CN117487758A - Application of Sub1 gene in disease treatment - Google Patents
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- CN117487758A CN117487758A CN202210878985.2A CN202210878985A CN117487758A CN 117487758 A CN117487758 A CN 117487758A CN 202210878985 A CN202210878985 A CN 202210878985A CN 117487758 A CN117487758 A CN 117487758A
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Abstract
The invention discloses a Sub1 gene serving as a target point for preparing a disease treatment drug, wherein the treatment comprises T cell treatment (CAR-T/TCR-T and the like) or other immunotherapy (immune checkpoint blocking and the like) for improving the T cell capacity, and the anti-tumor capacity of T cells can be promoted by up-regulating the expression quantity of the Sub1 gene.
Description
Technical Field
The invention belongs to the technical field of biology, in particular to immunotherapy, and particularly relates to an application of Sub1 genes in immune cells as targets in preparing medicines for treating diseases.
Background
CD8 + T cells are derived from bone marrow hematopoietic stem cells, mature in thymus through gene V (D) J rearrangement and male/female developmental stage, and circulate between blood and lymphoid organs after exiting thymus. The concept of using the immune system to treat tumors has emerged for centuries, in which CD8 is elevated + T cell killing of tumors is a popular area.
The composition of the tumor is complex, including tumor cells (approximately 30%), stromal cells (which provide nutrition for the survival of the tumor) and infiltrating immune cells (which are complex in composition and function, and have various types of cells that enhance and also reduce the ability to monitor immunity). There are a number of factors that are detrimental to CD8 + T cells act, gradually losing effector functions under the influence of the tumor microenvironment, such as tumor cells being able to produce enzymes that degrade tryptophan and arginine, and compete with immune cells for nutrients and oxygen, while also producing high concentrations of lactate. In addition, there are many immunosuppressive innate immune cells within the tumor, such as myeloid-derived suppressor cells, tumor-associated macrophages, and CD4 + FoxP3 + CD 8-lowering of regulatory T cells and the like + Killing function of T cells. Thus, in the treatment of some solid tumors, CD8 infiltrated in the tumor + T cell number or CD8/FoxP3 high ratio is also often used as an indicator of good prognosis.
Various therapeutic approaches to improve T cell immune monitoring have been tried in experiments and in clinic. For example, immune checkpoint therapies are those that enhance their effector functions by blocking the inhibitory pathways of T cells. anti-CTLA-4 monoclonal antibody Iilimiumab was approved by FDA in 2011 for treatment of melanoma.
However, therapies or drugs for enhancing antitumor ability by using overexpression of Sub1 gene in T cells have not been reported yet.
Disclosure of Invention
In one aspect, the invention provides an immune cell having a Sub1 gene over-expression and/or an agent that induces Sub1 gene over-expression.
Further, the above-mentioned Sub1 gene overexpression includes that the post-induction Sub1 gene expression amount is at least 2 times as large as the pre-induction Sub1 gene expression amount, and preferably, the post-induction Sub1 gene expression amount is 2 to 1000 times as large as the pre-induction Sub1 gene expression amount, for example, 3 times, 4 times, 5 times, 6 times, 8 times, 10 times, 15 times, 20 times, 30 times, 50 times, 100 times, 500 times, 1000 times, and the like.
Further, the agent is a viral vector expressing the SUB1 protein, more preferably, a retroviral vector.
Further, the immune cells are primary cells from the subject, preferably the immune cells are human cells, such as white blood cells or human peripheral blood mononuclear cells, more preferably the immune cells are T cells (including, cd4+ or cd8+ T cells, CAR-T cells) or NK cells, particularly preferably the immune cells are cd8+ T cells.
Further, the immune cells are derived from primary cells of a patient with cancer, wherein the cancer is lymphoma, chronic Lymphocytic Leukemia (CLL), B-cell acute lymphocytic leukemia (B-ALL), acute lymphocytic leukemia, acute myelogenous leukemia, non-hodgkin's lymphoma (NHL), diffuse Large Cell Lymphoma (DLCL), multiple myeloma, renal Cell Carcinoma (RCC), neuroblastoma, colorectal cancer, breast cancer, ovarian cancer, melanoma, sarcoma, prostate cancer, lung cancer, esophageal cancer, hepatocellular carcinoma, pancreatic cancer, astrocytoma, mesothelioma, head and neck cancer, and/or medulloblastoma.
