Method and composition for reprogramming tumor cells into conventional dendritic cells by in situ transdifferentiation

Abstract

Provided in the present invention is a transcription factor composition for reprogramming tumor cells into conventional dendritic cells (cDCs), particularly conventional type 1 dendritic cells (cDC1s), through in situ transdifferentiation. Through combined expression of specific transcription factors, provided in the present invention is a technique for reprogramming tumor cells into professional antigen-presenting cells (Tumor-APCs), thereby restoring the immunogenicity of the tumor cells and enhancing the immune response of the body against tumors. This technique has important clinical application prospects.

Description

Methods and compositions for reprogramming tumor cells into classical dendritic cells via in situ transdifferentiation. Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a cell and method for tumor immunotherapy. It reprograms tumor cells into specialized antigen-presenting cells (Tumor-APCs) through in situ transdifferentiation technology to restore tumor immunogenicity, combat tumor immune escape, and enhance the body's immune response to tumors. Background Technology

[0002] Dendritic cells (DCs) develop from hematopoietic stem cells (HSCs) and undergo precursor-to-type differentiation in the bone marrow. Most DCs differentiate from a common DC progenitor cell (CDP) and are mainly divided into classical dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs).

[0003] pDCs can secrete type I interferon. pDCs recognize viral or self-nucleic acid through Toll-like receptors TLR7 and TLR9, producing large amounts of interferon α (IFN-α) and interferon β (IFN-β) [1,2], which are crucial in controlling viral infections. cDCs are mainly divided into two subsets: classical type 1 dendritic cells (cDC1) and classical type 2 dendritic cells (cDC2). cDCs induce naive T cell activation by presenting antigenic peptides through MHC molecules. In addition, DCs can also secrete cytokines to enhance and regulate immune responses [3,4].

[0004] cDC2 cells have the functions of the general DC family, activating naive CD4+ T cells through MHC class II antigen presentation and co-stimulation. In human cDC1 cells, cDC1 specifically expresses CD141 and CLEC9A, as well as other markers to distinguish them from cDC2. cDC1 is specifically used for antigen cross-presentation or for presenting exogenous antigens on MHC class I to induce differentiation and activation of naive CD8+ T cells[5]. cDC2-regulated helper T cell activation results in a strong adaptive immune response to extracellular pathogens (including microbial and worm infections), and cDC1 has great potential in activating cytotoxic T lymphocyte (CTL)-mediated antitumor responses[6].

[0005] Cancer is caused by the accumulation of gene mutations leading to abnormal cell proliferation and malignant spread. Over time, these abnormal cells may acquire further genetic and epigenetic changes, enhancing their invasiveness and drug resistance, ultimately forming tumors. These characteristics of tumor cells enable them to evade the body's immune surveillance, thereby promoting disease progression.

[0006] The rise of tumor immunotherapy has brought about revolutionary changes in cancer treatment. Tumor immunotherapy alone or in combination with traditional therapies (such as chemotherapy, radiotherapy and surgery) has changed the landscape of cancer treatment. In particular, the application of immune checkpoint inhibitors (ICIs), such as drugs targeting programmed cell death protein 1 (PD-1) and its ligand (PD-L1), has become a means of treating a variety of cancers. These drugs enhance the immune response of T cells to tumors by blocking the PD-1 / PD-L1 pathway and relieving the immunosuppression of T cells by tumor cells.[7]

[0007] Despite the significant efficacy of immunotherapy in cancer treatment, ICI monotherapy only elicits a response in a small percentage of patients, and tumor cells evade the immune system through various mechanisms, leading to resistance to ICI [8,9,10]. Developing novel therapeutic strategies to improve the effectiveness of tumor immunotherapy is crucial. To enhance the efficacy of ICI, researchers are exploring various strategies to restore the immunogenicity of tumor cells or increase the infiltration of cDC1 cells in the tumor microenvironment

[0011] . This includes using genetic engineering, drug therapy, or cell reprogramming techniques to enable tumor cells to express more immune co-stimulatory molecules or enhance their ability to present tumor antigens [12,13]. Summary of the Invention

[0008] The first objective of this invention is to provide a transcription factor composition that, when transduced into cells, activates the cellular immune response by reprogramming the cells.

[0009] A second object of the present invention is to provide a construct or vector for transducing the transcription factor composition into the cells;

[0010] A third object of the present invention is to provide a method for reprogramming or inducing stem cells or differentiated cells or tumor cells or mixtures of these cells into dendritic cells or antigen-presenting cells;

[0011] A fourth object of the present invention is to provide reprogrammed or induced dendritic cells or antigen-presenting cells prepared by the method of the present invention;

[0012] A fifth object of the present invention is to provide a pharmaceutical composition comprising the reprogrammed or induced dendritic cells or antigen-presenting cells of the present invention;

[0013] The sixth objective of this invention is to provide a cancer vaccine.

[0014] According to a first aspect of the invention, a transcription factor composition for reprogramming cells in in situ transdifferentiation is provided, wherein the composition comprises a combination selected from the following transcription factors:

[0015] Examples include PU.1 (SEQ ID NO:1), IRF8 (SEQ ID NO:2), and ID2 (SEQ ID NO:5); PU.1 (SEQ ID NO:1), IRF8 (SEQ ID NO:2), and ZEB1 (SEQ ID NO:7); PU.1 (SEQ ID NO:1), BATF3 (SEQ ID NO:3), and ID2 (SEQ ID NO:5); PU.1 (SEQ ID NO:1), BATF3 (SEQ ID NO:3), and NFIL3 (SEQ ID NO:6) (SEQ ID NO:6); PU.1 (SEQ ID NO:1), IRF8 (SEQ ID NO:2), and ID2 (SEQ ID NO:5) (SEQ ID NO:5); PU.1 (SEQ ID NO:1), BATF3 (SEQ ID NO:3), and NFIL3 (SEQ ID NO:6) (SEQ ID NO:6); PU.1 (SEQ ID NO:1), IRF8 (SEQ ID NO:2), and NFIL3 (SEQ ID NO:6) (SEQ ID NO:4); PU.1 (SEQ ID NO:1), BATF3 (SEQ ID NO:3), and ZNF366 (SEQ ID NO:4) (SEQ ID NO:4); PU.1 (SEQ ID NO:1), BATF3 (SEQ ID NO:3), and ID2 (SEQ ID NO:5) (SEQ ID NO:6 ... ZEB1 (PB+ZEB1) shown in SEQ ID NO:7; and PU.1 shown in SEQ ID NO:1, BATF3 shown in SEQ ID NO:3, and TRIM33 (PB+TRIM33) shown in SEQ ID NO:8. More preferably, the composition comprises a combination of at least three transcription factors, the combination of which is a combination of PU.1 shown in SEQ ID NO:1, BATF3 shown in SEQ ID NO:3, and NFIL3 shown in SEQ ID NO:6, or a combination of PU.1 shown in SEQ ID NO:1, BATF3 shown in SEQ ID NO:3, and TRIM33 shown in SEQ ID NO:8; wherein the reprogrammed cells are stem cells or differentiated cells or tumor cells and mixtures thereof that are reprogrammed or induced into dendritic cells or antigen-presenting cells.

[0016] The transcription factor composition of the present invention, wherein the stem cells or differentiated cells or tumor cells and mixtures thereof are: pluripotent stem cells or multipotent stem cells, differentiated cells, tumor cells and mixtures thereof, preferably mammalian cells, such as mouse or human cells.

[0017] According to a second aspect of the invention, a construct or vector is provided comprising a nucleic acid encoding the above-described transcription factor composition; preferably, the construct or vector comprises a combination of at least three transcription factors, wherein the sequences of the at least three transcription factors are: PU.1 encoded by SEQ ID NO:9, BATF3 encoded by SEQ ID NO:11, and NFIL3 encoded by SEQ ID NO:14, or PU.1 encoded by SEQ ID NO:9, BATF3 encoded by SEQ ID NO:11, and TRIM33 encoded by SEQ ID NO:16.

[0018] In a preferred embodiment of the present invention, the construct or vector of the present invention, wherein the combination of the at least three transcription factors is: a combination of PU.1 encoded by SEQ ID NO:9, BATF3 encoded by SEQ ID NO:11, and NFIL3 encoded by SEQ ID NO:14, or a combination of PU.1 encoded by SEQ ID NO:9, BATF3 encoded by SEQ ID NO:11, and TRIM33 encoded by SEQ ID NO:16.

