5 DNA polymerase, 2mM MgCl ₂ , 0.2mM dNTP mixture, 20ng plasmid template, 0.25μM forward primer and 0.25μM reverse primer. The PCR reaction program was: pre-denaturation at 98°C for 30 s, denaturation for 10 s, annealing at 58°C for 20 s, extension at 72°C for 30 cycles, a total of 30 cycles, and finally extension at 72°C for 5 min. The PCR product was electrophoresed in 1% agarose, and the PCR fragment was recovered with an Omega gel extraction kit.

Table 1 Primer sequence list.

CAR F and CAR R amplify the CD22AB CAR sequence, PD-1 EM F and PD-1 EM R amplify the PD-L1 blocking protein, and P2A F is used to insert the PD-1 extracellular sequence at the 5' end of P2A.

The nucleotide sequence of P2A is:

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

CD4/8/28 TM M R is used to construct different transmembrane sequences to the 3' end of PD-1 extracellular sequence, the vector does not contain ZsGreen reporter gene, CD4 TM BR, CD8 TM NR and CD28 TM BR are used to integrate different The transmembrane sequence is constructed to the 3' end of the PD-L1 blocking protein sequence, and the vector contains the ZsGreen reporter gene. The sequences of the transmembrane regions are:

The nucleotide sequence of CD4 TM is:

gccctgattgtgctggggggcgtcgccggcctcctgcttttcattgggctaggcatcttcttc, amino acid sequence of CD4TM: 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 Figure 1 represents the 5' long terminal repeat sequence; LS represents the leader sequence; Anti-22 represents the coding sequence of the single-chain antibody of the anti-CD22 antibody-22AB; CD8 TM represents the coding sequence of the CD8 transmembrane domain; 41- BB and CD3ζ represent the coding sequence of co-stimulatory signal molecule 41-BB and intracellular signal transduction domain CD3ζ, respectively; 2A represents the coding sequence of Thoseaasigna virus 2A peptide; PD-1 EM+CD4 TM, PD-1 EM+CD8 TM and PD-1 EM+CD28 TM represent the coding sequence of the combination of the extracellular domain of PD-1 and the transmembrane domain of CD4, CD8 and CD28, respectively; IRES represents the coding sequence of the internal ribosome entry site; ZsGreen1 represents a Coding sequence for fluorescent protein.

Three plasmid vectors were constructed, plasmid CD22ABPDR 4 ZS, in which the extracellular domain of PD-1 was combined with the CD4 transmembrane domain; plasmid CD22ABPDR 8 ZS, in which the extracellular domain of PD-1 was combined with the CD8 transmembrane domain combination; and the plasmid CD22ABPDR 28 ZS in which the extracellular domain of PD-1 is combined with the CD28 transmembrane domain.

The above three plasmid vectors CD22ABPDR 4 ZS, CD22ABPDR 8 ZS and CD22ABPDR 28 ZS were transfected into 293T cells respectively.

Next, the transfected 293 T cells were cultured and harvested for FACS detection.

FACS detection includes the following steps:

CD22 expression on tumor cell lines was stained using FITC anti-human CD22 antibody. CAR expression in transduced T cells was measured using flow cytometry with CD22-Fc protein and secondary antibody 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/CY7-conjugated anti-human CD8. For T cell activation, stain with APC-conjugated anti-human CD8 and PE-conjugated CD27, PE-conjugated CD28, PE-conjugated CD69, on CAR T cells or control T cells. 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/CY7-conjugated CD62L. For the 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 performed in 100 μl of FACS buffer (PBS + 3% FBS) per 1×10 ⁶ cells at 4° C. in the dark for one hour for the primary antibody and 30 minutes for the secondary antibody. Cytokines were detected using a pool of magnetic beads coated with PE-conjugated anti-human IL2, IL4, IL6, IL10, TNF, and IFNγ antibodies of varying fluorescence intensities. All samples, except for commercially available magnetic beads, were filtered through 70 μl cell strainers before loading into the flow cytometer. Flow cytometric analysis was performed using BD Accuri ^™ C6 or Beckman CytoFLEX S.

Fluorescence-activated cell sorting (FACS) was used to detect the expression of CAR, PDR and ZsGreen1 in 293 T cells transfected by the above three vectors CD22ABPDR 4 ZS, CD22ABPDR 8 ZS and CD22ABPDR 28 ZS, respectively.

