CN107858352B - shRNA-cluster sequence, expression vector and application thereof - Google Patents

Abstract

The invention provides a shRNA-cluster sequence, an expression vector expressing the shRNA-cluster sequence and CAR molecules, and applications of the shRNA-cluster sequence and the CAR molecules. The invention down-regulates the expression of three receptors of PD-1, Tim-3 and Lag-3 or down-regulates the expression of PD-1 receptor through the sequence specificity of miRNA-cluster source. The invention utilizes the T cell modified by the CAR molecule and inhibits the expression of a plurality of inhibitory receptors on the surface of the T cell, thereby enhancing the inhibitory property of the T cell for antagonizing the microenvironment of the solid tumor, further powerfully mediating the killing of the tumor and being better used for the immunotherapy against the solid tumor.

Description

shRNA-cluster sequence, expression vector and application thereof

Technical Field

The invention relates to a shRNA-cluster sequence, an expression vector and application thereof.

Background

Since the CAR-T immunization technology achieved a result in 2011 in clinical trials of acute lymphoid leukemia against CD19 antigen and was selected by the Science journal in 2013 as the first ten technological breakthroughs in the year, the technology achieved a consistent high ranking from the scientific world to the financial world.

More than 300 million new tumor patients diagnosed every year in China, the probability of postoperative recurrence of most tumors exceeds 50%, the median survival time is less than 5 years, and the current traditional treatment means has important significance in treating most refractory and recurrent tumors and developing CAR-T cells which can specifically infiltrate and effectively antagonize the microenvironment of solid tumors so as to mediate effective killing of the solid tumors. Compared with the fruitful results of CAR-T cells in blood type tumors, the CAR-T cells have great individual difference in phase I clinical tests aiming at solid tumors, and only a few successful cases exist, so that the key for overcoming the infiltration of CAR-T to the solid tumors and avoiding premature failure is to overcome the immunotherapy of the solid tumor cells.

Based on the development of immunotherapy for solid tumors, scientists developed a large number of monoclonal drugs capable of specifically recognizing surface antigens of solid tumors, and these protein drugs provided support for the development of CAR-T immunocyte therapies for solid tumors, such as the monoclonal drugs trastuzumab (herceptin) for treating Her-2 positive colon cancer and ovarian cancer, which were developed into CAR-T immunocytes capable of specifically recognizing and killing Her-2 positive tumors since 2010, and nimotuzumab, cetuximab and panitumumab for treating various tumors that are positive for EGFR, which were developed into CAR-T immunocytes with related scfvs as membrane ectodomains, and developed a large number of clinical trials. However, most clinical trials for solid tumors are not ideal except for a few with good prognostic outcome, and there is a wide debate on using CAR-T immune cell therapy to eliminate solid tumors in patients. In addition to lymphoma, in a clinical trial using CAR-T cells specific for theIL13R α 2 antigen in a patient treated with a 50 year old brain glioma, the clinician eventually observed the disappearance of the tumor after a total of 16 cell reinfusions into the tumor site and cranium of the patient over 220 days. Clinical trials of current CAR-T immune cell therapy in the removal of solid tumors such as pancreatic cancer, prostate cancer, etc. have demonstrated the safety of relevant CAR-T cells and limited killing of tumor cells by several groups. For this reason, the inhibitory property of the tumor microenvironment makes CAR-T cells unable to inhibit and kill tumor cells as soluble monoclonal antibody drugs, so the focus of CAR-T technology development for solid tumors should be focused on enhancing the infiltration capacity of CAR-T cells for solid tumors and the tolerance capacity to hypoxic conditions in the tumor microenvironment. Therefore, the design of the next generation CAR-T immune cell technology which can fully infiltrate the tumor microenvironment, maintain self-renewal for a long time and simultaneously tolerate a hypoxic environment, further enhance the killing of solid tumors and continuously have the function of immune monitoring in vivo is the key for attacking various solid tumors by utilizing the immune cell therapy.

Disclosure of Invention

The invention aims to provide a shRNA-cluster sequence, an expression vector for expressing the shRNA-cluster sequence and a CAR molecule, and application of the shRNA-cluster sequence and the CAR molecule.

