CN114277056A

CN114277056A - Preparation method of renal cancer antigen CA9 protein specific cytotoxic T lymphocyte

Info

Publication number: CN114277056A
Application number: CN202111598162.6A
Authority: CN
Inventors: 段训威; 肖桂清; 杨槟煌; 王礼兴; 陈怀宇; 吴文林
Original assignee: Quanzhou Normal University
Current assignee: Quanzhou Normal University
Priority date: 2021-12-24
Filing date: 2021-12-24
Publication date: 2022-04-05

Abstract

The invention provides a preparation method of a renal cancer antigen CA9 protein specific cytotoxic T lymphocyte, belonging to the technical field of immune cell therapy. The method of the invention comprises the following steps: preparing a rAAV2-siSOCS1-CA9 recombinant virus vector carrying target genes of an immune suppression negative control gene siSOCS1 and CA 9; separating and culturing DC cells in mononuclear cells; infecting the recombinant virus vector of rAAV2-siSOCS1-CA9, inducing the DC cell to mature, and obtaining iAPA-CA9-DC cell; the iAPA-CA9-DC cells and T cells are mixed and cultured to obtain iAPA-CA9-DC/CTL cells with antigen specificity, namely, renal carcinoma protein CA9 specific cytotoxic T lymphocytes activated by dendritic cells. The method of the invention cultures and expands a large amount of tumor antigen specific T cells to achieve the purpose of treating cancer.

Description

Preparation method of renal cancer antigen CA9 protein specific cytotoxic T lymphocyte

Technical Field

The invention belongs to the technical field of immune cell therapy, and particularly relates to a preparation method of a renal cancer antigen CA9 protein specific cytotoxic T lymphocyte. The cytotoxic T lymphocyte is activated by dendritic cells modified by rAAV2 carrying an immune negative regulatory gene SOCS1 gene and a carbonic anhydrase IX gene, has CA9 tumor antigen specificity, and can be used for immune cell therapy against renal cell carcinoma.

Background

The most common space occupying lesion in adult kidney, Renal Cell Carcinoma (RCC), accounts for approximately 2% of all tumors and increases worldwide by 1.5-5.9% every year. The first diagnosis of some patients is late stage or has far-end metastasis, and kidney cancer has multiple drug-resistant genes which are not sensitive to radiotherapy and chemotherapy and are worse after recovery, the median survival time is less than 12 months, and the 5-year survival rate is less than 10%. At present, surgical resection is the best treatment method, but the surgical resection of micro and metastatic lesions is difficult to perform, and the recurrence rate of postoperative patients is as high as 20-40%; in addition, some patients are effectively treated by cytokines such as IL-2, IFN-alpha and the like, but the toxic effect of the drugs is very obvious.

The occurrence and development of RCC are related to the functional deficiency of T lymphocytes in patients. Particularly, the humoral immunity and the cellular immunity of the RCC patients are generally low at the late stage, and the RCC cells cannot be recognized and killed by T cells. Because the number of Dendritic Cells (DC) of a tumor-bearing host is small, RCC antigen can not be effectively presented, and the RCC cells secrete cytokines and adhesion molecules such as VEGF, IL-10, TGF-beta, PGE2 and the like, so that the mature differentiation of the dendritic cells is stopped at a juvenile stage or is transformed to other endothelial cells, and the low expression of MHC-II molecules and certain co-stimulatory molecules causes the DC to seriously reduce or even lose the capability of presenting antigen and activating T cells.

At present, in vitro experiments prove that lymphocytes for separating peripheral blood of a patient have good reaction capability on various foreign antigens. Therefore, the adoptive immune cell therapy (DC/CTL) of the tumor is expected to be a better RCC treatment method, the action mechanism is that the DC loads RCC antigen in vitro, activates specific CTL through the interaction of MHC/antigen peptide-TCR, and is returned to an organism to start anti-RCC immune response so as to eliminate RCC. At present, DC is loaded by tumor cell lysate, but the DC has the defects that the types of non-tumor related antigens are multiple, autoimmune diseases are easy to induce, and the immunogenicity is low due to multiple antigens; the other stimulating inducer adopts tumor antigen peptide to sensitize DC, which has good specificity and can be immunized for many times, but has the defects that the antigen peptide is generally expensive in price and short in half-life period, and good CTL can be obtained only by immunization for many times.