Further, the immune cell further comprises a recombinant receptor expressed on the surface of the immune cell or a polynucleotide encoding the recombinant receptor, wherein the recombinant receptor specifically binds to an antigen, and wherein the immune cell is capable of inducing cytotoxicity, proliferation and/or secretion of cytokines upon binding of the recombinant receptor to the antigen.
Further, wherein the recombinant receptor is a recombinant T cell receptor or a chimeric antigen receptor.
Further, wherein the recombinant receptor specifically binds one or more antigens independently selected from the group consisting of: RORl, her2, ll-CAM, CD19, CD20, CD22, CEA, hepatitis B surface antigen, folate receptor antibody, CD23, CD24, CD30, CD33, CD38, CD276, CD44, EGFR, EGP-2, EGP-4, EPHa2, erbB3, erbB4, FBP, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R, kdr, lewis Y, L1 cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D ligand, NY-ESO-1, MART-1, gp100, tumor embryo antigen, TAG72, VEGF-R2, carcinoembryonic antigen, prostate specific antigen, PSMA, estrogen receptor, progestin receptor, ephrinB2, CD123, CS-1, c-Met, GD2, GE A3, CE7, cyclin A1, BCMA 12, interleukin A12.
In a further aspect the invention provides a method of increasing cytotoxicity of an immune cell or increasing infiltration of an immune cell into a tumour, the method comprising treating the immune cell with an agent which causes overexpression of the Sub1 gene or increases the activity of the Sub1 protein in the cell.
Further, the agent is selected from the group consisting of:
1) A viral vector expressing a SUB1 protein;
2) Small molecule drugs with the function of improving the expression of Sub1 genes in CD8+ T cells;
3) Small molecule drugs or antibodies with effect of increasing SUB1 protein activity in cd8+ T cells
Further, the agent is a viral vector expressing the SUB1 protein, more preferably, a retroviral vector.
Further, the above-mentioned Sub1 gene overexpression includes that the post-induction Sub1 gene expression amount is at least 2 times as large as the pre-induction Sub1 gene expression amount, and preferably, the post-induction Sub1 gene expression amount is 2 to 1000 times as large as the pre-induction Sub1 gene expression amount, for example, 3 times, 4 times, 5 times, 6 times, 8 times, 10 times, 15 times, 20 times, 30 times, 50 times, 100 times, 500 times, 1000 times, and the like.
In a further aspect the invention provides the use of an agent targeting Sub1 genes and/or Sub1 proteins in immune cells in the manufacture of a medicament for the treatment of a disease.
Further, the agent allows overexpression of Sub1 gene or increases the activity of Sub1 protein in cells.
Further, the agent is selected from the group consisting of:
1) A viral vector expressing a SUB1 protein;
2) Small molecule drugs with the function of improving the expression of Sub1 genes in CD8+ T cells;
3) A small molecule drug or antibody having the effect of increasing the activity of SUB1 protein in cd8+ T cells.
In a further aspect, the invention provides the use of an immune cell having a Sub1 gene over-expression and/or an agent that induces Sub1 gene over-expression in the preparation of a medicament for the treatment of a disease.
In a further aspect the invention provides a method of treatment of a disease comprising administering to a subject in need thereof an agent or immune cell which is an immune cell having a Sub1 gene over-expression and/or an agent which induces Sub1 gene over-expression.
Further, the agent is selected from the group consisting of:
1) A viral vector expressing a SUB1 protein;
2) Small molecule drugs with the function of improving the expression of Sub1 genes in CD8+ T cells;
3) A small molecule drug or antibody having the effect of increasing the activity of SUB1 protein in cd8+ T cells.
Further, the disease treatment method of the present invention comprises: (1) obtaining immune cells from a patient; (2) Treating immune cells with an agent that induces overexpression of Sub1 genes; (3) administering the immune cells to the patient.
Further, the disease is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or cancer.
Further wherein the disease is a cancer or tumor selected from leukemia, lymphoma, chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), non-hodgkin's lymphoma, acute myelogenous leukemia, multiple myeloma, refractory follicular lymphoma, mantle cell lymphoma, indolent B-cell lymphoma, B-cell malignancy, colon cancer, lung cancer, liver cancer, breast cancer, prostate cancer, ovarian cancer, skin cancer, melanoma cancer, bone cancer, brain cancer, epithelial cancer, renal cell cancer, pancreatic adenocarcinoma, hodgkin's lymphoma, cervical cancer, colorectal cancer, glioblastoma, neuroblastoma, ewing's sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma, and/or mesothelioma.