In order to achieve the purpose, the invention adopts the technical scheme that: an shRNA-cluster sequence, the nucleotide sequence of the shRNA-cluster sequence is shown as SEQ ID NO. 1 or SEQ ID NO. 2. The invention down-regulates the expression of three receptors of PD-1, Tim-3 and Lag-3 or down-regulates the expression of PD-1 receptor through the sequence specificity of miRNA-cluster source.

The invention provides an expression vector, which is obtained by connecting a sequence shown as SEQ ID NO. 1 or SEQ ID NO. 2 and a coding sequence of a CAR molecule to a basic vector; the expression vector expresses the shRNA-cluster sequence and the CAR molecule. The expression vector obtained by connecting the sequence shown as SEQ ID NO. 1 and the coding sequence of the CAR molecule to a basic vector is called PTL-CAR molecule; the expression vector obtained by connecting the sequence shown as SEQ ID NO. 2 and the coding sequence of the CAR molecule to a basic vector is called a P-CAR molecule, the sequence shown as SEQ ID NO. 1 or the sequence shown as SEQ ID NO. 2 and the CAR molecule are driven by two different promoters respectively, and the CAR molecule can specifically recognize a targeted antigen. The structure of the P-CAR molecule is shown in the upper panel of figure 1; the structure of the PTL-CAR molecule is shown in the lower panel of FIG. 1.

Preferably, the base vector is a lentiviral vector.

The invention provides a modified CAR-T cell, wherein the T cell is obtained by transferring the expression vector into a CD8+ T cell. PTL-CAR molecule engineered CD8+ T cells are referred to as PTL-CAR-T cells, and P-CAR molecule engineered CD8+ T cells are referred to as P-CAR-T cells. After in vitro long-term culture, the effects of killing and secreting cytokines of the PTL-CAR modified CD8+ T cell mediated expression Her2 cell line SK-OV3 are more obvious.

The invention provides a preparation method of the modified CAR-T cell, which comprises the following steps:

(1) collecting peripheral blood mononuclear cells and separating CD8+ T cells enriched in the peripheral blood mononuclear cells, and activating the CD8+ T cells by using anti-CD3, anti-CD28 and IL-2;

(2) CD8+ T cells were activated 48 hours later at 1 ml/1X 10⁶Adding the slow virus expression vector ofclaim 3 into the proportion of the cells for infection, simultaneously adding polybrene solution, and continuing to culture after centrifugation; and after 8-12 hours, performing a second round of infection to obtain the modified CAR-T cell.

Preferably, the anti-CD3 is used at a concentration of 1 μ g/mL, the anti-CD28 is used at a concentration of 1 μ g/mL, and the IL-2 is used at a concentration of 10 ng/mL.

Preferably, the concentration of the polybrene solution is 8. mu.g/mL.

The invention provides a method of expanding the engineered CAR-T cell of claim 4, comprising the steps of: (1) suspending the engineered CAR-T cells in fresh medium and adding IL-2 and IL-7 to maintain the cellular state; (2) ondays 3 and 5 after transformation, complete RMPI1640 medium was added to the cells to maintain the cell concentration at2X 10 according to the cell state and proliferation⁶Ml, supplemented with IL-2 and IL-7, continued culture and timely passage, further expansion of cells.

Preferably, the concentration of IL-2 is 10ng/mL and the concentration of IL-7 is 10 ng/mL.

The invention provides application of the shRNA-cluster sequence in preparation of a preparation for removing solid tumor cells.

The invention provides the use of an expression vector as described above or an engineered CAR-T cell as claimed in claim 4 in the preparation of a formulation for the elimination of solid tumour cells.

The invention provides a method for improving the function of a CAR-T cell, wherein the method is used for down-regulating the expression of three receptors of PD-1, Tim-3 and Lag-3 of the CAR-T cell or the expression of the PD-1 receptor of the CAR-T cell through shRNA.

The invention provides application of PD-1 receptor or combination of three receptors of PD-1, Tim-3 and Lag-3 as a target point in improving CAR-T cell function.

