Dendritic cell formulation and preparation method therefor

Abstract

The present invention relates to a novel dendritic cell formulation and a preparation method therefor. The cell formulation comprises PD-L1-negative dendritic cells and / or dendritic cells to the surface of which a CD40 agonist is bound. The preparation method comprises: blocking and / or activating dendritic cells with a treatment agent, wherein the treatment agent comprises a PD-L1 antibody and / or a CD40 agonist. Specifically, the method comprises: during or after dendritic cell culture, adding the treatment agent to block and / or activate the dendritic cells. The provided dendritic cell formulation, compared to conventional dendritic cells, has better cell maturity, a better antigen-presenting ability, and a stronger ability to activate and expand antigen-specific T cells, and has a wide application prospect in the field of cancer immunotherapy.

Description

A dendritic cell preparation and its preparation method Technical Field

[0001] This application belongs to the field of somatic cell therapy and relates to a novel dendritic cell preparation with the ability to activate and expand T cells and its preparation method.

[0002] Cross-references

[0003] This application claims the benefit of Chinese Patent Application No. CN2024106748147, filed on May 28, 2024, and Chinese Patent Application No. CN202410675731X, filed on May 28, 2024, the contents of which are incorporated herein by reference in their entirety.

[0004] The sequence listing file submitted at the same time

[0005] The entire contents of the following XML file are incorporated herein by reference in their entirety: Computer-readable format (CRF) sequence list (name: TFH01057PCT-Sequence listing.xml, date: 20240708, size: 5.56KB). Background Technology

[0006] Immunotherapy is a hot topic in contemporary medical research, and somatic cell therapy, as an emerging treatment method, plays a crucial role in this field. The basic principle of somatic cell therapy is to obtain specific types of cells from the patient, culture and process them in vitro to acquire special biological functions, and finally reinfuse these modified, active cells into the patient to achieve the desired therapeutic effect. Among various immune cell therapies, dendritic cells (DCs) are widely used in tumor immunotherapy, infectious disease prevention and control, and the treatment of autoimmune diseases due to their excellent antigen-presenting capabilities.

[0007] Dendritic cells (APCs) are a type of antigen-presenting cell discovered by Canadian scholar Steinman in 1973. They are named for the numerous dendritic or pseudopodia-like projections they produce when mature. Widely distributed in blood, liver, spleen, lymph nodes, and other non-immune organs and tissues, dendritic cells are currently the most potent professional antigen-presenting cells (APCs) known, and the only APCs capable of stimulating a naive T-cell response. They activate the body's T-cell immune response by efficiently capturing and processing exogenous antigens and interacting with T cells through antigen-presenting molecules on their surface. Dendritic cells play a central role in the initiation, regulation, and maintenance of the immune response within the immune system, thus playing a crucial role in the human immune system.

[0008] The preparation of dendritic cell drugs is a crucial technology. A common method involves first obtaining monocytes from the patient's peripheral blood, then inducing and activating them in vitro to enhance their antigen-presenting function, and finally reinfusing these activated dendritic cells into the patient. Because dendritic cells are extremely limited in number in the human body, accounting for less than 1% of white blood cells in peripheral blood, isolating them from vivo is not only time-consuming and laborious but also yields very small quantities, insufficient to meet large-scale clinical needs. Therefore, in vitro induction and culture are necessary. However, existing preparation methods still have some shortcomings, such as:

[0009] 1. Current induction culture methods suffer from low cell yield, poor cell viability, and low efficiency in stimulating T cells. Dendritic cells often fail to achieve sufficient activation and maturation during in vitro culture, resulting in insufficient antigen presentation capacity and difficulty in effectively stimulating T cell immune responses.

[0010] 2. Existing antigen loading methods need further optimization to improve the efficiency of dendritic cells in capturing and processing antigens, and enhance their ability to stimulate T cells.

[0011] 3. There is no standardized protocol for the in vitro culture of dendritic cells. Different laboratories and clinical centers use different culture conditions and process parameters, which affects the quality controllability of the formulation.

[0012] Meanwhile, recent research has found that dendritic cells express high levels of PD-L1 on their surface. These PD-L1 molecules bind to the dendritic cells' own B7.1 molecules, leading to dendritic cell dysfunction and inhibiting their ability to activate T cells.

[0013] Other studies have found that CD40 is a costimulatory factor and protein receptor distributed on antigen-presenting cells (APCs). When CD40 binds to CD154 (also known as CD40L) on helper T cells, the antigen-presenting cells are activated, triggering a series of downstream responses. The expression of CD40 on monocytes, macrophages, dendritic cells, and B cells plays an important role in immune cell function and anti-tumor immunity.

[0014] Therefore, the development of dendritic cell preparations with stronger activity, enhanced T cell activation capacity and antigen presentation function, as well as stable dendritic cell culture methods, is currently a research focus.

[0015] Summary of the Invention

[0016] In view of the aforementioned technical problems to be solved, the applicant has filed this application.