Figure 2A-Figure 2C respectively show the results of FACS detection of cultured transfected 293 T cells, and Figure 2A shows the expression of CAR in 293 T cells transfected by vectors CD22ABPDR 4 ZS, CD22ABPDR 8 ZS and CD22ABPDR 28 ZS Case. The FACS count of the CAR expressed by the 293 T cells transfected with the vector CD22ABPDR 4 ZS (called CD22AB CAR) indicated a value of 90.6; the FACS count of the CAR expressed by the 293 T cells transfected with the vector CD22ABPDR 8 ZS (called CD22ABCAR) The indicated value is 72.5; the FACS count of the CAR expressed by the 293 T cells transfected with the vector CD22ABPDR 28 ZS (called CD22AB CAR) indicates that the value is 87.5.

Figure 2B shows the expression of PDR in 293 T cells transfected with vectors 22ABPDR 4 ZS, 22ABPDR 8 ZS and 22ABPDR 28 ZS, respectively. The FACS counting value of the PDR expressed by the 293 T cells transfected with the vector CD22ABPDR 4 ZS was 89.6; the FACS counting value of the PDR expressed by the 293 T cells transfected with the vector CD22ABPDR 8 ZS was 58.5; The FACS counting value of PDR expressed by the transfected 293 T cells was 96.9.

Figure 2C shows the expression of ZsGreen1 in 293 T cells transfected with vectors CD22ABPDR 4 ZS, CD22ABPDR 8 ZS and CD22ABPDR 28 ZS, respectively. The FACS counting value of ZsGreen1 expressed by the 293 T cells transfected with the vector CD22ABPDR 4 ZS was 88.0; the FACS counting value of the ZsGreen1 expressed by the 293 T cells transfected with the vector CD22ABPDR 8 ZS was 90.2; The FACS counting value of ZsGreen1 expressed by the transfected 293 T cells was 84.9.

By Western blot (Fig. 2D), we can see that CD22ABPDR 28 can be highly expressed in 293T cells. Since 293T is a human cell, it can be inferred that CD22ABPDR 28 can be applied to subsequent human PBMC cells. Therefore, CD28TM can effectively promote the combined expression provided by the present invention. We used this vector abbreviated CD22ABPDR28 for the rest of the experiments.

Western blotting steps:

Transfect 1ug of the above plasmid into 293T cells in a 6-well plate. After 48 hours, wash the cell layer twice with PBS, add 100ul RIPA to each well, incubate on ice for 30min, transfer the lysate to a 1.5ml centrifuge tube, 12000rpm Centrifuge for 15 minutes. Transfer the supernatant to a new 1.5ml EP tube and store at -20°C for later use.

The protein was measured by BCA assay method, 5ul of protein solution was diluted to 25ul with PBS, 200ul of BCA working solution was added, and after incubation at 37°C for 30min, the absorbance value was measured at OD562, and then the protein concentration was calculated through the standard curve according to the absorbance value.

Then absorb 35ug of total protein, add a certain volume of SDS loadingbuffer, heat at 95°C for 5min, centrifuge the sample at 12000rpm for 5min, then load all the samples into SDSpage wells, electrophoresis at 80V for 30min, then separate protein samples at 120V for 45min, and then separate the protein samples on the gel The samples on the surface were transferred to PVDF membrane under the conditions of semi-dry transfer 20v 30min. After electroporation, the membrane was blocked in 5% skimmed milk powder at room temperature for 1 h. Incubate overnight at 4°C with mouse anti-human PD-1 antibody (1:500) and mouse anti-human β-Actin (1:5000), wash with PBST three times, and incubate with HRP-anti-mouse Fc (1:5000) at room temperature 1h. After washing with PBST three times, the color was developed with Super Signal West Pico Chemiluminescent Substrate and photographed with BioRadChemiDoc MP Imaging System.

Example 2

In this example, T cells co-expressing CAR and PDR with CD28 were respectively constructed. Refer to Figure 3 for the construction of the inserted gene and plasmid. Four kinds of plasmid vectors ZS, PDR, CD22AB and CD22ABPDR were constructed by conventional technical means in the field. Among them, the vector ZS is a control vector containing nucleic acids encoding IRES and ZsGreen1; the vector PDR is a control vector containing nucleic acids encoding PD-1 EM and CD28 TM; the vector CD22AB is a control vector containing nucleic acids encoding Anti-CD22, CD8 TM, 41-BB, CD3ζ, IRES and ZsGreen1 nucleic acid control vector; and the vector CD22ABPDR is the same as the vector CD22ABPDR 28 ZS in Example 1.