The invention provides application of shRNA (short hairpin ribonucleic acid) which can down regulate three receptors of PD-1, Tim-3 and Lang-3 of CAR-T cells or down regulate shRNA expressed by PD-1 receptors of CAR-T cells in improving the functions of the CAR-T cells.

The invention has the beneficial effects that:

compared with the reported CAR-T cell, the PTL-CAR-T cell-treated mouse tumor size of the completely modified PTL-CAR-T cell-treated mouse is obviously smaller than that of the CAR-T cell, and the survival rate of the PTL-CAR-T cell-treated mouse is obviously better than that of the CAR-T cell. PTL-CAR-T cells can be specifically activated and secrete cytotoxicity-related cytokines IFN-gamma in large quantities to strongly mediate tumor necrosis.

Compared with the reported CAR-T cells, PTL-CAR-T cells mediate the necrosis effect of a solid tumor expressing CD19 positivity in NSG mice treating CD19 positive Raji cell-loaded tumors, and the size of the tumor of the mice in a related treatment group is obviously smaller than that of the CAR-T cells.

Drawings

FIG. 1 is a schematic structural diagram of the constructed PTL-CAR molecule.

FIG. 2 is a schematic diagram of the structure of pLKO-IRES-GFP lentiviral vector.

FIG. 3 is a graph of PTL-Her2-CAR inhibiting expression of inhibitory receptors on CD8+ CAR-T cells cultured in vitro for long periods of time.

FIG. 4 is a comparison of IFN- γ secretion from PTL-Her2-CAR and P-Her2-CAR engineered CD8+ T cells mixed with a Her2 positive target cell line, respectively.

FIG. 5 is a comparison of killing effect of PTL-Her2-CAR and P-CAR engineered CD8+ T cells mixed with a Her2 positive target cell line, respectively.

FIG. 6 shows the size of SK-OV3 tumor bearing mice and mouse survival following treatment with PTL-Her2-CAR-T and Her2-CAR engineered CD8+ T cells.

FIG. 7 is the capacity of infiltrating CD8+ T cells and releasing IFN- γ in SK-OV3 tumor-bearing mice following treatment with PTL-Her2-CAR-T and Her2-CAR engineered CD8+ T cells.

FIG. 8 shows normal organ-infiltrating immune cells of SK-OV3 tumor-bearing mice after treatment with PTL-Her2-CAR-T and Her2-CAR engineered CD8+ T cells.

FIG. 9 is a change in size of Raji tumor-bearing mouse tumors following PTL-CD19-CAR-T and CD19-CAR engineered CD8+ T cell therapy.

Detailed Description

The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Example 1

An shRNA-cluster sequence co-transcribed with CAR molecule, a technology for down-regulating the expression of three receptors of PD-1, Tim-3 and Lag-3 or down-regulating the expression of PD-1 receptor by miRNA-cluster derived sequence specificity, wherein the nucleic acid nucleotide sequence is shown as SEQ ID NO:1 or SEQ ID NO:2, respectively. SEQ ID NO: the structure of the shRNA-cluster molecule shown in the figure 1 is shown in the lower graph in the figure 1; SEQ ID NO: the structure of the shRNA-cluster molecule shown in FIG. 2 is shown in the upper diagram of FIG. 1.

Example 2

Firstly, shRNA-cluster-CAR molecule recombination pLKO.3G lentiviral vector: according to SEQ ID NO:1 or SEQ ID NO:2, the synthesized shRNA-cluster is subjected to double enzyme digestion through EcoR I and Pac I and is inserted into a pLKO.3G lentiviral vector (shown in figure 2), and the specific steps of enzyme digestion and connection refer to the conventional method in the field; GFP protein in the plasmid is replaced by a molecular cloning means of homologous recombination to obtain CAR molecules, and the pLKO.3G lentiviral vector co-transcribing shRNA-cluster and CAR sequences is obtained after recombination.