[0019] In one specific embodiment, the construct or vector of the present invention, wherein the combination of the three transcription factors is arranged in a 5′ to 3′ order, for example: PU.1, BATF3, NFIL3; or PU.1, BATF3, TRIM33.

[0020] Preferably, the vector is a viral vector.

[0021] More preferably, the viral vector is selected from retroviral vectors, adenovirus vectors, lentiviral vectors, herpesvirus vectors, poxvirus vectors, or adeno-associated virus vectors.

[0022] Preferably, the construct vector containing the transcription factor composition can be the structure shown in Figure 13 or Figure 14.

[0023] According to a third aspect of the present invention, a method is provided for reprogramming or inducing stem cells, differentiated cells, tumor cells, or mixtures of these cells into dendritic cells or antigen-presenting cells, comprising the following steps:

[0024] a) Transducing cells using the constructs or vectors described in this invention; and

[0025] b) Culture virus-transduced cells in a cell culture medium that supports the growth of dendritic cells or antigen-presenting cells.

[0026] In the method described in this invention, it is preferable to culture the cells transduced by the combination of the transcription factors for at least 9 days.

[0027] In the method described in this invention, the cells are selected from pluripotent stem cells, multipotent stem cells, differentiated cells, tumor cells, or mixtures thereof;

[0028] Preferably, the differentiated cells are selected from endoderm-derived cells, mesodermal-derived cells or ectoderm-derived cells, pluripotent stem cells including mesenchymal stem cells, hematopoietic stem cells, intestinal stem cells, multipotent stem cells and cell lines.

[0029] The tumor cells mentioned include: epithelial tumors, soft tissue and bone tumors, hematopoietic and lymphatic system tumors, central nervous system tumors, head and neck tumors, thoracic tumors, digestive system tumors, genitourinary system tumors, breast tumors, skin tumors, endocrine tumors, germ cell tumors, and other rare and unclassified tumors.

[0030] More preferably, the cell is a mammalian cell, such as human or mouse fibroblasts, or human or mouse differentiated cells or cancer cells, or mammalian hematopoietic lineage cells, including monocytes, hematopoietic stem cells and progenitor cells, or mesenchymal stem cells or pluripotent stem cells.

[0031] According to a fourth aspect of the invention, induced dendritic cells or induced antigen-presenting cells obtained by the method described herein are provided.

[0032] Preferably, the induced antigen-presenting cells of the present invention contain antigens that are: tumor antigens, autoantigens, allergens, or antigens from pathogenic or infectious organisms.

[0033] According to a fifth aspect of the invention, a pharmaceutical composition is provided comprising a therapeutically effective amount of the induced dendritic cells or the induced antigen-presenting cells of the invention and a pharmaceutically acceptable excipient.

[0034] The pharmaceutical composition of the present invention may further comprise an antiviral agent, an analgesic, an anti-inflammatory agent, a chemotherapeutic agent, a radiotherapy agent, an antibiotic, a diuretic, or a mixture thereof.

[0035] In a preferred embodiment of the present invention, the pharmaceutical composition of the present invention is further used in combination with an immune checkpoint inhibitor, which yields better results than using the inhibitor alone. Therefore, the pharmaceutical composition of the present invention may further contain an immune checkpoint inhibitor, such as PD-1.

[0036] According to a sixth aspect of the invention, a cancer vaccine is provided, comprising a selection from the transcription factor composition, the pharmaceutical composition, the induced dendritic cells, the induced antigen-presenting cells, or mixtures thereof.

[0037] Furthermore, the present invention also provides the use of the aforementioned transcription factor composition, the aforementioned pharmaceutical composition, the aforementioned induced dendritic cells, the aforementioned induced antigen-presenting cells, or mixtures thereof in the preparation of a therapeutic agent for tumors.

[0038] The present invention also provides treatments for cancer or tumors, including administering the pharmaceutical compositions of the present invention, the induced dendritic cells, the induced antigen-presenting cells, or mixtures thereof to patients in need.

[0039] This invention transdifferentiates tumor cells to give them characteristics similar to classic dendritic cells (cDCs) and specialized antigen presentation capabilities. This transdifferentiation transforms tumor cells into cells that can activate the patient's own immune system, thereby activating a more effective anti-tumor immune response, particularly activating tumor antigen-specific cytotoxic T lymphocytes (CTLs) to attack tumors. This allows more patients to benefit from immune checkpoint inhibitor therapy. Attached Figure Description

[0040] Figure 1A shows the expression of CD45 and HLA-DR on the surface of U87 cells using FACS.

[0041] Figure 1B shows the collection of U87 cells on day 9 of reprogramming. FACS analysis was used to determine the proportion of fully reprogrammed (CD45+HLA-DR+) and partially reprogrammed (CD45+HLA-DR-, CD45-HLA-DR+) cells.

[0042] Figure 2A shows the expression of CD45 and HLA-DR on the surface of T98G cells using FACS.

[0043] Figure 2B shows the collection of T98G cells on day 9 of reprogramming. FACS was used to detect the proportion of fully reprogrammed (CD45+HLA-DR+) and partially reprogrammed (CD45+HLA-DR-, CD45-HLA-DR+) cells.

[0044] Figures 3A-3E show the expression of cDC1-specific markers CD11c and CLEC9A on the surface of T98G cells on day 9 of reprogramming, detected by FACS, and some replicates were performed.

[0045] Figure 4A: T98G cells CD8 on day 9 of reprogramming + CMV Tetramer + Cell proliferation rate;

[0046] Figure 4B: T98G cells on day 9 of reprogramming secrete CXCL10 under LPS stimulation.

[0047] Figures 5A-5D: T98G cells induced by the transcription factor composition for 9 days. Cell cycle progression-related genes PCNA and MCM6 were suppressed, while tumor suppressor genes RB1 and CDKN1A were reprogrammed and activated.

[0048] Figures 6A and 6B: Validation of the association between the reprogramming efficiency of candidate transcription factor combinations and cell stemness in human tumor cells with different expression levels of the cell stemness indicators CD44 and CD133;

[0049] Figures 6C and 6D: The candidate compositions PB+NFIL3 and PB+TRIM33 showed good transdifferentiation efficiency in non-small cell lung cancer A549, glioma T98G, melanoma cell lines A375 and HT144, and also showed good transdifferentiation efficiency in some pancreatic cancer Panc01 and oral squamous cell carcinoma cell lines SCC-25.

[0050] Figures 6E-6H: The expression of candidate compositions PB+NFIL3 and PB+TRIM33 in cDC1-specific markers CLEC9A and CD11c, and the expression of CD80, CD86, and CD40 in CD45+HLA-DR+ cells also correspond to transdifferentiation efficiency.

[0051] Figures 7A-7D: Validation of the reprogramming efficiency of the candidate transcription factor combination PBN and PBT on the corresponding mouse cells.

[0052] Figure 8A: Management and administration records of experimental animals after tumor tissue inoculation;

[0053] Figures 8B-8D: The candidate composition, delivered in the form of an adenovirus (ADV) vector, can activate a good anti-tumor immune response in a mouse melanoma B16-F10 CDX model;

[0054] Figures 8E-8H: The candidate composition, delivered in the form of an adenovirus vector, showed an increase in the proportion of CD8+ T cells in both peripheral blood and TIL assays;

[0055] Figures 8I-8J: The candidate composition, delivered as an adenovirus vector, activated an antitumor immune response in a mouse melanoma B16-F10 CDX model, and an increase in CD45+MHC CLASS II+ was observed compared with the control virus group.

[0056] Figure 9A: Management and administration records of experimental animals after tumor tissue inoculation;

[0057] Figure 9B: Delivery of the candidate composition in the form of an adeno-associated virus (AAV) vector did not produce a significant antitumor effect.

[0058] Figure 10A: Management and administration records of experimental animals after tumor tissue inoculation;

[0059] Figures 10B-10C: The candidate composition, delivered in the form of an adenovirus (ADV) vector, was able to activate a good antitumor immune response in a mouse Lewis lung cancer cell LLC CDX model;

[0060] Figure 10D: An increase in the proportion of CD8+ T cells was detected in the high-dose group during TIL assay.

[0061] Figure 11A: Management and administration records of experimental animals after tumor tissue inoculation;

[0062] Figure 11B: Delivery of the candidate composition in the form of an adeno-associated virus (AAV) vector did not produce a significant antitumor effect.

[0063] Figure 12A: Management and administration records of experimental animals after tumor tissue inoculation;

[0064] Figure 12B: The candidate composition PBN reprogrammed B16-F10 compared with the control virus-infected B16-F10 showed a certain degree of tumor suppression ability (TGI = 26.76%), and its inhibitory effect was not weaker than that of the control PIB group (TGI = 32.78%).