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

The transfected human peripheral blood lymphocytes were cultured and harvested for FACS detection and Q-PCR identification.

The preparation method of PBMC cells is as follows: Transfer 30-50ml of residual blood in Buffy Coat to a new 50ml centrifuge tube, dilute to 200ml with PBS, after mixing, transfer 28ml of diluted blood sample to the centrifuge tube, gently add 21ml of Ficoll, Set the speed of the centrifuge to increase speed 3 and reduce speed 0, centrifuge at 400g for 30min. Transfer the white blood cell layer to a new 50ml centrifuge tube, add 10 times PBS to wash twice, and centrifuge at 100g for 10min. Then PBMCs were resuspended with FBS containing 10% DMSO, counted and diluted to a cell concentration of 2×107/ml, and stored in liquid nitrogen tanks for future use.

The construction of CAR T cells is shown in the following steps: PBMCs are recovered and cultured in a medium containing IL2-containing RIPM+10% FBS+P.S.+Glutamine, and Dynabeads are added at a ratio of 1:1. After 24 hours, add the virus into PBMCs containing 5ug/ml polybrene according to MOI=5~10, control the cell concentration at a final concentration of about 2×106, centrifuge at 570g at 32°C for 1 hour, repeat the infection once in 48 hours, and change the medium after 72 hours until there is no polybrene In the culture medium of RIPM+10%FBS+P.S.+Glutamine containing IL2, the magnetic beads were removed after one week, and half of the medium was changed every other day until the required amount was reached.

Isolation of PBMCs: PBMCs used in the present invention were obtained from healthy donors. PBMCs were isolated from the intermediate buffy coat using Ficoll-Paque PLUS following the manufacturer's instructions. Purified PBMCs were obtained by lysing red blood cells and removing NK cells.

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

After further detecting changes in PD-1 expression, referring to Figure 4B, we could see that PD-1 expression was downregulated day by day after activation. On day 12, the expression of PD-1 was undetectable, which means that PD-1 was not expressed when PBMC were cultured in vitro after 12 days.

Example 3

This example verifies the effect of the constructed CAR T cells.

(1) Construct a cell line expressing PD-L1.

To characterize the function of PDRs expressed on CAR T cells, tumor cells need to express PD-L1. Because the tumor cell lines of Raji, K562, Daudi, and BV173 do not express endogenous PD-L1, the inventors used lentivirus to construct PD-L1-expressing tumor cell lines on the basis of Raji, K562, Daudi, and BV173. Tumor cell lines Raji-PD-L1, K562-PD-L1, Daudi-PD-L1 and BV173-PD-L1. Through flow cytometry analysis, it was determined that all constructed cell lines could stably and highly express PD-L1 (see Figure 5).

(2) Expression detection of key activation markers.

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

It can be seen from FIG. 6A that the ratios of CD4 and CD8 expressed on human PMBCs transfected with plasmid vectors 971 and 971 PDR are similar. It can be seen from FIG. 6B that although human PMBCs transfected with plasmid vector 971 showed higher CD8 ⁺ T cells, there was no difference in the expression of the activated cell marker CD27. However, Figure 6C and Figure 6D show that human PMBC transfected with plasmid 971 has lower CD28 expression and higher CD69 expression than human PMBC transfected with the other three plasmids.

From these results, transfection of plasmid 971 into human PBMCs can reduce the expression of CD28 but increase the expression of CD69, whereas plasmid PDR can restore the expression of CD28 and down-regulate the expression of CD69. In conclusion, the introduction of PDR plasmid basically does not affect the activation function of human PBMC.

(3) The effect of PDR on the differentiation function of CD22 CAR T cells.

The PDR expressed in human peripheral blood lymphocytes transfected with the vector 971PDR can effectively target tumor cells expressing PD-L1, and PDR can compete with endogenously expressed PD-1 to bind to PD-L1 on target cells, thereby Enhances the activation of CAR T cells. CAR T cells targeting tumor cells can promote the differentiation of T cells into different subtypes of T cells.

Human PMBCs transfected by 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, the cells were collected and stained with antibodies. After the CD8+ T cells were circled, the effector cells CD62L-CD45RA+ were analyzed, and it was found that the proportion of effector T cells in human PMBC transfected with the plasmid vector CD22AB was significantly higher than that transfected with the plasmid vector CD22ABPDR The proportion of effector T cells in human PMBC (see Figure 7B). In Figure 7A, CD62L+CD45RA+ expression double-positive is NaiveT cells, and CD45RA+CD62L- is effector cells, indicating the ability of cells to differentiate into effector cells, further indicating that PDR can effectively target tumor cells expressing PD-L1 and promote T cell differentiation.