Compared with the prior art, the application provided by the invention is based on the fact that the Sub1 gene is an important factor for regulating the anti-tumor immunity of the T cells, and the anti-tumor capability of the T cells can be promoted by up-regulating the expression quantity of the Sub1 gene. Applications of the invention include T cell therapy (CAR-T/TCR-T, etc.) or other immunotherapy (immune checkpoint blockade, etc.) that follows the elevation of T cell capacity.
Drawings
FIG. 1 is a graph showing the expression of Sub1mRNA in MigR1 blank T cells and MigR1-Sub1T cells;
FIG. 2 is a graph showing the expression of the SUB1 protein in MigR1 blank T cells and migR1-SUB1T cells;
FIG. 3 is a statistical plot of the frequency of B16-OVA tumor cell death in a media co-incubated with MigR1 blank T cells and migR1-sub1T cells and B16-OVA tumor cells;
FIG. 4 is a FACS analysis of IFN-gamma cytokine production in overexpressed Sub1T cells after 4 hours peptide stimulation;
FIG. 5 is a schematic illustration of the experimental design of example 1; FIG. 6 is a graph of tumor growth; FIG. 7 is a tumor image on day 14; fig. 8 is tumor weight.
FIGS. 9-11 are frequency graphs of CD8+CD45.1+GFP+, PD1+ and Tim3+OT-I CD8T cells from day 14 tumors using flow cytometry analysis.
FIG. 12 is a diagram showing the detection of Sub1 expression in ShRNA vector and ShRNA-Sub1T cells by RT-PCR;
FIG. 13 is a statistical plot of the frequency of B16-OVA tumor cell death by flow cytometry after 24h co-incubation of ShRNA-blank and ShRNA-sub1T cells with B16-OVA tumor cells in culture;
FIGS. 14 and 15 are statistical graphs of IFN-gamma (FIG. 14) and TNF-a (FIG. 15) cytokines, respectively, from FACS analysis 4 hours after peptide stimulation of Sub1 knockout T cells;
FIG. 16 is a schematic illustration of the experimental design of example 2; FIGS. 17-19 are graphs of tumor growth of B16-OVA melanoma mice with ShRNA vector or ShRNA-sub1 CD45.1 OT-I CD8T cells adoptive transfer (FIG. 17), tumor images on day 14 (FIG. 18), tumor weights (FIG. 19);
FIG. 20 is a flow cytometry analysis of the frequency of TNF-a+CD45.1+OT-I CD8T cells in tumor tissue on day 14 of B16-OVA (left), statistically showing the percentage of TNF-a+CD45.1+OT-I CD8T cells in tumor-infiltrating CD8+ cells (right) (E, n=10).
FIG. 21 is a graph showing the statistics of the expression level of Sub1 in CD8+ T cells in peripheral lymphoid tissues of Sub1-ko mice and wild-type mice;
FIG. 22 is a graph showing the statistics of the expression amounts of CD4+ and CD8+ T cells in thymus by wild-type and Sub1-KO T cells;
FIG. 23 statistical graphs of the expression amounts of wild type and Sub1-KO T cells in inguinal lymph node, mesenteric CD4+ and CD8+ T cells.
FIG. 24 is a schematic illustration of the experimental design of example 3;
FIGS. 25-27 are tumor growth curves (FIG. 25), day 14 tumor images (FIG. 26) and tumor weights (FIG. 27) of Sub1tKO or wild-type control mice injected with melanoma cells;
FIGS. 28-31 are frequency analyses of CD4+ and CD8+ T cells (FIG. 28), interferon-gamma CD8+ T cell effect (FIG. 29) and blocked PD1+CD8+ T cells (FIG. 30) and Tim3+CD8+ T cells (FIG. 31) in Sub1tKO mice and wild type controls 14 days of tumor B16-OVA transplantation (WT cases: n=10; sub1tko: n=14/genotype).
Detailed Description
Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Further, one skilled in the art can engage and combine the different embodiments or examples described in this specification.
Experimental method
1. Preparation of tumor model organisms
B16-F10/B16-F10-ova(2×10 5 ) Female C57BL/6 mice of 6 weeks of age were implanted on left and right dorsal sides. After 7 days, the tumor reaches 50-100mm 3 About, 7 mice (n=7) were injected with anti-ctl-4 antibody 1 time every 3 days. Mice tumor size was monitored every 3 days during dosing. The tumor volume is measured by a caliper, and the calculation formula is as follows: volume = length x width 2 X 0.52. Mice were observed continuously until they were sacrificed. Tumor tissue was isolated for further experiments.