[0065] Figures 12C-12F: After the administration of the drug, an increase in CD8+OVAtetramer+ was detected in the peripheral blood of both the PBN and PIB groups.

[0066] Figure 13: ADV plasmid map:

[0067] 13A: PBN; 13B: PIB; 13C: PBT

[0068] Figure 14: AAV plasmid map;

[0069] 14A: PBN; 14B: PIB. Detailed implementation method:

[0070] Methods and Materials

[0071] Source of materials:

[0072] Unless otherwise specified, all materials and reagents used in the following experiments were purchased commercially.

[0073] Origin of human glioma cells U87 and T98G (from Shanghai Academy of Sciences)

[0074] Example 1: Transcription Factor Composition

[0075] Eight transcription factors inducing DC differentiation were identified through literature review (Table 1). These factors were cloned into lentiviral vectors, each containing at least one or two transcription factor nucleic acid sequences. Tumor cell transdifferentiation was induced using a combination of two or three transcription factors, which could be one of the following combinations:

[0076] PU.1 and IRF8 (PI), PU.1 and BATF3 (PB), PU.1, IRF8 and BATF3 (PIB), PU.1, IRF8 and ZNF366 (PI+ZNF366), PU.1, IRF8 and ID2 (PI+ID2), PU.1, IRF8 and NFIL3 (PI+NFIL3), PU.1, IRF8 and ZEB1 (PI+ZEB1), PU.1, IRF8 and TRIM33 (PI+TRIM33), PU.1, BATF3 and ZNF366 (PB+ZNF366), PU.1, BATF3 and ID2 (PB+ID2), PU.1, BATF3 and NFIL3 (PB+NFIL3 or PBN), PU.1, BATF3 and ZEB1 (PB+ZEB1), PU.1, BATF3 and TRIM33 (PB+TRIM33 or PBT).

[0077] Table 1

[0078] Table 2. Sequence Information

[0079] Example 2: Screening candidate transcription factor combinations via viral transduction

[0080] This embodiment provides a method for infecting U87 and T98G cells with a virus.

[0081] The lentiviruses packaged with transcription factors were sourced from Heyuan Biotechnology Co., Ltd., and the viruses were stored at -80°C.

[0082] Observe the growth status of U87 and T98G cells under a microscope. Cells are ready for experimentation when they reach 90% aggregation. Discard the cell culture medium and wash the cells once with PBS (phosphate-buffered saline) to remove serum and nutrients, preparing for subsequent trypsin digestion. Add an appropriate amount of 0.25% trypsin solution and incubate the cells at 37°C for 1-2 minutes until complete detachment. Add an equal volume of complete culture medium to stop trypsin digestion, then transfer the cell suspension to a 15ml centrifuge tube and centrifuge at 400g for 5 minutes, collecting the cell pellet. Resuspend the cells in an appropriate amount of complete culture medium, adjusting the U87 cell density to 8E4 cells / ml or the T98G cell density to 4E4 cells / ml. Then seed 500µl of the cell suspension into 24-well plates and incubate overnight at 37°C in a CO2 incubator to allow cell attachment and growth. The next day, discard the culture medium in the 24-well plates, add 250µl of fresh complete culture medium to each well, and incubate the cells at 37°C in a CO2 incubator for later use. Prepare a 16 μg / ml polybrene co-infection solution using serum-free complete culture medium. (Polybrene is a polycationic polymer that enhances virus-cell binding and improves infection efficiency.) Prepare a virus suspension using this co-infection solution, and infect U87 cells with an MOI of 50 or T98G cells with an MOI of 100. Then, adjust the volume of the virus suspension to 250 μL. Add the virus suspension to prepared 24-well plates and incubate the cells in a 37°C CO2 incubator.

[0083] U87 and T98G cells were selected to test the reprogramming efficiency of the transcription factor candidate composition. First, the expression of CD45 and HLA-DR on the surface of U87 and T98G cells was detected. The growth status of U87 / T98G cells was observed under a microscope. Cells were collected when the cell aggregation reached 90%, washed once with PBS, and then cultured at 4 × 10⁻⁶ cells / cells. 6 Resuspend each sample / ml in PBS / 1% FBS (FACS buffer), transfer 50 μL / well to a 96-well U-plate, and then add 50 μL of 1:50 APC-Cy in FACS buffer to each well. TM 7MouseAnti-Human CD45(BD,557833) and PerCP-Cy TM 5.5 Anti-human HLA-DR (BD, 552764) flow cytometry antibody. Incubate 96-well plates at 4°C in the dark for 30 minutes, then wash twice with 200 μL / well FACS buffer. Resuspend cells in 100 μL FACS buffer and analyze using a BD Celesta flow cytometer.

[0084] U87 cells have high immunogenicity and highly express HLA-DR (Figure 1A), while T98G cells have low immunogenicity and hardly express CD45 and HLA-DR (Figure 2A).

[0085] After transducing U87 / T98G tumor cells with a candidate composition containing three transcription factors, cells were collected on day 9, washed once with PBS, and then cultured at 4 × 10⁻⁶ cells / day. 6 Resuspend each sample / ml in PBS / 1% FBS (FACS buffer), transfer 50 μL / well to a 96-well U-plate, and then add 50 μL of 1:50 APC-Cy in FACS buffer to each well. TM 7MouseAnti-Human CD45(BD,557833) and PerCP-Cy TM 5.5 Anti-human HLA-DR (BD, 552764) flow cytometry antibody. 96-well plates were incubated at 4°C in the dark for 30 minutes, and washed twice with 200 μL / well FACS buffer. Cells were resuspended in 100 μL FACS buffer, and the expression of CD45 and HLA-DR (mouse MHC-II) on the surface of reprogrammed tumor cells was analyzed using a BD Celesta flow cytometer (Figure 1B, Figure 2B). CD45 + HLA-DR + The cell populations were named Tumor-APCs to quantify cell reprogramming efficiency. Using the reprogramming efficiency of a combination of PU.1, IRF8, and BATF3 (PIB) transcription factors on tumor cells as a reference, in U87 cells, the superior or equivalent transcription factor combinations to PIB were PI+ID2, PI+ZEB1, PB+ID2, and PB+NFIL3. In T98G cells, the superior or equivalent transcription factor combinations to PIB were PI+NFIL3, PB+ZNF366, PB+ID2, PB+NFIL3, PB+ZEB1, and PB+TRIM33.

[0086] Example 3: Reprogrammed cells induced by a transcription factor composition acquired the cDC1 phenotype and showed upregulated expression of cell surface co-stimulatory molecules.

[0087] T98G cells were collected 9 days after induction with the transcription factor composition in Example 2, and the cells were then cultured at 4 × 10⁻⁶ cells / day. 6 Resuspend each cell / ml in PBS / 1% FBS (FACS buffer), and transfer 50 μL / well to a 96-well U-plate. When detecting the cDC1 phenotype, add 50 μL of 1:50 APC-Cy in FACS buffer to each well. TM 7MouseAnti-Human CD45(BD,557833)、PerCP-CyTM 5.5 Anti-human HLA-DR (BD, 552764), APC Mouse Anti-human CD11c (BD, 560895) and PE Rat Anti-Human Clec9A (Invitrogen, 12-3709-42) flow cytometry antibodies.

[0088] When detecting co-stimulated molecular phenotypes, add 50 μL of 1:50 APC-Cy diluted in FACS buffer to each well. TM 7Mouse Anti-Human CD45(BD,557833)、PerCP-Cy TM 5.5 Anti-human HLA-DR (BD, 552764), BV421 Mouse Anti-Human CD80 (BD, 566263), APC Mouse Anti-Human CD86 (BD, 560956) and PE Mouse Anti-Human CD40 (BD, 568580) flow cytometry antibodies.

[0089] The 96-well plates were incubated at 4°C in the dark for 30 minutes, and washed twice with 200 μL / well FACS buffer. The cells were resuspended in 100 μL of FACS buffer, and the expression of cDC1-specific markers CLEC9A and CD11c in CD45+HLA-DR+ cells and the expression of CD80, CD86, and CD40 in CD45+HLA-DR+ cells were analyzed using a BD Celesta flow cytometer (Figures 3A, 3B, 3C, 3D, and 3E).

[0090] The candidate transcription factor compositions PB+ZNF366, PB+ID2, PB+NFIL3, PB+ZEB1, and PB+TRIM33 significantly upregulated the expression of CLEC9A and CD11c compared to the control empty vector virus (NC). The candidate transcription factor compositions PB+ZNF366, PB+ID2, PB+NFIL3, PB+ZEB1, and PB+TRIM33 significantly upregulated the expression of CD80, CD86, and CD40 compared to the control empty vector virus (NC).