As the research on the loaded DC is deepened, researchers find that CTL can be promoted to fully exert the anti-tumor effect by preparing the transgenic DC and inducing the CTL in vitro. Compared with tumor whole antigen and antigen lysate, the DC/CTL prepared by tumor transgenosis has better specificity and stronger anti-tumor immune response; compared with tumor antigen peptide, the tumor transgene can be expressed continuously, and the preparation cost of DC/CTL is effectively reduced. At present, rAAV has the characteristics of strong infection capacity, good safety, low immunogenicity, long-term in vivo expression of foreign genes and the like, is regarded as one of the most promising gene transfer vectors, and is widely applied to gene therapy and vaccine research in the world. The tumor cell targeted therapy technology (ACTL) based on DC is that DC is transduced by rAAV which is reconstructed by gene recombination to carry specific tumor-associated antigenic determinant gene, and CTL which specifically kills tumor cells is stimulated to generate. Research shows that in the aspect of CTL response capacity, rAAV with antigen is superior to protein load, the efficiency of DC transfection is up to more than 90%, and the rAAV has the function of up-regulating DC.

The method for preparing the DC/CTL by the tumor transgenosis is safe and has few side effects, and has incomparable high targeting property for RCC of tiny and metastatic lesions by a traditional means. However, in practice, the antigen-loaded DC/CTL alone often fails to elicit a sufficiently strong anti-tumor immune response, and thus the therapeutic effect is not as desirable as expected. This is because in order to avoid the attack of the host immune system, tumors develop various escape mechanisms, and in the antigen presentation stage, factors such as low immunogenicity of tumor antigens, reduced expression of MHC molecules, deletion of coordinative stimulatory molecules, secretion of suppressive cytokines, and the like may exist. Therefore, certain methods must be used to modify and treat DCs to enhance their antigen presentation and T cell immune priming capabilities.

Cytokine signaling inhibitory factor-1 (SOCS 1) is a negative regulator of JAK-STAT signaling pathway, negatively regulates cytokine signaling pathway and TLR signaling pathway, and can inhibit intracellular signaling of IL, IFN and other factors. By silencing or interfering withSOCS1The expression of the gene can promote DC maturation and change the tumor tolerance state. Chen et al propose enhancing DC function by inhibiting the inhibition of immune-presentation attutors (iAPA). The iAPA is a novel tumor immune cell treatment technology, and the core is that small interfering ribonucleic acid (siRNA) is adopted to specifically block key negative regulatory factors in DC, so that the gene expression of the iAPA is silenced, an organism is induced to break through the immune tolerance of the iAPA to the tumor, the antigen specific immune response is enhanced, and the clinical curative effect of tumor immunotherapy is further improved.

In addition, carbonic anhydrase IX (CAIX or CA 9) is a glycoprotein distributed on cell and nuclear membranes. Oosterwi JK et al first obtained murine CA9 monoclonal antibody, and immunohistochemistry was performed on most types of RCC using monoclonal antibody, with 88% of the antigen expressed and most tissues not. The gene is located at 9p12-13, 10898bp in total, is homologous with MN and G250 genes, has the same gene conformation, expresses proteins distributed on cell membranes and nuclear membranes, a mature body is glycoprotein encoded by 459 amino acids, wherein 38 amino acids (aal-37) form an N-terminal Signal Peptide (SP), 377 amino acids (aa 38-414) form an extracellular region, 20 amino acids (aa 415-434) form a transmembrane region, and 25 amino acids (aa 435-459) form the C-terminal of a cell. The extracellular region is divided into two parts: a proteoglycan domain (PG) and a carbonic anhydrase domain (CA). Wherein the CA region is located in the cell membrane and comprises 257 amino acids (aal 35-391), and 59 amino acids (aa 53-111) between SP and CA have 38% homology with the human keratin adhesion region. Therefore, the tumor associated antigen CA9 can be used as an RCC tumor marker.