2. Isolation and flow cytometry of tumor infiltrating lymphocytes
Tumor Infiltrating Lymphocytes (TILs) were isolated by excision of tumor tissue, removal of subcutaneous adipose tissue. Cutting the tissue into 1mm 3 The blocks were digested with 2% FCS RPMI 1640 medium containing 1mg/mL collagenase D (Worthington Biochemical Corp.) and 0.01mg/mL DNase I (Millipore Sigma Corp.) at 37℃for 30min. The isolated tissue was entered into roswell park souvenir institute (RPMI) -1640 medium (Corning inc., corning) using a 70 μm cell filter (Falcon, corning). TILs were isolated using a Percoll gradient (GE Healthcare) and suspended in 2% fetal bovine serum (FBS; biowest) added. Single cell suspensions were stained with fluorescent-labeled dye antibodies and subjected to CD8 by flow cytometry + T cell classification.
3. Generation and detection of T hybridoma cell activation
The NFAT-GFP 58 a- β -hybridoma cell line was provided by Huang Huang doctor. To reconstruct TCRs, cdnas of tcra and tcrp (tcra-p 2A-tcrp) generated by overlapping oligonucleotide anneals were ligated using the self-cleaving sequence of 2A and placed into a modified MigR1 reverse transcription vector in which IRES-GFP was replaced by IRES-mCD 8. Retroviral vectors were then transfected into 293FT packaging cells with PEI. The virus supernatant was transfected with 8. Mu.g/ml polystyrene (polybrene) and spun at 32℃for 90min. Transfection efficiency was monitored by detecting mCD3 surface expression on day 2. 8 hybridomas with dominant clonotype TCRs were produced, and the V.alpha.and V.beta.sequences of these TCRs were retrieved from single cell TCR sequences. Injection 8×10 before 10 days 6 B6 mice spleen cells from LT3-B16 melanoma cells were enriched positively for CD11c+ cells using MACS LS column (Miltenyi). 1X 10 4 Hybridoma cells and 2×10 5 APCs and antigen were incubated for 2 days. Flow cytometry detection analyzed GFP induction in cd3+ hybridoma cells.
4. Cell separation for subsequent analysis
The spleen was mechanically disrupted by a 1ml syringe, filtered through a 70 μm filter, and erythrocytes were lysed with potassium ammonium chloride buffer (erythrocyte lysate). Cells were washed 2 times with cold RPMI 1640 medium containing 2. Mu.M glutamine, 100U/ml penicillin/streptomycin and 5-10% FCS. Liver tumor and B16 tumor tissues were destroyed mechanically and isolated with scissors (complete RPMI pre-chilled with 1-2 ml). The separated tissue pieces were transferred to a 70- μm filter (placed in a 60mm dish with 1-2ml pre-chilled complete RPMI) and further separated from a 3ml syringe. The cell suspension was filtered through a 70 μm sieve. The tumor homogenate was spun at 400g for 5min at 4 ℃. The microspheres were resuspended in 15ml FCS 3% HBSS, 500. Mu.l (500U) heparin and 8.5ml Percoll were added, mixed upside down, and 500g spun at 4℃for 10min. The particles were lysed with potassium ammonium chloride buffer and the cells were further processed for subsequent use.