[0091] Example 4: Reprogramming of transcription factor compositions to produce functional Tumor-APCs, triggering CD8+ T cell responses, and increasing the secretion of chemokine CXCL10 under LPS induction.

[0092] Whether Tumor-APCs acquire cDC1 cell function was determined by assessing cytokine release and in vitro stimulation to stimulate CMV antigen-specific CD8+ T cell expansion.

[0093] The candidate transcription factor composition can reprogram tumor cells into cDC1-like antigen-presenting cells, cross-presenting exogenous antigens to CD8+ T cells and expanding antigen-specific CD8+ T cells. T98G cells were harvested on day 9 of reprogramming and then cultured at a rate of 1×10⁻⁶. 5 Cells were resuspended at 1 μg / ml in MEM complete medium containing 1 μg / ml LPS, and transferred 500 μl / well to 24-well plates. The cells were incubated overnight at 37°C in a 5% CO2 incubator. The next day, cells were washed twice with 1 ml PBS and cultured again in MEM complete medium containing 20 μg / ml CMV peptide (NLVPMVATV), and incubated for 3 hours in a CO2 incubator. After 3 hours, cells were washed twice with 1 ml PBS and then incubated with 1.5 × 10⁶ cells / well. 6 PBMC (CMV) + HLA-A2 + Cells were co-cultured for 12 days in RPMI 1640 complete medium containing 10 ng / ml rhIL-2, with 50% of the old medium replaced with fresh medium during this period. On day 12, suspension cells were collected and resuspended in PBS / 1% FBS (FACS buffer). 50 μL / well was transferred to 96-well U-plates, and 50 μL of 1:50 APC anti-human CD8 (BD, 980904) and 1:25 Tetramer / PE HLA-A*02:01 CMVpp65 (NLVPMVATV) flow cytometry antibody diluted in FACS buffer were added to each well. The 96-well plates were incubated at 4°C in the dark for 30 minutes, and washed twice with 200 μL / well of FACS buffer. Cells were resuspended in 100 μL of FACS buffer, and CD8 were analyzed using a BD Celesta flow cytometer. + CMV Tetramer + Cell proliferation ratio (Figure 4A). The candidate transcription factor combination PB+NFIL3 and PB+ZEB1 significantly activated the expansion of CMV antigen-specific CD8+ T cells relative to the empty vector virus (TP004).

[0094] Tumor-APCs secreted the chemokine CXCL10 upon LPS stimulation (Figure 4B). T98G cells were harvested on day 9 of reprogramming and then... (The cells were then...) 6Cells / ml were resuspended in MEM complete medium containing 1ug / ml LPS, and 100ul / well was transferred to 96-well U plates and incubated overnight at 37°C in a 5% CO2 incubator. The next day, the cells were centrifuged at 400g for 5 minutes, and the supernatant was collected and processed using LEGENDplex cytokine assay kits. Experimental data were acquired using a BD Celesta flow cytometer and analyzed using LEGENDplex software.

[0095] Example 5: Reprogramming tumor cells with transcription factor compositions to reduce their tumorigenicity

[0096] Forced expression of a transcription factor composition within tumor cells reduced cancer cell proliferation and promoted mitotic arrest. T98G cells induced with the transcription factor composition for 9 days were collected and then... 6 Resuspend each APC-Cy at 1:50 concentration in PBS and place in a 5 ml flow cytometry tube. Then add an equal volume of 1:50 APC-Cy diluted in PBS buffer. TM 7MouseAnti-Human CD45(BD,557833)、PerCP-Cy TM 5.5 Anti-human HLA-DR (BD, 552764) flow cytometry antibody. Incubate at 4°C in the dark for 30 minutes, then wash twice with 1 ml PBS buffer. Resuspend cells in 1 ml PBS buffer and sort CD45+HLA-DR+ positive cell populations using a BD flow cytometer. PureLink was used. TM RNA was extracted from cells using an RNA micro-extraction kit (Thermo, 12183016), and RNA concentration was measured using Nanodro. Subsequently, the RNA was reverse transcribed into cDNA according to the manufacturer's instructions (BeyoRT). TM II. cDNA Synthesis Kit (D7170M). Finally, using cDNA as a template, quantitative real-time PCR was performed. Primer design was completed on Primer 5.0, and the reaction system followed the manufacturer's instructions. T98G cells were used as the control group for qPCR data processing and analysis, and 2^(2^(280-2000)) data were obtained from each experimental group. (-△△Ct) value.

[0097] Cell cycle progression-related genes PCNA and MCM6 were observed to be suppressed, while tumor suppressor genes RB1 and CDKN1A were reprogrammed and activated (Fig. 5A, B, C, D). Compared with the empty virus control group (NC), reprogrammed tumor cells reduced the tumorigenicity of cancer cells.

[0098] Based on the screening results and functional studies of Tumor-APCs, the transcription factor combinations that are superior to or equivalent to PIB were identified as: PB+NFIL3 (PBN) and PB+TRIM33 (PBT).

[0099] Example 6: The candidate transcription factor composition can induce reprogramming of various human tumor cells, enabling them to acquire DC and cDC1 phenotypes to varying degrees and upregulate the expression of co-stimulatory molecules.

[0100] In this embodiment, we hypothesize that the efficiency of reprogramming cells by the selected candidate transcription factor combinations is related to cell stemness

[0014] . Human tumor cells with different expression levels of cell stemness indicators CD44 and CD133 were selected and verified (Figure 6A, B). Lentiviral PB+NFIL3 and PB+TRIM33 were used to reprogram these human tumor cells respectively.

[0101] Breast cancer cell lines MD-MB-231 (culture medium: DMEM + 10% + 1% P / S) and HCC1937 (culture medium: RPMI) The following cell lines were used: RPMI 1640 (10% + 1% P / S); human non-small cell lung cancer cell line A549 (F-12K + 10% + 1% P / S); human oral squamous cell carcinoma cell lines HSC-3 (DMEM + 10% + 1% P / S) and SCC-25 (DMEM / F12 + 10% FBS + 400 ng / mL Hydrocortisone + 1% P / S); human pancreatic cancer cell lines Panc01 (DMEM + 10% + 1% P / S) and MIAPaCa-2 (DMEM + 10% FBS + 1% P / S + 2.5% horse serum). All of these cells were sourced from Shanghai Fuheng Biotechnology Co., Ltd. Human gastric cancer cells SNU-16 (RPMI 1640 + 10% + 1% P / S) and GCIY (DMEM + 10% + 1% P / S) were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0102] During tumor cell stemness verification, cells were harvested when the cell aggregation reached 90%, and the cells were cultured at a rate of 4 × 10⁶ cells / year. 6Cells were resuspended at 50 μL / well in PBS / 1% FBS (FACS buffer) and transferred to 96-well U-plates. PE-anti-human CD133 (Biolegend, 397903) and FITC-anti-human CD44 (Biolegend, 397518) were added. The 96-well plates were incubated at 4°C in the dark for 30 minutes. Cells were washed twice with 200 μL / well FACS buffer. Cells were resuspended in 100 μL FACS buffer, and the expression of CD133 and CD44 on the surface of tumor cells was analyzed using a BD Celesta flow cytometer.

[0103] The reprogramming method follows Example 2, using cells with a confluence of 90% for experimentation. The cell culture medium is discarded, and the cells are washed once with PBS. An appropriate amount of 0.25% trypsin solution is added, and the cells are digested at 37°C for 1-2 minutes until complete detachment. An equal volume of complete culture medium is added to terminate digestion, and the cell suspension is transferred to a 15ml centrifuge tube and centrifuged at 400g for 5 minutes to collect the cell pellet. The cells are resuspended in an appropriate amount of complete culture medium to a cell density of 4E4 cells / ml. 500µl of the cell suspension is then seeded into 24-well plates and incubated overnight at 37°C in a CO2 incubator to allow cell attachment and growth. The next day, the culture medium in the 24-well plates is discarded, and 250µl of fresh complete culture medium is added to each well. The cells are then incubated at 37°C in a CO2 incubator for later use. Prepare a 16 μg / ml polybrene co-infection solution using serum-free complete culture medium. Use this co-infection solution to prepare a virus suspension at an MOI of 100, adjusting the volume to 250 μL. Add the virus suspension to prepared 24-well plates and incubate the cells at 37°C in a CO2 incubator.