Based on the technical research results, the method for killing RCC cells by immunity is a very promising therapeutic means, and the key point is the selection of DC stimulating antigens and the optimization of the method, so that the DC is enhanced, and T cell specific immunity is started. The ACTL technology endows the DC/CTL with high targeting property, the iAPA technology can effectively solve the self tolerance problem of an organism and obviously improve the antigen presenting capability of the DC, and if the ACTL technology and the iAPA technology can be jointly used, the antigen specific immune response of the DC/CTL is expected to be obviously enhanced. In the research, rAAV2 is adopted to mediate RNAi to down-regulate SOCS1 gene expression, and the combination of CA9 and S-Flagellin stimulates DC cells to mature, induces specific DC/CTL to specifically kill anti-CA 9 positive RCC. The significance lies in that the targeting property of CTL to RCC is effectively enhanced, the damage to autologous cells is avoided, and clinically, the iAPA technology is applied, so that the growth of tumors can be effectively inhibited, the cells are prevented from being transferred remotely, and the life cycle of a patient is prolonged.

Disclosure of Invention

The invention aims to provide a preparation method of a renal cancer antigen CA9 protein specific cytotoxic T lymphocyte aiming at the defects of the prior art. Under the premise of collecting a small amount of peripheral blood of a patient, the invention cultures and expands a large amount of tumor antigen specific T cells to specifically kill the tumor cells.

In order to achieve the purpose, the invention adopts the following technical scheme:

a preparation method of a renal cancer antigen CA9 protein specific cytotoxic T lymphocyte comprises the following steps:

(1) preparing a rAAV2-siSOCS1-CA9 recombinant virus vector carrying siSOCS1 and CA9 target genes;

(2) infecting, differentiating and inducing DC cells to mature by the rAAV2-siSOCS1-CA9 recombinant virus vector to obtain iAPA-CA9-DC cells;

(3) the iAPA-CA9-DC cells and T cells are mixed and cultured to obtain iAPA-CA9-DC/CTL cells, namely the dendritic cell activated CA9 tumor antigen specific cytotoxic T lymphocytes.

The target gene sequences carrying the target siSOCS1 and CA9 in the step (1) are sequentially as follows: a U6 promoter sequence, a SOCS1 siRNA sequence, a SOCS1 siRNA reverse sequence, a CMV promoter sequence, a CA9 protein gene sequence, an IRES sequence, a Flagellin Flagellin gene sequence; the sequence of the SOCS1 siRNA is shown as SEQ ID NO.1, the reverse sequence of SOCS1 siRNA is shown as SEQ ID NO.2, the sequence of SOCS1 siRNA and the reverse sequence of SOCS1 siRNA are complementary in reverse direction, and both ends of a gene segment are respectively provided with BamH 1/HindIII enzyme cutting sites; the CA9 protein gene sequence, the IRES sequence and the Flagellin Flagellin gene sequence are respectively shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5; the target gene is assembled with an adenovirus vector to obtain the rAAV2-siSOCS1-CA9 recombinant virus vector.

Infecting, differentiating and inducing mature DC cells by the rAAV2-siSOCS1-CA9 recombinant virus vector in the step (2) to obtain iAPA-CA9-DC cells; the method specifically comprises the following steps:

1) according to whole blood: physiological saline: the lymphocyte separation liquid is in a ratio of 1:1:1, the centrifugal force is 650g, the centrifugal time is 15min, the speed of rise is 4, the speed of fall is 0, a mononuclear cell layer above the lymphocyte separation liquid is extracted after centrifugation, and washing is carried out twice;

2) adjustment of monocyte density to 5X 10⁶cell/mL, as per 10⁶ cell/cm²The cell number of (2) was added to a culture flask at 37 ℃ with 5% CO₂Culturing for 2-4h in a concentration incubator, slightly shaking the culture bottle, shaking the non-adherent cells, sucking away the upper non-adherent cells, centrifugally collecting, and freezing;