5. Retroviral transduction
Frame (in-frame) fusion of mouse Sub1 cDNA without stop codon with coding sequence of Green fluorescent protein (mGFP) using self-cleaving sequence of P2AAnd (5) combining. Amplifying the SUB1-mGFP by PCR cloning, cloning the SUB1-mGFP into the MigR1 retroviral vector by using restriction enzymes BglII and SalI, and generating the MigR1-SUB1-mGFP. Retroviral transduction of CD8 with mig1-sub1-mGFP and mGFP-only control mig1-mGFP + T cells, steps are as follows:
day 1, retroviral packaging cell line 293FT was transfected with PEI; isolation of CD45.1 on day 2 + OT-I mouse lymphocytes stimulated with OVA peptide; on day 3, cells were infected with retrovirus MigR1-SUB1-mGFP or a placebo vector (MigR 1-mGFP). Activated lymphocytes in the presence of 50U/ml IL -2 And 5. Mu.g/ml polybrene, transferred to a 24-well plate and spun at 2500rpm for 90min at 30 ℃. The transduced T cells were cultured for an additional 3 days, with fresh IL added every other day -2 (100U/ml). Cells with high GFP expression were classified using FACS for adoptive transfer or downstream transcriptome analysis. GFP (Green fluorescent protein) + T cell transfer number was 1.5X10 6 。
MSCV-LTRmiR30-PIG (LMP) is a retroviral vector based on MSCV. Puromycin resistant vectors are used to express shrrnamir from the retroviral LTR promoter. GFP was used as a marker for retroviral integration. LMP vectors were purchased from Mark M davis' lab. The shRNA primer of Sub1 sequence was designed with sigma software, and then the primer was synthesized. The synthesized 97mer oligo was subjected to PCR, and cloned into LMP vector using restriction enzymes EcoRI and XhoI to obtain LMP-shRNsub1. DNA purified from the sequence verified clone was used to package retroviral particles in 293FT cells. Mu.g of plasmid (empty plasmid or shRNAs plasmid) and 6. Mu.g of packaging plasmid pcl-ECO 6. Mu.g, 50. Mu.L PEI, 800. Mu.L opti-MEM were co-cultured with the culture broth for 48h, and the supernatant (virus-enriched solution) was collected and concentrated. Activated lymphocytes were obtained in the virus supernatant after resuspension.
Will be 1.5X10 5 Each activated lymphocyte was co-infected with 500. Mu.L of virus-enriched solution and 8. Mu.g/mL of polyether on a 24-well plate and spun (spun) at 2500rpm for 90min at 30 ℃. The transduced T cells were cultured for an additional 3 days, and fresh IL-2 (100U/ml) was added every other day. Sorting high GFP expressing cells using FACS for adoptive transfer or downstream transcriptome analysis. EXAMPLE 1 test of overexpression of Sub1 for increasing anti-tumor Effect of CD8+ T cells
The Ova-specific OT-I CD8+ T cells were transferred into Ova-expressing B16-OVA tumor mice, demonstrating tumor growth control. CD8T cells were isolated from CD45.1OT 1 mice. Transformation of retroviral vector with Green Fluorescent Protein (GFP) encoding full Length SUB1 or blank empty vector with GFP into Effect CD45.1 + OT 1T cells to generate MigR1-sub1-GFP and CD8 of MigR1-GFP + Effector T cells. Following transfection, effector T cells were incubated with IL-2 for 3 days (without any TCR stimulation) and GFP expression was flow sorted.
The experimental results are shown in FIGS. 1-4. In the case of RT-PCR detection of Sub1mRNA expression in MigR1 blank T cells and migR1-Sub1-GFP T cells, it can be seen that the amount of Sub1mRNA expression in migR1-Sub1T cells is significantly higher than that in blank control; in addition, FIG. 2 shows that the protein expression of Sub1 in Migr1 blank T cells and Migr1-Sub1T cells is significantly higher than that in blank control, indicating that the overexpression of Sub1 has been successfully achieved.
FIG. 3 shows that MigR 1-blank T cells and MigR1-sub1T cells were co-incubated with B16-OVA tumor cells in the medium, and it can be seen that the number of dead cells in the medium in which MigR1-sub1T cells were co-incubated with B16-OVA tumor cells was significantly higher than in the blank, and that the frequency of B16-OVA tumor cell death was significantly higher in flow cytometry analysis (FACS).
FIG. 4 is a graph showing IFN-gamma cytokine production in overexpressed Sub1T cells after 4h of FACS analysis of peptide stimulation, statistics of IFN-gamma + T cells occupy CD8 + T cell percentage (mean ± SD, n=3); it can be seen that the percentage of IFN-. Gamma. + T cells in the overexpressed Sub1T cells was significantly higher than in the blank.
FIG. 5 is a schematic illustration of the experimental design of example 1, where subjects were C57BL/6J mice 6-8 weeks old, B16-OVA tumor cells were inoculated subcutaneously on day 0, migR1-sub1-GFP and MigR1-GFP (blank) CD45.1OT-1T cells were adoptively transferred after day 7d, and tumor weight, volume, tumor size plots were counted on day 14 and flow cytometry FACS analysis. Figure 6 is a graph showing the growth curve of a tumor,FIG. 7 is a photograph of the tumor at day 14, and FIG. 8 is a statistical plot of tumor weight (g), as can be seen from the above 3 statistical plots of results, with transfected MigR1-GFP (blank) CD8 + Compared with tumor-bearing mice of T cells, the method transfects MigR1-sub1-GFP CD8 + The tumor growth speed of the tumor-bearing mice of the T cells is obviously reduced.