[0104] The cDC1 phenotype and co-stimulatory molecular phenotype detection methods were described in Example 3. Tumor cells induced by the transcription factor composition for 9 days were collected and the cells were cultured at 4 × 10⁻⁶ cells / day. 6 Resuspend each cell / ml in PBS / 1% FBS (FACS buffer), and transfer 50 μL / well to a 96-well U-plate. When detecting the cDC1 phenotype, add 50 μL of 1:50 APC-Cy in FACS buffer to each well. TM 7MouseAnti-Human CD45(BD,557833)、PerCP-Cy TM5.5 Anti-human HLA-DR (BD, 552764), APC Mouse Anti-human CD11c (BD, 560895) and PE Rat Anti-Human Clec9A (Invitrogen, 12-3709-42) flow cytometry antibodies.

[0105] When detecting co-stimulated molecular phenotypes, add 50 μL of 1:50 APC-Cy diluted in FACS buffer to each well. TM 7MouseAnti-Human CD45(BD,557833)、PerCP-Cy TM 5.5 Anti-human HLA-DR (BD, 552764), BV421 MouseAnti-Human CD80 (BD, 566263), APC MouseAnti-Human CD86 (BD, 560956) and PE MouseAnti-Human CD40 (BD, 568580) flow cytometry antibodies.

[0106] Incubate the 96-well plates at 4°C in the dark for 30 minutes, then wash twice with 200 μL / well FACS buffer. Resuspend the cells in 100 μL of FACS buffer and analyze the expression of cDC1-specific markers CLEC9A and CD11c in CD45+HLA-DR+ cells and the expression of CD80, CD86, and CD40 in CD45+HLA-DR+ cells using a BD Celesta flow cytometer.

[0107] The study found a strong correlation between transdifferentiation efficiency and tumor cell stemness. High expression of CD44 was observed in non-small cell lung cancer (A549), pancreatic cancer (Panc01), gastric cancer (GCIY and SNU-16), glioma (T98G), and melanoma (A375 and HT144), with correspondingly high expression of CD133. The candidate compositions PB+NFIL3 and PB+TRIM33 demonstrated good transdifferentiation efficiency in non-small cell lung cancer (A549), glioma (T98G), and melanoma cell lines A375 and HT144, and also showed good transdifferentiation efficiency in some pancreatic cancer (Panc01) and oral squamous cell carcinoma cell lines (SCC-25) (Figures 6C and D).

[0108] The expression of cDC1-specific markers CLEC9A and CD11c, and the expression of CD80, CD86, and CD40 in CD45+HLA-DR+ cells, also corresponded to transdifferentiation efficiency (Fig. 6E, F, G, H).

[0109] Example 7: The candidate composition can also reprogram the cDC1-related phenotype on different mouse cells and enhance the expression of co-stimulatory molecules, but its reprogramming efficiency is lower than that of human cells.

[0110] In this embodiment, melanoma, non-small cell lung cancer, pancreatic cancer, and glioma cell lines with high reprogramming efficiency from Example 6 were selected and verified on the corresponding mouse cells. We found that they also showed a certain degree of reprogramming efficiency (Figure 7A), which was lower than that of human cells.

[0111] B16-F10 (mouse melanoma cell line, cultured in RPMI 1640 + 10% + 1% P / S), LLC (mouse Lewis lung cancer cell line, cultured in DMEM + 10% + 1% P / S), Panc02 (mouse pancreatic cancer cell line, cultured in RPMI 1640 + 10% + 1% P / S), GL261-Luc (mouse glioma cell line, cultured in DMEM + 10% + 1% P / S); all of the above cells were obtained from the Cell Bank of the Chinese Academy of Sciences.

[0112] The mouse lentiviruses PIB, PBN, and PB+T were obtained from Heyuan Biotechnology Co., Ltd., and the viruses were stored at -80°C.

[0113] For the reprogramming of mouse tumor cells, the protocol was as described in Example 2. Cells were cultured until they reached 90% confluence for the experiment. The culture medium was discarded, and the cells were washed once with PBS. An appropriate amount of 0.25% trypsin solution was added, and the cells were digested at 37°C for 1-2 minutes until complete detachment. An equal volume of complete culture medium was added to terminate the digestion. The cell suspension was then transferred to a 15ml centrifuge tube and centrifuged at 400g for 5 minutes to collect the cell pellet. The cells were resuspended in an appropriate amount of complete culture medium to a cell density of 1E5 cells / ml. 500µl of the cell suspension was then seeded into 24-well plates and incubated overnight at 37°C in a CO2 incubator to allow cell attachment and growth. The next day, the culture medium in the 24-well plates was discarded, and 250µl of fresh complete culture medium was added to each well. The cells were then incubated at 37°C in a CO2 incubator for later use. Prepare a 16 μg / ml polybrene co-infection solution using serum-free complete culture medium. Use this co-infection solution to prepare a virus suspension at an MOI of 100, adjusting the volume to 250 μL. Add the virus suspension to prepared 24-well plates and incubate the cells at 37°C in a CO2 incubator.

[0114] The cDC1 phenotype and co-stimulatory molecular phenotype detection methods were described in Example 3. Tumor cells induced by the transcription factor composition for 9 days were collected and the cells were cultured at 4 × 10⁻⁶ cells / day. 6Resuspend each cell / ml in PBS / 1% FBS (FACS buffer), and transfer 50 μL / well to a 96-well U-plate. When detecting the cDC1 phenotype, add 50 μL of Alexa diluted in FACS buffer to each well. Flow cytometry antibodies were prepared from 700 anti-mouse CD45 Antibody (Biolegend, 103128), PE anti-mouse IA / I-EA Antibody (Biolegend, 107608), Pacific Blue anti-mouse CD11c Antibody (Biolegend, 117322), and APC anti-mouse CD370 (CLEC9A, DNGR1) Antibody (Biolegend, 143506). All antibody concentrations were diluted to 1 μg / ml.

[0115] When detecting co-stimulated molecular phenotypes, add 50 μL of Alexa diluted in FACS buffer to each well. 700anti-mouse CD45 Antibody (Biolegend, 103128), PE anti-mouse IA / IE Antibody (Biolegend, 107608), PerCP / Cyanine5.5 anti-mouse CD80 Antibody (Biolegend, 104722), Brilliant Violet421 TM Flow cytometry antibodies were prepared for anti-mouse CD86 Antibody (Biolegend, 105031) and APC anti-mouse CD40 Antibody (Biolegend, 124612), with all antibody concentrations diluted to 1 μg / ml.

[0116] Incubate the 96-well plates at 4°C in the dark for 30 minutes, then wash twice with 200 μL / well FACS buffer. Resuspend the cells in 100 μL of FACS buffer and analyze the expression of cDC1-specific markers CLEC9A and CD11c in CD45+HLA-DR+ cells and the expression of CD80, CD86, and CD40 in CD45+HLA-DR+ cells using a BD Celesta flow cytometer.

[0117] We found that the reprogramming efficiency of DCs in mouse cells was lower than that in human cells. Among them, mouse glioma cells GL261-Luc showed relatively good reprogramming efficiency. Although the CD45+HLA-DR+ phenotype of melanoma cells was lower than that of glioma cells, they showed a high increase in the co-stimulatory molecule CD80. Although the reprogramming efficiency of LLC and Panco2 was extremely low, the candidate transcription factor combination PBN and PBT group was observed to be better than or no less than that of the PIB control group (Figures 7A, 7B, 7C, 7D). Example 8: The candidate composition activated a good anti-tumor immune response in the mouse melanoma B16-F10 CDX model, significantly inhibited tumor growth, increased the proportion of peripheral blood and tumor-infiltrating CD8+ T cells, prolonged the survival of mice, and showed better anti-tumor effects when used in combination with PD-1 than single drugs.

[0118] This example explores the dosing regimens and results of the candidate compositions in different forms of carriers in a mouse melanoma B16-F10 CDX model.

[0119] We explored the antitumor effects of single-agent and combined use of two different vectors (ADV and AAV2, from Heyuan Biotechnology Co., Ltd.) to deliver candidate transcription factor compositions in a mouse melanoma B16-F10 CDX model. Peripheral blood was collected 3 days after administration to detect the proportion of CD8+ T cells, and TILs (tumor-infiltrating immune cells) were collected on day 5 after administration to detect the proportion of tumor-infiltrating CD8+ T cells.