3) the original flask was supplemented with Lymphocyte serum-free medium KBM581 (KBM 581) medium containing 2 IU/mL of DC cell culture factor (Novoprotein), and incubated at 37 ℃ with 5% CO₂Culturing in a concentration incubator;

4) after overnight culture, gently shaking the culture bottle at the 2 nd day, shaking the non-adherent cells, sucking away the upper layer of the non-adherent cells, centrifugally collecting, and freezing again;

5) adding the 581 culture medium containing the DC cell culture factor into the DC cell culture bottle again, and continuously culturing for 3 d; culturing to 5d, completely replacing the culture medium with KBM581 culture medium containing 2 IU/mL DC cell maturation factor, and continuing culturing for 48 h; collecting suspension cells, calculating the total amount of the cells, infecting by using the rAAV2-siSOCS1-CA9 recombinant virus vector according to MOI 100, adding the rAAV2-siSOCS1-CA9 recombinant virus vector to infect for 2h, changing the solution, and continuously culturing for 48h to obtain the iAPA-CA9-DC cells.

In the step (3), the specific steps of mixed culture of the iAPA-CA9-DC cells and the T cells are as follows:

1) adding stimulating factor CD₃ Monoclonal antibody 50 ng/ml, CD ₂₈50 ng/mL of monoclonal antibody and IL-21000U/mL of monoclonal antibody stimulate the proliferation of T cells;

2) after the T cells are cultured for 48h by stimulating proliferation, iAPA-CA9-DC cells and the T cells are mixed and cultured according to the proportion of 1:5, and the cell density is adjusted to 1 multiplied by 10⁶cell/mL, adding KBM581 culture medium containing IL 2; iAPA-CA9-DC/CTL cells were obtained.

A cancer therapeutic agent comprising the above-mentioned renal cancer antigen CA9 protein-specific cytotoxic T lymphocyte.

The application of the kidney cancer antigen CA9 protein specific cytotoxic T lymphocyte in cancer treatment.

The application of the cancer treatment drug containing the kidney cancer antigen CA9 protein specific cytotoxic T lymphocyte in cancer treatment.

Advantageous effects

The DC cell is infected by rAAV2-siSOCS1-CA9 recombinant virus vector with siSOCS1 and CA9 target genes, the DC cell is subjected to gene modification to obtain a renal cancer antigen CA9 protein specific DC cell, the CA9 specificity is transferred to the T cell by the DC iAPA-CA9-DC cell of the in vitro antigen presenting cell, and a large number of CA9 specific cytotoxic T lymphocyte iAPA-CA9-DC/CTL cells are obtained through amplification, so that the aim of treating cancer is fulfilled.

Drawings

FIG. 1 shows the construction of rAAV2-siSOCS1-CA 9.

FIG. 2 flow cytometry detected the expression of CA9 in transfected DCs. A. A control group; B. rAAV2-siSOCS1-CA9 transfection group.

Figure 3 flow cytometry detected hla.a 2402/CA9 tetramer specificity of CTLs. A. A control group; B. rAAV2-siSOCS1-CA9 transfection group

FIG. 4. iAPA-CA9-DC/CTL cell proliferation potency was compared with that of T cell.

FIG. 5. iAPA-CA9-DC/CTL cell and T cell phenotypic changes. A. CD (compact disc)₃ ⁺/CD₄ ⁺；B. CD₃ ⁺/CD₈ ⁺；C. CD₃ ⁺/CD₅₆ ⁺. *P < 0.05, **P < 0.01 vs T cells。

Detailed Description

In order to explain the technical contents of the invention, the objects and effects achieved by the invention in detail, the following description is given with reference to the embodiments.