This means that PDR expressed on human PBMC cells transfected with plasmid CD22ABPDR can effectively target PD-L1 expressed on tumor cells. PDR expressed on human PBMC cells transfected with plasmid CD22ABPDR can target PD-L1 on tumor cells expressing PD-L1, which helps human PBMC cells transfected by plasmid CD22ABPDR to target tumor cells and increase The anti-tumor efficacy of transfected human PBMC cells also accelerated the differentiation of human PBMC cells transfected by the plasmid CD22ABPDR into terminal effector cells. This result suggests that the PDR expressing CAR T cells can serve as a second target for PD-L1-expressing tumor cells. Therefore, T cell activation can be enhanced by CD22ABPDR CAR T cells targeting both CAR and PD-1. Targeting tumor cells by CAR T cells can improve the anti-tumor efficacy of CD22ABPDR on the one hand, and accelerate the differentiation of CD22ABPDR CAR T cells into terminal effector cells on the other hand.

(4) The co-expression of PDR and CAR enhanced the tumor killing effect of CAR T cells.

CAR T cells can directly target antigen-expressing tumor cells through methods not limited to MHC complexes.

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

T cells were cultured in RPMI medium + 10% fetal bovine serum + 100 units/ml + 1% double antibody human interleukin-2, and tumor cells were cultured in RPMI medium + 10% fetal bovine serum. The tumor-killing effect of the respective hot peripheral blood lymphocytes was analyzed by using Calcein-AM. It can be seen from Figure 8 that for the tumor cell lines of Raji, Daudi and BV173 highly or moderately expressing the CD22 antigen, the human peripheral blood lymphocytes transfected by the vector CD22ABPDR have enhanced activity relative to the human peripheral blood lymphocytes transfected by the vector CD22AB Anti-tumor efficiency, K562 does not express CD22. This confirmed that co-expression of PD-1 PDR in CAR T cells enhanced the tumor killing effect of CAR T cells.

Cytotoxicity assay method:

The lytic effect of CAR T cells on tumor cells was analyzed with Calcein-AM (Invitrogen, C3100MP). Incubate target cells in a 10 μM working solution of Calcein-AM at a density of 1 × 106 cells/ml for 30 min at 37°C. 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 were co-cultured with effector CAR T or mock T cells. Transfer 100 μl per well to a 96-well plate as a target. CAR T cells and target cells were added to 96-well plates in triplicate at dilutions ranging from 20/1 to 2.5/1 in a total volume of 200 μl per well. Set up spontaneous target tumor control wells and maximal release target control wells, and add 100 μl of medium to each well to a total volume of 200 μl. Then, set up a set of negative controls, and add 200 μl of medium as negative controls. The plate was then placed in a humidified atmosphere incubator at 37 °C with 5% _CO2 .

After incubation for 3 hours at 37 °C in 5% _CO2 , 2 μl triton X-100 was added to the positive wells. Continue incubation for 1 h at 37 °C in 5% _CO2 , then centrifuge the plate at 1500 rpm for 5 min. Next, transfer 100 μl of cell lysate supernatant to a white opaque 96-well plate. Fluorescence was measured at 495/515 using a PerkinElmer Multimode Reader. Calculate the percentage of tumor cell lysis using the following formula:

Test sample: the value of fluorescein released by killing the tumor; spontaneous sample: the fluorescein naturally released by tumor cells; the maximum value of the sample: all the fluorescein in the tumor cells; the maximum value of the medium: the background in the medium.

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

Assays for cytokine secretion include:

Cytokine release was detected using BD Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit II. In complete RPMI-1640, 1 × ¹⁰⁶ effector T cells were co-cultured with 1 × ¹⁰⁵ target cells. After 24 hours, the medium was collected and cytokines were measured.

Specifically, add a vial of lyophilized standard spheres 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, transfer 300 μl of the highest standard solution to a 1:2 dilution tube containing 300 μl of assay buffer and mix well. Then, serially perform serial dilutions from 1:2 to 1:4 and so on up to 1:256 by transferring 300 μl. Set up a negative control tube that includes 300 μl of assay buffer.