Figures 9-11 are frequency of cd8+cd45.1+gfp+, pd1+ and tim3+ot-I CD8T cells in tumors on day 14 of B16-OVA using flow cytometry (left), statistics show the percentage increase of cd45.1+gfp+ot-I CD8T cells in tumor infiltrating cd8+ cells in tumors of MigR1-sub1-GFP group (right) (E, n=10); while the decreased percentage of pd1+ and tim3+ OT-I CD8T cells in tumor-infiltrating cd8+ cells suggests decreased expression of protein receptors PD1 and Tim3 associated with T cell depletion. The data are representative of three independent experiments. Summary data are expressed as mean ± s.e.m., P-values were determined by a double t student t-test.
In addition, CD8 has a response to MigR1-GFP (blank) + Compared with T cells, the effect of tumor infiltration of the MigR1-sub1-GFP group, CD8+CD45.1+GFP+T cells, although not proliferation, had improved viability (FIG. 9).
Thus, as can be seen from FIGS. 1-11, migR1-sub1-GFP CD8 + The effector T cells have stronger in-vitro cytotoxicity on B16-OVA tumor cells, and adoptive transfer of CD8 over-expressing Sub1 in vivo + After effector T cells, the effect of delaying tumor growth is remarkable, and it can be inferred that overexpression of Sub1 in T cells can directly enhance the anti-tumor capability.
Example 2 test of Knockdown (Knockdown) Sub1 on attenuation of anti-tumor effects of CD8+ T cells
The experimental results are shown in FIGS. 12-20. Wherein FIG. 12 shows normalized raw liquid drop count housekeeping genes, gapdh, for RT-PCR detection of Sub1 expression data in ShRNA vector and ShRNA-Sub1T cells; n=3. As can be seen from FIG. 12, the expression level of Sub1mRNA was significantly higher than that of shRNA-Sub1 in the blank, indicating that the effect of knocking down the Sub1 gene was successfully achieved.
FIG. 13 is a statistical plot of the frequency of B16-OVA tumor cell death after 24h by flow cytometry analysis of ShRNA-vector and ShRNA-sub1T cells co-incubated with B16-OVA tumor cells in culture medium. As can be seen from FIG. 13, the percentage of dead cells was significantly lower in the shRNA-Sub1 cell co-incubated with the B16-OVA tumor cells.
FIGS. 14 and 15 are FACS analysis of ShRNA-vector and ShRNA-sub1T cells, respectively, for IFN-gamma (FIG. 14) and TNF-a (FIG. 15) cytokine production 4 hours after peptide stimulation. According to CD8 + The percentage statistics of T cells showed IFN- γ+t (fig. 14) and TNF-a+t cell percentages (fig. 15) (mean ± SD, n=3). As can be seen from fig. 14-15, the percentage of both IFN- γ+ T cells and TNF- α+ T cells in shRNA-Sub1T cells was significantly lower than the blank.
FIG. 16 is a schematic illustration of the experimental design of example 2, where subjects were C57BL/6J mice 6-8 weeks old, B16-OVA tumor cells were inoculated subcutaneously at day 0, CD45.1+ OT-1T cells were adoptively transferred after day 7, and tumor weights, volumes, pictures and flow cytometry analysis were performed on day 14. FIG. 17 is a graph showing tumor growth in B16-OVA melanoma mice adoptively transferred with either shRNA vector or ShRNA-sub1 CD45.1 OT-I CD8T cells, FIG. 18 is a photograph of the tumor taken on day 14, FIG. 19 is a statistical plot of tumor weight (g), and from the above 3 results (from tumor growth curves, weight and photograph images, respectively) statistical plots, the results were compared with transfected ShRNA-vector CD8 + Transfection of shRNA-Sub1 CD8 compared to tumor-bearing mice with T cells (i.e., a blank) + The tumor growth speed of the tumor-bearing mice of the T cells is obviously accelerated.
FIG. 20 is a graph showing the frequency of occurrence of tumor necrosis factor a (TNF-a) produced by adoptively transferred CD8T cells in tumor tissue on day 14 of B16-OVA by flow cytometry (left), and statistics show the percentage of TNF-a+CD45.1+OT-I CD8T cells in tumor infiltrating CD8+ cells (right) (E, n=10). The data are representative of three independent experiments. Summary data are expressed as mean ± s.e.m., P-values were determined by a two-tailed student t-test.