One growth state of HEK293T cells was plated on average in 10cm dishes treated with polylysine (cell density approximately 6.5X 10)⁴/cm²) It is required that the cells are uniformly distributed individually. After approximately 24 hours of culture, the confluency of cells should be close to 80%, at which point each dish was exchanged with 12ml of complete medium and 3.75ul chloroquine (100mM, 4000X) was added. After changing the solution, preparing a calcium phosphate-DNA mixed solution according to the table 1, reversing the solution for several times, uniformly mixing the solution, standing the solution for 1 minute, quickly adding the solution into each dish of cell culture solution by 3 ml/dish, shaking the solution uniformly while adding the solution, and quickly adding the solution drop by drop.

Table 1 shows the formulation of calcium phosphate-DNA mixed liquid system

At 12 hours post-transfection, the cell confluence should be close to 100% when each dish is exchanged with 12ml fresh medium and 90. mu.l sodium butyrate (1M, 100X) is added. About 48 hours after transfection, collect all 34ml HEK293T cell supernatant, filter with 0.45 μm filter, then pack into 50ml centrifuge tube, add PEG-NaCl-PBS mixed solution according to the following table 2 proportion, reverse the mixing, then stand at 4 ℃ for 1.5 hours, during every 20-30 minutes mixing once or reverse the mixing, then stand at 4 ℃ overnight.

TABLE 2

Centrifuging the mixed solution at 4 ℃ for 10 minutes at 7000g until white precipitate is visible on the tube wall, carefully removing all supernatant, adding a proper volume (300 ul-3 ml) of fresh culture medium, slightly shaking to dissolve the precipitate to obtain the shRNA-cluster-CAR molecule recombinant virus concentrate, and immediately using or subpackaging and storing at-80 ℃.

Bis, shRNA-cluster-CAR in CD8⁺Expression profile in T cells: isolation of peripheral blood mononuclear cells from peripheral blood samples followed by isolation of enriched CD8 by biotin conjugated CD8 antibody⁺T lymphocytes, counted, centrifuged. Resuspension in RPMI1640 complete medium, then2X 10⁶Cell concentration per ml, plated evenly onto cell culture plates. Cells were stimulated with anti-CD3 (final concentration of 1. mu.g/ml), anti-CD28 (final concentration of 1. mu.g/ml) antibody and IL2 (final concentration of 10ng/ml), and after 48 hours, the cells were harvested for infection with pseudoviruses.

And taking a proper amount of target cell suspension with good growth state, and putting the target cell suspension into a centrifuge tube for centrifugation for 5 minutes at 300 g. The supernatant was discarded at 1 ml/1X 10⁶The pseudovirus concentrated solution is added into the proportion of the cells, Polybrene with the final concentration of 8 mu g/ml is added at the same time, and the mixture is gently blown and evenly mixed. Transferring the cell suspension into a culture dish, centrifuging at 37 ℃ and 350 Xg for 90 minutes, and after the centrifugation is finished, putting the cell suspension back into the incubator to continue culturing. After about 8-12 hours, a second round of infection was performed, as above, after about 12 hours, the cells were resuspended in fresh medium by centrifugation, virus was washed out of the medium with PBS, and cells were maintained by adding IL-2 and IL-7 at final concentrations of 10 ng/ml. And continuously culturing and carrying out passage in time to further expand the cells. Onday 3 after infection, the cells were supplemented with complete RMPI1640 medium to maintain the cell concentration at2X 10 according to the cell state and proliferation⁶And/ml. And IL-2 and IL-7 were supplemented to a final concentration of 10 ng/ml. On day 5 post infection, expansion of cells continued according to theday 3 protocol. And determining the down-regulated CD8 of the shRNA-cluster-CAR in vitro long-term culture by using the expression of the fluorescent protein murine Fab detected by flow⁺Efficiency of T cell inhibitory receptor expression (figure 3). On day 5 post infection, cells were used for subsequent experimental testing or continued culture and frozen. Hereinafter, for convenience, the sequence represented by SEQ ID NO:1, the shRNA-cluster-CAR plasmid constructed by the method is called PTL-CAR in the following experiments and is composed of SEQ ID NO:2 the constructed shRNA-cluster-CAR-T plasmid is called P-CAR in the following experiments.