[0120] When administering and inoculating the candidate transcription factor composition using an adenovirus vector, B16-F10 cells were cultured for 2-3 generations, then harvested. The cells were digested with an appropriate amount of trypsin, and digestion was terminated with 8-10 times the volume of complete culture medium. After mixing the cells, they were centrifuged at 1200 rpm for 5 min, and the supernatant was discarded. PBS was added and repeated twice to wash away the serum. An appropriate amount of PBS was added to resuspend the cells, and they were counted. Cells (1x10⁻⁶) were then... 5 (100 μL / mouse) was injected subcutaneously on the right side of C57BL / 6J mice until the tumor volume reached an average of 30-90 mm. 3At the age of 6, the mice were randomly divided into groups of 6: solvent control group, control virus group, composition control group and target composition low-dose group (1E8 IFU / mouse), control virus group, composition control group and target composition medium-dose group (1E9 IFU / mouse), target composition high-dose group (1E10 IFU / mouse), composition control group and target composition low-dose group combined with PD-1 antibody group (1E8 IFU / mouse + 6mpk / mouse), composition control group and target composition medium-dose group combined with PD-1 antibody group (1E9 IFU / mouse + 6mpk / mouse), and PD-1 monotherapy group (6mpk / mouse).

[0121] Day 0 was designated as grouping day, and medication was initiated on this day. Dosing intervals were every two days, for a total of four doses, administered via intratumoral injection. The mouse PD-1 antibody source was BioXcell, administered every three days, for a total of three doses, via intraperitoneal injection. Throughout the experiment, the use and observation of experimental animals were conducted in accordance with AAALAC animal use and management regulations. After tumor tissue inoculation, the experimental animals were observed daily, measured twice weekly using calipers, and tumor volume and mouse weight changes were recorded, along with medication administration records (Figure 8A).

[0122] When administering and inoculating the candidate transcription factor composition using the adeno-associated virus vector AAV2, B16-F10 cells were cultured for 2-3 passages, then harvested. The cells were digested with an appropriate amount of trypsin, and digestion was terminated with 8-10 times the volume of complete culture medium. After mixing the cells, they were centrifuged at 1200 rpm for 5 min, and the supernatant was discarded. PBS was added and repeated twice to wash away the serum. An appropriate amount of PBS was added to resuspend the cells, and they were counted. Cells (1x10⁻⁶) were then... 5 (100 μL / mouse) was injected subcutaneously on the right side of C57BL / 6J mice until the tumor volume reached an average of 30-90 mm. 3 Animals were randomly divided into groups of six: a solvent control group, a control virus group, a composition control group, and a medium-dose target composition group (1E9 IFU / animal); and a control virus group, a composition control group, and a high-dose target composition group (1E10 IFU / animal). Grouping was designated Day 0, and drug administration began on that day. Dosing was administered every two days for a total of four times via intratumoral injection. Throughout the experiment, the use and observation of the experimental animals were conducted in accordance with AAALAC animal use and management regulations. After tumor tissue inoculation, the animals were observed daily, and measurements were taken twice weekly using calipers. Tumor volume and mouse weight changes were recorded, and drug administration records were maintained (Figure 9A).

[0123] When detecting peripheral blood CD8+ T cells, 50 μL of orbital blood was collected from mice using a capillary tube, and 1xRBC Lysis Buffer (eBioscience) was added. TM 1 ml / tube (RBC:blood = 10:1) was added to the cell culture medium and incubated at room temperature for 3 min. The cells were then centrifuged at 1200 rpm for 5 min, and washed once. 500 μl of 1xRBC Lysis Buffer was added again, and the cells were incubated at room temperature for 1 min. After centrifugation to remove the supernatant, 10 times the volume of culture medium was added again and washed once more at 1200 rpm for 5 min. The cells were resuspended in 100 μl of PBS and transferred to a 96-well round-bottom plate. Alexa... 700 anti-mouse CD45 Antibody (Biolegend, 103128), PE anti-mouse CD3 Antibody (Biolegend, 100206), APC / Cyanine7 anti-mouse CD8a Antibody (Biolegend, 100714), all antibody concentrations were adjusted to 1ug / ml.

[0124] When detecting TILs, tumor-infiltrating lymphocytes were isolated using the Tumor Dissociation Kit, mouse (Miltenyi Biotec, 130-096-730) according to its instructions. The cells were then transferred to 96-well round-bottom plates. First, BD Horizon... TM Fixable Viability Stain 780 (565388) was diluted 1:1000, and 50 μL was added to each well. The mixture was incubated at room temperature for 10 min (viability staining). After incubation, the sample was centrifuged at 350 g for 5 min and washed twice with 200 μL of PBS. After removing the supernatant, BD Pharmaceuticals was added. TM Purified RatAnti-Mouse CD16 / CD32 (Biolegend, 156603), adjusted to a concentration of 1 μg / ml, was added to each well with 50 μl and incubated at 4°C for 15 min to block the FC receptor. Then, Brilliant Violet 510 was added. TM anti-mouse CD45 Antibody (Biolegend, 103138), Alexa 700anti-mouse CD3Antibody (Biolegend, 100216), Brilliant Violet 650 TManti-mouse CD8aAntibody (Biolegend, 100742), PerCP / Cyanine5.5 anti-mouse CD4Antibody (Biolegend, 116012), PE anti-mouse CD49b (pan-NK cells) Antibody (Biolegend, 108908), APC anti-mouse / human CD44Antibody (Biolegend, 103012), Pacific Blue TM Anti-mouse CD62LA antibody (Biolegend, 104424); all antibody concentrations were adjusted to 1 ug / ml.

[0125] To verify in vivo transdifferentiation, cells were isolated using the Tumor Dissociation Kit, mouse (Miltenyi Biotec, 130-096-730) following the manufacturer's instructions, and the antibody Brilliant Violet 510 was added. TM Anti-mouse CD45 Antibody (Biolegend, 103138), PE anti-mouse IA / I-EA antibody (Biolegend, 107608), all antibody concentrations were adjusted to 1ug / ml.

[0126] Incubate the 96-well plate at 4°C in the dark for 30 minutes, then wash twice with 200 μL / well FACS buffer. Resuspend the cells in 100 μL of FACS buffer and analyze the proportion of CD8+ in CD45+CD3+ and the increase in CD45+ MHC CLASS II+ using a BD Celesta flow cytometer.

[0127] We observed that delivery of the candidate composition in the form of an adeno-associated virus (AAV) vector did not produce a significant antitumor effect (Fig. 9B), but delivery in the form of an adenovirus (ADV) vector activated a good antitumor immune response in a mouse melanoma B16-F10 CDX model. The medium-dose monotherapy group (1E9 IFU / mouse) and the high-dose monotherapy group (1E10 IFU / mouse) significantly inhibited tumor growth compared to the solvent group and the control virus group, and their efficacy was no less than that of the PIB control group (Fig. 8B, C, D). In the low-dose (1E8 IFU / mouse) and medium-dose combination with PD-1 groups, we observed a stronger inhibitory effect than PD-1 monotherapy, which was statistically significant, and we observed cured individuals (Fig. 8C, 8D). We detected an increase in the proportion of CD8+ T cells in peripheral blood and TIL assays (Fig. 8E, F, G, H), demonstrating that our candidate composition, delivered as an adenovirus vector, can activate an anti-tumor immune response in a mouse melanoma B16-F10 CDX model. In addition, compared with the control virus group, we observed an increase in CD45+ MHC CLASS II+ (Fig. 8I, 8J).

[0128] Example 9: The adenovirus vector candidate composition showed good antitumor efficacy in a mouse Lewis lung cancer cell LLC CDX model, and when used in combination with PD-1, it showed better antitumor effect than single drug.

[0129] This example explores the administration regimens and results of the candidate compositions in different forms of carriers in a mouse Lewis lung cancer cell LLC CDX model.

[0130] The study explored the antitumor effects of single-drug and combined-drug administration of candidate transcription factor compositions delivered by two different vectors (ADV and AAV9, from Heyuan Biotechnology Co., Ltd.) in a mouse Lewis lung cancer cell LLC CDX model. Peripheral blood samples were collected 3 days after administration to detect the proportion of CD8+ T cells in peripheral blood. On day 5 after administration, tumor-infiltrating immune cells (TILs) were collected to detect the proportion of tumor-infiltrating CD8+ T cells.