Thus, as can be seen from FIGS. 12-20, sub1 Knockdown reduced the ability of CD8T cells to produce tumor necrosis factor, reduced their ability to kill tumors, and it was concluded that Knockdown (KnockDown) Sub1 in T cells could attenuate their anti-tumor ability.
Example 3 test of knockdown (Knockout) Sub1 on significantly reduced anti-tumor effects of CD8+ T cells
Sub1-floxed mice derived from cre-recombinase transgenic mice CD4 promoter were prepared. Sub1mRNA expression was significantly reduced in Zhou Pizang CD8+ T cells outside Sub1 flox/flox CD4-Cre transgenic (Sub 1-ko) mice as observed by qRT-PCR (FIG. 21).
The expression differences of the Wild Type (WT) and Sub1-KO T cells in the thymus were not significant for CD4+ and CD8+ T cells. There was also no significant difference in the number of CD44hiCD62Lhi in cd8+ T cells in thymus between WT mice and Sub1-KO mice (fig. 22). However, the number of CD44hiCD62Lhi cells was lower in spleen cd8+ T cells of Sub1-KO mice and the numbers of cd4+ and cd8+ T cells of CD45.2+ T cells was lower compared to WT mice. Other tissues, exemplified by Inguinal Lymph Node (iLN), also significantly reduced the percentage of cd4+ T cells in Sub1-KO CD45.2+ T cells compared to WT, but no significant difference in cd8+ T cells between WT and Sub1-KO (fig. 23).
The experimental results are shown in FIGS. 24-27. Wherein FIG. 24 is a schematic illustration of experimental design of the implanted B16-ova mice. After 7d tumor implantation, tumor size was measured with calipers every 2 days, and volume was calculated according to the formula: volume=0.52×length×width×width (unit: mm). FIGS. 25-27 are views of an injection 2X 10 5 Tumor growth curves (FIG. 25), day 14 tumor images (FIG. 26) and tumor weights (FIG. 27) of Sub1tKO or wild-type control mice of B16-OVA melanoma cells. As can be seen from the above figures, subcutaneous melanoma Sub1-KO and wild type mice are shown in fig. 24; after 14 days, tumor growth was significantly delayed in WT mice B16-OVA melanoma compared to Sub1-KO (FIGS. 25-27), and the data showed that Sub1-KO mice had serious drawbacks in establishing anti-tumor immunity.
FIGS. 28-31 are frequency analyses of CD4+ and CD8+ T cells (FIG. 28), interferon-gamma CD8+ T cell effects (FIG. 29) and PD1+CD8+ T cells (FIG. 30) and Tim3+CD8+ T cells (FIG. 31) in Sub1tKO mice and wild type controls 14 days of tumor B16-OVA transplantation (WT case: n=10, sub1tKO: n=14/genotype). The data are representative of three independent experiments. Flow cytometer data are represented in multiple mice as representative graph (left) and summary graph (mean ± s.e.m.) (right, each circle represents one mouse). The P value was determined by two-way analysis of variance with Bonferroni correction (B), two-tailed student t test (E-H). It can be seen that the response of injured T cells to tumors in Sub1KO mice showed reduced cd8+ and interferon-gamma production in effector cd8+ T cells (fig. 28, fig. 29). Tumor infiltrating Sub 1-defect cd8+ T cells also showed an increase in PD1 and Tim3 expression levels (fig. 30, fig. 31). Similar phenomena were also observed in the spleen and lymph node attached sites.
Thus, as can be seen from FIGS. 28-31, sub1 Knockout reduced the anti-tumor function of CD8T cells, and it can be inferred that the anti-tumor ability was significantly reduced in T cell Sub1 gene conditional Knockout (Knockout) mice. In summary, the Sub1 gene is an important factor for regulating the anti-tumor immunity of T cells, and the anti-tumor capability of the T cells can be promoted by up-regulating the Sub1 in the future. The principle can be applied to T cell therapy (CAR-T/TCR-T and the like) or other immunotherapy (immune checkpoint blocking and the like) for improving the capacity of T cells, or used for preparing anti-tumor medicaments.
It should be noted and appreciated that various modifications and improvements of the invention described in detail above can be made without departing from the spirit and scope of the invention as claimed in the appended claims. Accordingly, the scope of the claimed subject matter is not limited by any particular exemplary teachings presented.