Prior to mixing, each cytokine capture bead suspension was vortexed vigorously, then 100 μl of capture beads were added to individual tubes. Vortex vigorously again to mix the beads well and centrifuge at 200 x g for 5 min. Then, add an equal volume of assay buffer to resuspend the mixed beads and add 50 μl of mixed beads to 10 new tubes. Then, add 50 μl of human cytokine standard dilution or negative control to all tubes containing 50 μl of mixed magnetic beads and 50 μl of PE detection reagent. The tubes were incubated and kept in the dark for three hours at room temperature. After one wash with wash buffer, the beads were resuspended in 300 μl wash buffer for flow cytometry analysis.

Data were analyzed using FlowJo. After ringing PE-conjugated anti-human IL2, IL4, IL6, IL10, TNF and IFNγ antibody-coated magnetic beads with different fluorescence intensities, the average fluorescence intensity of PE was measured by software. The standard curve was drawn with MFI as the horizontal coordinate and cytokine concentration as the vertical coordinate.

Samples were taken from supernatants co-cultured with 1 × ¹⁰⁵ targeted tumor cells in complete RPMI-1640. The bead mixture needs to be pretreated to detect cytokines in FBS-containing media. After transferring and mixing the magnetic beads, the mixture was centrifuged at 200 xg for 5 minutes. Discard the supernatant and resuspend the mixed beads 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, add an equal volume of assay buffer to resuspend the mixed beads.

Samples were split in triplicate and centrifuged at 1500 rpm for five minutes to discard cell debris, and 50 μl of the supernatant was transferred to a new tube. Then, 50 μl of mixed magnetic beads and 50 μl of PE detection reagent were added to all tubes and incubated for 3 hours at room temperature in the dark. After one wash with wash buffer, the beads were resuspended in 300 μl of wash buffer for flow cytometry analysis.

Data were analyzed using FlowJo. After circling the magnetic beads coated with PE-conjugated anti-human IL2, TNFα antibodies with different fluorescence intensities, the average fluorescence intensity of PE was calculated by software. Then, the concentration of each cytokine released into the supernatant was calculated using the standard curve.

Human peripheral blood lymphocytes transfected with the vector CD22ABPDR and the tumor cell line BV173-PD-L1 were co-cultured at a ratio of 10:1.

Co-culture conditions include: CD22AB PDR or CD22ABCAR T cells and tumor cell line BV173 or BV173-PD-L1 were co-cultured in RPMI medium + 10% fetal bovine serum + 1% double antibody for 24 hours at a ratio of 10:1. Cell culture conditions are: 37 °C 5% _CO2 incubator.

One group of co-culture systems was added with 50nM anti-human PD-1 antibody. After 24 hours, the supernatant was collected to analyze the changes of cytokines. The results showed that the addition of anti-PD-1 antibody did not rescue the expression of IL2 and TNFα when co-cultured with BV173-PD-L1 (see, Figure 9A).

In addition, 50 nM anti-human PD-L1 antibody was added in another co-culture system. After 24 hours, the supernatant was collected to analyze the changes of cytokines. The results showed that the addition of anti-PD-L1 antibody rescued the expression of IL2 and TNFα when co-cultured with BV173-PD-L1 (see, Figure 9B).

This means that PD-L1 expressed on CAR T cells induces suppression of the expression of cytokines IL2 and TNFα. Therefore, the use of anti-PD-L1 antibodies can further promote the tumor-killing effect of T cells co-expressing PDR and CAR.

In summary, comparing the different functions between CAR and PDR+CART cells by co-culture with tumor cell lines with or without PD-L1 expression, it was found that in terms of cytotoxicity, cell type differentiation, expression of cell markers and cytokines There are many differences.

The results showed that PDR co-expressed in CAR T cells could significantly inhibit the secretion of IL2 and TNFα compared with CD22 CAR. Cytokine suppression was independent of tumor cells with or without PD-L1 expression. When we analyzed PD-L1 expression on CAR T cells, we could see that PD-L1 could be up-expressed 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 there is some effect on the cytokine secretion of CART cells, we found that only anti-PD-L1 antibodies can simultaneously Blocking the binding of PD-L1 to PD-1 and CD80 suggests that PD-L1 expressed on CAR T cells can inhibit the function of CAR T cells.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

SEQUENCE LISTING

<110> University of Macau

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

application

<160> 4

<170> PatentIn version 3.5

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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

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

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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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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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gtgcacacga gggggctgga cttcgcctgt gatatctaca tctgggcgcc cttggccggg 960

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ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 180

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 240

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Claims (12)

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.

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