Example 3

A polypeptide consisting of SEQ ID NO:1 shRNA-cluster-CAR plasmid constructed and targeted to Her2, designated as PTL-Her2-CAR in the following experiments, consisting of SEQ ID NO:2 shRNA-cluster-CAR-T plasmid constructed and targeted to Her2 this was called P-Her2-CAR in the following experiments.

PTL-Her2-CAR and P-Her2CAR engineered CD8⁺And (3) comparing the effects of the T cells after being amplified and cultured in vitro and respectively mixed with a target cell line to release IFN-gamma: transduction of CD8 with the same conditions⁺T lymphocytes are then mixed with a Her2 protein expressing target cell line (SK-OV3) in ELIspot plates pre-coated with IFN-gamma antibodies, and the relationship between the expression of inhibitory receptors and Her2-CAR-T cell function is explored by testing their ability to secrete IFN-gamma to target cells. The results show that PTL-Her2-CAR-T cells have a stronger capacity to secrete IFN- γ in response to stimulation by target cells than P-CAR-T cells, i.e., downregulation of multiple inhibitory receptors enhances the function of Her2-CAR-T cells than downregulation of the inhibitory receptors alone enhances the function of Her2-CAR-T cells (FIG. 4).

II, comparison of killing effects: we will transduce CD8 empty and PTL-Her2-CAR, P-Her2-CAR molecules⁺T effector cells are subjected to in vitro amplification culture, equal amount of cells are respectively frozen and stored at five time points of 5 days, 10 days, 15 days, 20 days and 25 days in vitro culture, after a fifth batch of cells are frozen and stored, the cells are simultaneously recovered and cultured for 24 hours, and then three effector cells and a target cell line are respectively cultured in a mode of 8: 1 for 24 hours, and detecting the killing activity of Her2-CAR-T lymphocytes by a method of detecting the release of Lactate Dehydrogenase (LDH). The experimental results show that: five time-points of control effector cells Mock (No-load transduced CD 8) cultured in vitro⁺T lymphocytes) had no killing effect on the target cell line, and at three time points ranging from 5 days to 15 days in vitro, the killing of the target cells by PTL-Her2-CAR-T cells was not significantly different from that of P-Her2-CAR-T cells, and the killing of the target cells gradually decreased with increasing culture time. At two time points of 20 days and 25 days in vitro culture, the killing efficacy of the two effector cells on the target cells is continuously reduced, the killing efficacy of the P-Her2-CAR-T cells on the target cells is almost disappeared, the PTL-Her2-CAR-T cells still have limited killing capacity on the target cells, and the two effector cells and the target cells are cultured in vitroThere was a significant difference in killing of the target cells (figure 5).

Example 4

First, the oncolytic effect of the PTL-Her2-CAR-T cell therapy and the survival rate of mice: 6-8 weeks of NSG mice 21 mice subcutaneously inoculated with SK-OV3 cells containing 40% Corning high concentration basementmembrane matrix gel 1 × 10⁶Tumor-bearing NSG mice were randomized into three groups: PTL-Her2-CAR-T cell treatment group, Her2-CAR-T cell treatment group, Mock-T cell treatment control group, 7 per group. When the tumor reaches 500mm³Thereafter, by means of tail vein reinfusion, each mouse adoptively immunizedcells 5X 10⁵And (4) respectively. The experimental result shows that after adoptive immune cells, the size of tumor-bearing mice of an experimental group in which PTL-Her2-CAR-T cells are adoptively inoculated to tail veins is obviously smaller than that of mice of an experimental group in which the Her2-CAR-T cells are adoptively inoculated to mice of a control group in which the Mock-T cells are adoptively inoculated to the tail veins, and the survival rate of the mice of the PTL-Her2-CAR-T cell treatment group is also obviously higher than that of the mice of the Her2-CAR-T cell treatment group and the mice of the Mock-T cell treatment group, and the experimental result proves that the PTL-Her2-CAR-T has obvious and specific killing effect on the Her2 positive tumors and can effectively improve the survival rate of the mice (figure 6).