Applicant states that the foregoing is a further detailed description of the invention in connection with certain preferred embodiments and that the practice of the invention is not to be construed as limited thereto. It will be apparent to those skilled in the art that several simple deductions or substitutions may be made without departing from the spirit of the invention, and these should be considered to be within the scope of the invention.
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Claims (14)
1. an immune cell having a Sub1 gene over-expression and/or an agent that induces Sub1 gene over-expression.
2. The immune cell of claim 1, wherein the Sub1 gene overexpression comprises a post-induction Sub1 gene expression level that is at least 2-fold greater than a pre-induction Sub1 gene expression level.
3. The immune cell of claim 1 or 2, wherein the agent is a viral vector expressing a SUB1 protein, more preferably a retroviral vector.
4. An immune cell according to any one of claims 1-3, which is a primary cell from a subject, preferably which is a human cell, such as a leukocyte or a human peripheral blood mononuclear cell, more preferably which is a T cell or an NK cell, particularly preferably which is a cd8+ T cell.
5. The immune cell of any one of claims 1-4, wherein the immune cell is derived from a primary cell of a cancer patient.
6. The immune cell of any one of claims 1-5, further comprising a recombinant receptor expressed on the surface of the immune cell or a polynucleotide encoding the recombinant receptor, wherein the recombinant receptor specifically binds to an antigen, and wherein the immune cell is capable of inducing cytotoxicity, proliferation, and/or secretion of cytokines upon binding of the recombinant receptor to an antigen.
7. The immune cell of claim 6, wherein the recombinant receptor is a recombinant T cell receptor or a chimeric antigen receptor.
8. The immune cell of claim 7, wherein the recombinant receptor specifically binds one or more antigens independently selected from the group consisting of: RORl, her2, ll-CAM, CD19, CD20, CD22, CEA, hepatitis B surface antigen, folate receptor antibody, CD23, CD24, CD30, CD33, CD38, CD276, CD44, EGFR, EGP-2, EGP-4, EPHa2, erbB3, erbB4, FBP, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R, kdr, lewis Y, L1 cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D ligand, NY-ESO-1, MART-1, gp100, tumor embryo antigen, TAG72, VEGF-R2, carcinoembryonic antigen, prostate specific antigen, PSMA, estrogen receptor, progestin receptor, ephrinB2, CD123, CS-1, c-Met, GD2, GE A3, CE7, cyclin A1, BCMA 12, interleukin A12.
9. A method of increasing cytotoxicity of immune cells or increasing infiltration of immune cells into a tumour, the method comprising treating immune cells with an agent which over-expresses Sub1 genes or increases the activity of Sub1 proteins in the cells.
10. Use of a reagent targeting the Sub1 gene and/or Sub1 protein in immune cells for the manufacture of a medicament for the treatment of a disease, said reagent allowing overexpression of the Sub1 gene or increasing the activity of the Sub1 protein in the cells.
11. The use of claim 10, wherein the agent is selected from the group consisting of:
1) A viral vector expressing a SUB1 protein;
2) Small molecule drugs with the function of improving the expression of Sub1 genes in CD8+ T cells;
3) A small molecule drug or antibody having the effect of increasing the activity of SUB1 protein in cd8+ T cells.
12. Use of an immune cell according to claims 1-8 for the preparation of a medicament for the treatment of a disease.
13. The use according to any one of claims 10-12, wherein the disease is an infectious disease or disorder, an autoimmune disease, an inflammatory disease or a tumor, cancer.
14. The method of claim 13, wherein the disease is a cancer or tumor selected from leukemia, lymphoma, chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), non-hodgkin's lymphoma, acute myelogenous leukemia, multiple myeloma, refractory follicular lymphoma, mantle cell lymphoma, indolent B-cell lymphoma, B-cell malignancy, colon cancer, lung cancer, liver cancer, breast cancer, prostate cancer, ovarian cancer, skin cancer, melanoma cancer, bone cancer, brain cancer, epithelial cancer, renal cell carcinoma, pancreatic adenocarcinoma, hodgkin's lymphoma, cervical cancer, colorectal cancer, glioblastoma, neuroblastoma, ewing's sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma, and/or mesothelioma.
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| CN202210878985.2A Pending CN117487758A (en) | 2022-07-25 | 2022-07-25 | Application of Sub1 gene in disease treatment |
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