Example 1 preparation of Kidney cancer antigen CA9 protein-specific cytotoxic T lymphocytes

The preparation method of the kidney cancer antigen CA9 protein specific cytotoxic T lymphocyte comprises the following steps:

(1) preparing rAAV2-siSOCS1-CA9 recombinant virus vector carrying siSOCS1 and CA9 target genes:

the target gene sequence in the recombinant virus vector is as follows: u6 promoter-SOCS 1 siRNA and SOCS1 siRNA reverse sequence-CMV promoter-CA 9 gene-IRES sequence-Flagellin Flagellin gene sequence, rAAV2-siSOCS1-CA9 construction scheme as shown in figure 1. The sequence of the SOCS1 siRNA is shown as SEQ ID NO.1, the reverse sequence of SOCS1 siRNA is shown as SEQ ID NO.2, the sequence of SOCS1 siRNA and the reverse sequence of SOCS1 siRNA are complementary in reverse direction, and both ends of a gene segment are respectively provided with BamH 1/HindIII enzyme cutting sites; the CA9 protein gene sequence, the IRES sequence and the Flagellin Flagellin gene sequence are respectively shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5; the target gene is assembled with an adeno-associated virus vector type 2 (AAV 2) to obtain the rAAV2-siSOCS1-CA9 recombinant virus vector. The assembly of the target gene and the adeno-associated virus vector is completed by Hemanshun Biotechnology Ltd.

(2) Isolation, differentiation, maturation and infection of DC cells

Mixing whole blood, physiological saline and lymphocyte separation solution at a ratio of 1:1:1, centrifuging at a centrifugal force of 650g for 15min, and increasing and decreasing at a speed of 4 and 0. After centrifugation, the mononuclear cell layer above the lymphocyte separation solution is extracted and washed twice.

Differentiation and Induction of DC cells and preparation of iAPA-CA 9-DC: adjustment of monocyte density to 5X 10⁶cell/ml according to 10⁶ cell/cm²The cell number of (2) was added to a culture flask at 37 ℃ with 5% CO₂Culturing for 2h in a concentration incubator, slightly shaking the culture bottle, shaking the non-adherent cells, sucking away the upper layer of the non-adherent cells, centrifugally collecting, and freezing. KBM581 medium containing 2 IU/mL IL-2, 50 ng/mL rmGM-CSF, and 25 ng/mL rmIL-4 was added to the original flask at 37 deg.C with 5% CO₂Culturing in a concentration incubator. After overnight culture, gently shaking the culture bottle at 2d, shaking the non-adherent cells, sucking away the upper layer of non-adherent cells, centrifugally collecting, and freezing again. Adding KBM581 culture medium containing 2 IU/mL IL-2, 50 ng/mL rmGM-CSF and 25 ng/mL rmIL-4 into the DC cell culture bottle again, and continuing to culture for 3 d; when the culture reached 5d, the medium was completely changed to KBM581 medium containing 1.2. mu.g/mL of LPS, and the culture was continued for 48 hours. Collecting suspension cells, calculating the total amount of the cells, infecting rAAV2-siSOCS1-CA9 recombinant virus vectors according to MOI 100, adding rAAV2-siSOCS1-CA9 recombinant virus vectors, infecting for 2h, changing into fresh KBM581 culture medium containing 2 IU/mL IL-2, 50 ng/mL rmGM-CSF and 25 ng/mL rmIL-4, continuously culturing for 48h to obtain iAPA-CA9-DC cells, and identifying the recombinant virus infection condition of rAAV2-siSOCS1-CA9 by flow. The results are shown in FIG. 2, and the results in FIG. 2 show that the rAAV2-siSOCS1-CA9 recombinant virus-infected DC cell CA9 is successfully expressed compared with the control group DC cell which is not infected with the rAAV2-siSOCS1-CA9 recombinant virus. The results in FIG. 2 show that the rAAV2-siSOCS1-CA9 recombinant viral vector successfully transfects DC cells, and the iAPA-CA9-DC ratio is as high as 83.4% (FIG. 2B).

(3) Preparation of renal carcinoma antigen CA9 protein specific cytotoxic T lymphocyte.