[0131] When administering and inoculating the candidate transcription factor composition using an adenovirus vector, LLC cells were cultured for 2-3 passages, then harvested. The cells were digested with an appropriate amount of trypsin, and digestion was terminated with 8-10 times the volume of complete culture medium. After mixing the cells, they were centrifuged at 1200 rpm for 5 min, and the supernatant was discarded. PBS was added and repeated twice to wash away the serum. An appropriate amount of PBS was added to resuspend the cells, and they were counted. Cells (3 x 10⁻⁶ cells) were then... 5 (100 μL / mouse) was injected subcutaneously on the right side of C57BL / 6J mice until the tumor volume reached an average of 50-110 mm. 3At the age of 6, the mice were randomly divided into groups of 6: solvent control group, control virus group, composition control group and target composition low-dose group (1E8 IFU / mouse), control virus group, composition control group and target composition medium-dose group (1E9 IFU / mouse), target composition high-dose group (1E10 IFU / mouse), composition control group and target composition low-dose group combined with PD-1 antibody group (1E8 IFU / mouse + 6mpk / mouse), composition control group and target composition medium-dose group combined with PD-1 antibody group (1E9 IFU / mouse + 6mpk / mouse), and PD-1 monotherapy group (6mpk / mouse).

[0132] Day 0 was designated as grouping day, and medication was initiated on this day. Dosing intervals were every two days, for a total of four doses, administered via intratumoral injection. The mouse PD-1 antibody source was BioXcell, administered every three days, for a total of three doses, via intraperitoneal injection. Throughout the experiment, the use and observation of experimental animals were conducted in accordance with AAALAC's relevant regulations for animal use and management. After tumor tissue inoculation, the experimental animals were observed daily, measured twice weekly using calipers, and tumor volume and mouse weight changes were recorded, along with medication administration records (Figure 10A).

[0133] When administering and inoculating the candidate transcription factor composition using the adeno-associated virus vector AAV9, B16-F10 cells were cultured for 2-3 passages, then harvested. The cells were digested with an appropriate amount of trypsin, and digestion was terminated with 8-10 times the volume of complete culture medium. After mixing the cells, they were centrifuged at 1200 rpm for 5 min, and the supernatant was discarded. PBS was added and repeated twice to wash away the serum. An appropriate amount of PBS was added to resuspend the cells, and they were counted. Cells (3 x 10⁻⁶ cells) were then... 5 (100 μL / mouse) was injected subcutaneously on the right side of C57BL / 6J mice until the tumor volume reached an average of 50-110 mm. 3 Animals were randomly divided into groups of six: a solvent control group, a control virus group, a composition control group, and a medium-dose target composition group (1E9 IFU / animal); and a control virus group, a composition control group, and a high-dose target composition group (1E10 IFU / animal). Grouping was designated Day 0, and drug administration began on that day. Dosing was administered every two days for a total of four times via intratumoral injection. Throughout the experiment, the use and observation of the experimental animals were conducted in accordance with AAALAC animal use and management regulations. After tumor tissue inoculation, the experimental animals were observed daily, and measurements were taken twice weekly using calipers. Tumor volume and mouse weight changes were recorded, and drug administration records were maintained (Figure 11A).

[0134] When detecting TILs, tumor-infiltrating lymphocytes were isolated using the Tumor Dissociation Kit, mouse (Miltenyi Biotec, 130-096-730) according to its instructions. The cells were then transferred to 96-well round-bottom plates. First, BD Horizon... TM Fixable Viability Stain 780 (565388) was diluted 1:1000, and 50 μL was added to each well. The mixture was incubated at room temperature for 10 min (viability staining). After incubation, the sample was centrifuged at 350 g for 5 min and washed twice with 200 μL of PBS. After removing the supernatant, BD Pharmaceuticals was added. TM Purified RatAnti-Mouse CD16 / CD32 (Biolegend, 156603), adjusted to a concentration of 1 μg / ml, was added to each well with 50 μl and incubated at 4°C for 15 min to block the FC receptor. Then, Brilliant Violet 510 was added. TM anti-mouse CD45 Antibody (Biolegend, 103138), Alexa 700anti-mouse CD3 Antibody (Biolegend, 100216), Brilliant Violet 650 TM anti-mouse CD8a Antibody (Biolegend, 100742), PerCP / Cyanine5.5 anti-mouse CD4Antibody (Biolegend, 116012), PE anti-mouse CD49b (pan-NK cells) Antibody (Biolegend, 108908), APC anti-mouse / human CD44Antibody (Biolegend, 103012), Pacific Blue TM Anti-mouse CD62LA antibody (Biolegend, 104424); all antibody concentrations were adjusted to 1 ug / ml.

[0135] To verify in vivo transdifferentiation, cells were isolated using the Tumor Dissociation Kit, mouse (Miltenyi Biotec, 130-096-730) following the manufacturer's instructions, and the antibody Brilliant Violet 510 was added. TMAnti-mouse CD45 Antibody (Biolegend, 103138), PE anti-mouse IA / I-EA antibody (Biolegend, 107608), all antibody concentrations were adjusted to 1ug / ml.

[0136] Incubate the 96-well plate at 4°C in the dark for 30 minutes, then wash twice with 200 μL / well FACS buffer. Resuspend the cells in 100 μL of FACS buffer and analyze the CD8+ ratio of CD45+CD3+ on a BD Celesta flow cytometer.

[0137] Similar to the B16-F10 CDX model, we observed that delivery of the candidate composition in the form of an adeno-associated virus (AAV) vector did not produce a significant antitumor effect (Fig. 11B). However, delivery in the form of an adenovirus (ADV) vector activated a good antitumor immune response in a mouse Lewis lung cancer cell LLC CDX model. The medium-dose monotherapy group (1E9 IFU / mouse) and the high-dose monotherapy group (1E10 IFU / mouse) significantly inhibited tumor growth compared with the solvent group and the control virus group (TGI = 30.02%, 69.25%), and the efficacy was no less than that of the PIB control group (Fig. 10B, 10C). In the low-dose (1E8 IFU / mouse) and medium-dose (1E9 IFU / mouse) combination with PD-1, we observed a stronger inhibitory effect than PD-1 monotherapy, which was statistically significant (TGI = 32.99%, 52.46%). We detected an increase in the proportion of CD8+ T cells in the high-dose group in the TIL assay (Figure 10D), demonstrating that our candidate composition, delivered in the form of an adenovirus vector, can activate an anti-tumor immune response in a mouse Lewis lung cancer cell LLC CDX model.

[0138] Example 10: Cells reprogrammed from candidate transcription factor compositions have the function of dendritic cells (DCs), can present antigens in vitro, and inhibit tumor growth in a mouse melanoma B16-F10-OVACDX model.

[0139] This example explores the administration protocol and results of cell reprogramming of the candidate composition in a mouse melanoma B16-F10-OVA CDX model.

[0140] We explored the efficacy of lentivirus (derived from Heyuan Biotechnology Co., Ltd.) reprogrammed for 9 days in mice with melanoma B16-F10 cells in vitro, after co-incubation with OVA APeptide (257-264) (MCS, HY-P1489), in the form of Tumor-APC in a mouse melanoma B16-F10-OVACDX model (cells sourced from Southern Model Biotechnology Co., Ltd., culture medium: DMEM + 10% FBS + 1% P / S). Peripheral blood of mice was collected 3 days after drug administration to detect CD8+OVA tetramer+ to verify the presentation effect.

[0141] The B16-F10 in vitro reprogramming protocol was referenced in Example 2. Nine days after reprogramming, the cells were transferred to T25 flasks for culture. After the cells adhered, the cell supernatant was removed, and 5 ml of complete culture medium containing 10 ug / ml OVA peptide (SIINFEKL) and 10 ng / ml poly(I:C) (InvivoGen, tlrl-pic) was added to each flask.

[0142] When administering Tumor-APC and inoculating the B16-F10-OVACDX model, after culturing B16-F10-OVA cells for 2-3 passages, harvest the cells, digest them with an appropriate amount of trypsin, and terminate the digestion with 8-10 times the volume of complete culture medium. After mixing the cells, centrifuge at 1200 rpm for 5 min and discard the supernatant. Repeat the process twice with PBS to wash away serum, add an appropriate amount of PBS to resuspend the cells, and count them. Divide the cells (1x10⁻¹) into several sections. 5 (100 μL / mouse) was injected subcutaneously on the right side of C57BL / 6J mice until the tumor volume reached an average of 30-90 mm. 3 When the animals were large, they were randomly divided into groups of 3: control virus group, composition control PIB group, and target composition PBN group.

[0143] Day 0 was designated as the grouping day, and drug administration began on that day. Dosing was administered every 3 days for a total of 3 times. The administration method was intratumoral injection, with a dose of 2E5 cells / 30 μL / mouse per group. Three days after the end of drug administration, orbital blood was collected from the mice for analysis. Throughout the experiment, the use and observation of experimental animals were conducted in accordance with the relevant regulations for animal use and management of AAALAC. After tumor tissue inoculation, the experimental animals were observed daily, and measurements were taken twice weekly using calipers. Tumor volume and mouse weight changes were recorded, and drug administration records were maintained (Figure 12A).