Secondly, infiltration and effective killing of PTL-Her2-CAR-T cells to solid tumors: frozen sections of tumor tissue were prepared and experiments were performed by indirect immunofluorescence. We used IFN- γ as a marker to assess CAR-T cell function. The frozen section results show that the number of CAR-T cells infiltrated in the tumor of the mice in the PTL-Her2-CAR-T cell treated group was significantly greater than the tumor tissue of the mice in the experimental group of Her2-CAR cells as well as the control group of mice, and at the border of the tumor necrosis region, PTL-Her2-CAR-T tended to be more enriched in the border region of tumor necrosis and secreted a large amount of IFN- γ to further mediate necrosis of the tumor tissue (fig. 7).

Thirdly, the safety evaluation of PTL-Her2-CAR-T cells: immune cells are inhibited by expression of a series of "brake" genes, and the associated CAR-T cells cause severe toxic and side effects once they are off-target. To evaluate the safety of clinical trials, we collected the major organs of mice for HE staining, and the experimental results showed that PTL-Her2-CAR-T cells did not infiltrate and kill the major organs of mice, and the safety was preliminarily demonstrated (fig. 8).

Fourthly, in order to explore whether the enhancement of the infiltration and killing capacity of the CAR-T cells for specifically inhibiting the expression of three inhibitory receptors on the solid tumor is limited to the Her-2 positive SK-OV3 ovarian cancer cell line, the CAR-T cells capable of specifically targeting CD19 are established, and the feasibility of the CD19-CAR-T cells for eliminating the solid lymphoma is further evaluated. We established a lymphoma NSG mouse model inoculated subcutaneously with Raji cells, and the tumor of the mouse reached 200mm³At that time, the tail vein was adoptively followed by5X 10 of the CAR-T cells transduced under three identical conditions⁵One/only. Experimental results showed that subcutaneous tumors of mice in the treatment group of postero-venal adoptive PTL-CD19-CAR-T cells were significantly smaller than the treatment group of CD19-CAR-T cells and the control group of VC-CAR-T cells (fig. 9).

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Sequence listing

<110> Guangzhou Qianyang biomedical technology Co., Ltd

<120> shRNA-cluster sequence, and expression vector and application thereof

<130> 2017

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Claims (7)

1. An shRNA-cluster sequence is characterized in that the nucleotide sequence of the shRNA-cluster sequence is shown as SEQ ID NO. 1.

2. An expression vector, which is obtained by connecting a sequence shown as SEQ ID NO. 1 and a coding sequence of a CAR molecule to a basic vector; the expression vector expresses the shRNA-cluster sequence of claim 1 and a CAR molecule.

3. The expression vector of claim 1, wherein the base vector is a lentiviral vector.

4. An engineered CAR-T cell, wherein said T cell is derived from the expression vector of claim 2 or 3 introduced into a CD8+ T cell.

5. A method of making an engineered CAR-T cell according to claim 4, comprising the steps of:

(1) collecting peripheral blood mononuclear cells and separating CD8+ T cells enriched in the peripheral blood mononuclear cells, and activating the CD8+ T cells by using anti-CD3, anti-CD28 and IL-2;

(2) CD8+ T cells were activated 48 hours later at 1 ml/1X 10⁶Adding the slow virus expression vector of claim 3 into the proportion of the cells for infection, simultaneously adding polybrene solution, and continuing to culture after centrifugation; and after 8-12 hours, performing a second round of infection to obtain the modified CAR-T cell.

6. Amplification methodThe method of engineering a CAR-T cell of claim 4, comprising the steps of: (1) suspending the engineered CAR-T cells in fresh medium and adding IL-2 and IL-7 to maintain the cellular state; (2) on days 3 and 5 after transformation, complete RMPI1640 medium was added to the cells to maintain the cell concentration at 2X 10 according to the cell state and proliferation⁶Ml, supplemented with IL-2 and IL-7, continued culture and timely passage, further expansion of cells.

7. Use of an shRNA-cluster sequence according to claim 1, an expression vector according to claim 2 or 3 or an engineered CAR-T cell according to claim 4 for the preparation of a preparation for the elimination of solid tumor cells.

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