Adding stimulating factor CD₃ Monoclonal antibody 50 ng/ml, CD ₂₈50 ng/mL of monoclonal antibody and IL-21000U/mL of monoclonal antibody stimulate the proliferation of T cells. After the T cells are stimulated and cultured for 48h, the iAPA-CA9-DC cells and the T cells are harvested and mixed and cultured according to the proportion of 1:5, and the cell density is adjusted to be 1 multiplied by 10⁶cell/mL, 1000U/mLIL2 in KBM581, iAPA-CA9-DC/CTL cells were obtained. And (4) performing fluid infusion operation according to the cell growth condition, and periodically sampling and tracking the cell proliferation condition, the survival rate and the phenotype.

The results in fig. 3 show that the positive rate of hla. a. x 2402/CA9 tetramers of the T cells obtained by iAPA-CA9-DC cell stimulation, i.e., iAPA-CA9-DC/CTL cells (fig. 3B), was significantly different from the control group (fig. 3A).

FIG. 4 iAPA-CA9-DC/CTL comparison with T cell proliferation potency. The result shows that the proliferation capacity of the iAPA-CA9-DC/CTL cell is obviously higher than that of the T cell.

The results in FIG. 5 show that compared with the killing activity of T cells on target cells CA9-293FT, iAPA-CA9-DC/CTL cells have significantly enhanced specific cell killing toxicity of iAPA-CA9-DC/CTL cells on target cells CA9-293FT compared with non-specific CTL in a control group.

SEQUENCE LISTING

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<120> preparation method of renal cancer antigen CA9 protein specific cytotoxic T lymphocyte

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Claims

1. The preparation method of the kidney cancer antigen CA9 protein specific cytotoxic T lymphocyte is characterized by comprising the following steps:

(3) the iAPA-CA9-DC cell and the T cell are mixed and cultured to obtain the iAPA-CA9-DC/CTL cell, namely the kidney cancer antigen CA9 protein specific cytotoxic T lymphocyte.

2. The culture method according to claim 1, wherein: the target gene sequences carrying the target siSOCS1 and CA9 in the step (1) are sequentially as follows: a U6 promoter sequence, a SOCS1 siRNA sequence, a SOCS1 siRNA reverse sequence, a CMV promoter sequence, a CA9 protein gene sequence, an IRES sequence, a Flagellin Flagellin gene sequence; the sequence of the SOCS1 siRNA is shown as SEQ ID NO.1, the reverse sequence of the SOCS1 siRNA is shown as SEQ ID NO.2, the sequence of the SOCS1 siRNA and the reverse sequence of the SOCS1 siRNA are reversely complementary; the target gene is assembled with AAV2 as the adeno-associated virus 2 vector to obtain rAAV2-siSOCS1-CA9 recombinant virus vector.

3. The method of claim 1, wherein: in the step (2), the rAAV2-siSOCS1-CA9 recombinant virus vector is used for infecting, differentiating and inducing the DC cells to mature, and the rAAV2-siSOCS1-CA9 recombinant virus vector is used for infecting the DC cells according to MOI 100.

4. The method of claim 1, wherein: in the step (3), the mixed culture of the iAPA-CA9-DC cells and the T cells comprises the following specific steps:

1) adding stimulating factor CD₃Monoclonal antibody 50 ng/ml, CD₂₈50 ng/mL of monoclonal antibody and IL-21000U/mL of monoclonal antibody stimulate the proliferation of T cells;

2) after the T cells are cultured for 48h by stimulating proliferation, iAPA-CA9-DC cells and the T cells are mixed and cultured according to the proportion of 1:5, and the cell density is adjusted to 1 multiplied by 10⁶cell/mL, KBM581 media supplemented with 1000U/mL IL 2; iAPA-CA9-DC/CTL cells were obtained.

5. A renal cancer antigen CA9 protein-specific cytotoxic T lymphocyte prepared by the method of claim 1.

6. A cancer therapeutic agent comprising the renal cancer antigen CA9 protein-specific cytotoxic T lymphocyte of claim 5.

7. The renal cancer antigen CA9 protein-specific cytotoxic T lymphocyte of claim 5, for use in biological fields of non-disease diagnosis and treatment.

8. The use of the cancer therapeutic agent comprising the renal cancer antigen CA9 protein-specific cytotoxic T lymphocyte of claim 6 in the fields of non-disease diagnosis and therapeutic biology.