[0144] When detecting peripheral blood CD8+ OVAtetramer+, 50 μL of orbital blood was collected from mice using a capillary tube and 1 x RBC Lysis Buffer (eBioscience) was added. TM1 ml / tube (RBC:blood = 20:1) was added to the culture medium and incubated at room temperature for 3 min. The cells were then centrifuged at 1200 rpm for 5 min, and washed once. 500 μl of 1xRBC Lysis Buffer was added again, and the cells were incubated at room temperature for 1 min. After centrifugation to remove the supernatant, 100 μl of culture medium was added again, and the cells were washed once more. 100 μl of PBS was added to resuspend the cells, and the cells were transferred to a 96-well round-bottom plate. BD Pharmaceuticals was added to the culture medium. TM Purified Rat Anti-Mouse CD16 / CD32 (Biolegend, 156603) was added to each well at a concentration of 1 μg / ml, and incubated at 4°C for 15 min to block the FC receptor. Then, T-Select H-2Kb OVATetramer-SINFEKL-PE (MBL, TS-5001-1C) and APC / Cyanine7 anti-mouse CD8a Antibody (Biolegend, 100714) were added, and all antibody concentrations were adjusted to 1 μg / ml.

[0145] Incubate the 96-well plate at 4°C in the dark for 30 minutes, then wash twice with 200 μL / well FACS buffer. Resuspend the cells in 100 μL of FACS buffer and analyze the CD8+OVAtetramer+ ratio using a BD Celesta flow cytometer.

[0146] The candidate composition PBN reprogrammed B16-F10 showed a certain degree of tumor suppression ability compared with the control virus-infected B16-F10 (TGI = 26.76%), and its inhibitory effect was not weaker than that of the control PIB group (TGI = 32.78%) (Figure 12B). After the administration, we detected an increase in CD8+OVAtetramer+ in the peripheral blood of both the PBN group and the PIB group (Figure 12C, 12D); after repeating this result (n = 8),

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Claims

1. A transcription factor composition for reprogramming cells in in situ transdifferentiation, wherein the composition comprises a combination of at least three transcription factors selected from the following combinations: Such as PU.1 shown in SEQ ID NO:1, IRF8 shown in SEQ ID NO:2, and ID2(PI+ID2) shown in SEQ ID NO:5; Such as PU.1 shown in SEQ ID NO:1, IRF8 shown in SEQ ID NO:2, and ZEB1(PI+ZEB1) shown in SEQ ID NO:7; Such as PU.1 shown in SEQ ID NO:1, BATF3 shown in SEQ ID NO:3, and ID 2(PB+ID2) shown in SEQ ID NO:5; Such as PU.1 shown in SEQ ID NO:1, BATF3 shown in SEQ ID NO:3, and NFIL3(PB+NFIL3) shown in SEQ ID NO:6; Such as PU.1 shown in SEQ ID NO:1, IRF8 shown in SEQ ID NO:2, and NFIL3(PI+NFIL3) shown in SEQ ID NO:6; Such as PU.1 shown in SEQ ID NO:1, BATF3 shown in SEQ ID NO:3, and ZNF366(PB+ZNF366) shown in SEQ ID NO:4; Such as PU.1 shown in SEQ ID NO:1, BATF3 shown in SEQ ID NO:3, and ZEB1(PB+ZEB1) shown in SEQ ID NO:7; and Such as PU.1 shown in SEQ ID NO:1, BATF3 shown in SEQ ID NO:3, and TRIM33(PB+TRIM33) shown in SEQ ID NO:8; The reprogrammed cells mentioned above are stem cells, differentiated cells, tumor cells, or mixtures thereof that are reprogrammed or induced into dendritic cells or antigen-presenting cells.

2. The transcription factor composition according to claim 1, wherein the composition comprises: a combination of PU.1 shown in SEQ ID NO:1, BATF3 shown in SEQ ID NO:3 and NFIL3 shown in SEQ ID NO:6, or a combination of PU.1 shown in SEQ ID NO:1, BATF3 shown in SEQ ID NO:3 and TRIM33 shown in SEQ ID NO:

8.

3. The transcription factor composition according to claim 1, wherein the cell is derived from mammalian cells.

4. The transcription factor composition according to claim 3, wherein the cells are derived from humans.

5. A nucleic acid encoding the transcription factor composition according to any one of claims 1-4.

6. A construct or vector comprising the nucleic acid of claim 5.

7. The construct or vector according to claim 6, comprising nucleic acids encoding at least three transcription factors, wherein the sequences of said at least three transcription factors are: U.1 encoded by SEQ ID NO:9, BATF3 encoded by SEQ ID NO:11, and NFIL3 encoded by SEQ ID NO:14, or PU.1 encoded by SEQ ID NO:9, BATF3 encoded by SEQ ID NO:11, and TRIM33 encoded by SEQ ID NO:

16.

8. The construct or vector according to claim 7, wherein the combination of the at least three transcription factors is: a combination of PU.1 encoded by SEQ ID NO:9, BATF3 encoded by SEQ ID NO:11, and NFIL3 encoded by SEQ ID NO:14, or a combination of PU.1 encoded by SEQ ID NO:9, BATF3 encoded by SEQ ID NO:11, and TRIM33 encoded by SEQ ID NO:

16.

9. The construct or vector according to claim 6, wherein in the construct or vector, the combination of the three transcription factors is arranged in a 5′ to 3′ sequence order.

10. The construct or vector according to any one of claims 6-9, wherein the vector is a viral vector.

11. The construct or vector according to claim 10, wherein the viral vector is selected from retroviral vectors, adenovirus vectors, lentiviral vectors, herpesvirus vectors, poxvirus vectors, or adeno-associated virus vectors.

12. The construct or vector according to claim 11, wherein the viral vector is an adenovirus.

13. A method for reprogramming or inducing stem cells, differentiated cells, tumor cells, or mixtures thereof into dendritic cells or antigen-presenting cells, comprising the following steps: a) Transducing cells using the construct or vector according to any one of claims 6-12; and b) Culture the transduced cells in a cell culture medium that supports the growth of dendritic cells or antigen-presenting cells.

14. The method of claim 13, wherein the transduced cells of step a) are cultured for at least 9 days.

15. The method of claim 13, wherein the cell is a mammalian cell.

16. An induced dendritic cell obtained by the method of any one of claims 13-15.

17. An induced antigen-presenting cell obtained by the method of any one of claims 13-15.

18. The induced antigen-presenting cell of claim 17, wherein the antigen is selected from tumor antigens, autoantigens, allergens, and antigens from pathogenic or infectious organisms.

19. A pharmaceutical composition comprising a therapeutically effective amount of the induced dendritic cells of claim 16 or the induced antigen-presenting cells of claim 17, and a pharmaceutically acceptable excipient.

20. The pharmaceutical composition of claim 19, further comprising an antiviral agent, an analgesic, an anti-inflammatory agent, a chemotherapeutic agent, a radiotherapy agent, an antibiotic, a diuretic, or a mixture thereof.

21. The pharmaceutical composition according to any one of claims 19-20, further comprising an immune checkpoint inhibitor.

22. A cancer vaccine comprising a transcription factor composition selected from any one of claims 1-4, a nucleic acid as described in claim 5, induced dendritic cells as described in claim 16, induced antigen-presenting cells as described in any one of claims 17-18, a pharmaceutical composition as described in any one of claims 19-21, or a mixture thereof.

23. Use of the transcription factor composition of claim 1, the nucleic acid of claim 5, the construct or vector of claim 6, the induced dendritic cell of claim 16, the induced antigen-presenting cell of claim 17, the pharmaceutical composition of claim 19, or the cancer vaccine of claim 22 in the preparation of a therapeutic agent for tumors.

24. The use according to claim 23, further comprising combining the transcription factor composition, nucleic acid, construct or vector, induced dendritic cells, induced antigen-presenting cells, pharmaceutical composition or cancer vaccine with an immune checkpoint inhibitor.

25. A method of treating cancer, comprising administering to a patient in need a therapeutically effective amount of the transcription factor composition of claim 1, the nucleic acid of claim 5, the construct or vector of claim 6, the induced dendritic cells of claim 16, the induced antigen-presenting cells of claim 17, the pharmaceutical composition of claim 19, or the cancer vaccine of claim 22.

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