[0017] Based on an improved method for preparing dendritic cells, the applicant has proposed a novel, functionally enhanced dendritic cell formulation that improves the maturity and antigen-presenting capacity of dendritic cells, as well as their ability to activate and expand T cells. Specifically:

[0018] The technical solution of this application provides a novel dendritic cell preparation comprising PD-L1 negative dendritic cells and / or dendritic cells with CD40 agonists bound to their cell surface.

[0019] Furthermore, the proportion of PD-L1 negative cells in the cell preparation is ≥80% of the total cells in the preparation, and / or the proportion of cells with CD40 agonists bound to their cell surfaces is ≥80% of the total cells in the preparation.

[0020] Furthermore, the proportion of dendritic cells with a mature cell phenotype in the cell preparation is ≥50% of the total cells in the preparation.

[0021] More preferably, the proportion of dendritic cells with a mature cell phenotype in the cell preparation is ≥80% of the total cells in the preparation.

[0022] Furthermore, the proportion of CD80, CD86, HLA-DR, and CD83 positive cells in the cell preparation accounts for 50% or more of the total cells in the preparation.

[0023] More preferably, the proportion of CD80, CD86, HLA-DR, and CD83 positive cells in the cell preparation accounts for 80% or more of the total cells in the preparation.

[0024] Furthermore, the proportion of CD14-positive cells in the cell preparation is ≤20% of the total cells in the preparation.

[0025] More preferably, the proportion of CD14-positive cells in the cell preparation is ≤10% of the total cells in the preparation.

[0026] Furthermore, the sum of the proportions of CD3, CD19, and CD56 positive cells in the cell preparation is ≤20% of the total cells in the preparation.

[0027] More preferably, the sum of the proportions of CD3, CD19, and CD56 positive cells in the cell preparation is ≤10% of the total number of cells in the preparation.

[0028] More preferably, the sum of the proportions of CD3, CD19 and CD56 positive cells in the cell preparation is ≤5% of the total number of cells in the preparation.

[0029] Furthermore, the cell preparation contains PD-L1 antibodies bound to the surface of PD-L1-negative dendritic cells.

[0030] Furthermore, the PD-L1 antibody is selected from PD-L1 monoclonal antibodies or multispecific antibodies containing PD-L1.

[0031] More preferably, the PD-L1 antibody is selected from monoclonal antibodies Atezolizumab, Adebrelimab, Durvalumab, or Avelumab.

[0032] Furthermore, the CD40 agonist is selected from CD40 agonist antibodies or CD40L ligands, wherein the CD40 agonist antibodies include CD40 monoclonal antibodies and multispecific antibodies containing CD40, and the CD40L ligands include recombinant CD40L proteins and fusion proteins.

[0033] More preferably, the CD40 agonist is selected from CD40 agonist antibodies.

[0034] More preferably, the CD40 agonist antibody is selected from the monoclonal antibody Mitazalimab, Cifurtilimab, Sotigalimab, or the bispecific antibody Tecaginlimab (targeting CD40 and 4-1BB).

[0035] Furthermore, the dendritic cells are derived from human peripheral blood mononuclear cells.

[0036] Furthermore, the dendritic cells in the cell preparation described in this application are also loaded with tumor neoantigens or tumor-associated antigens.

[0037] Furthermore, the cell preparations described in this application also include cell cryopreservation solutions, cell protectants, or pharmaceutically acceptable carriers.

[0038] The technical solution of this application also provides a method for preparing dendritic cells, including: blocking and / or activating dendritic cells with a treatment agent, said treatment agent including PD-L1 antibody and / or CD40 agonist.

[0039] Furthermore, the blocking treatment includes blocking dendritic cells using a PD-L1 antibody, and the activation treatment includes activating dendritic cells using a CD40 agonist.

[0040] Furthermore, the PD-L1 antibody is selected from PD-L1 monoclonal antibodies or multispecific antibodies containing PD-L1.

[0041] More preferably, the PD-L1 antibody is selected from monoclonal antibodies Atezolizumab, Adebrelimab, Durvalumab, or Avelumab.

[0042] Furthermore, the CD40 agonist is selected from CD40 agonist antibodies or CD40L ligands, wherein the CD40 agonist antibodies include CD40 monoclonal antibodies and multispecific antibodies containing CD40, and the CD40L ligands include recombinant CD40L proteins and fusion proteins.

[0043] More preferably, the CD40 agonist is selected from CD40 agonist antibodies.

[0044] More preferably, the CD40 agonist antibody is selected from monoclonal antibodies Mitazalimab, Cifurtilimab, Sotigalimab, or bispecific antibodies Tecaginlimab.

[0045] Furthermore, the working concentration of the PD-L1 antibody is 1-50 μg / mL, and / or the working concentration of the CD40 agonist is 0.1-10 μg / mL.

[0046] More preferably, the working concentration of the PD-L1 antibody is 10-20 μg / mL, and / or the working concentration of the CD40 agonist is 1-5 μg / mL.

[0047] Furthermore, the blocking and / or activation treatment includes the step of adding the treatment agent to the dendritic cells and co-incubating, preferably, the co-incubation conditions are: temperature 35-38°C, 5% carbon dioxide.

[0048] Furthermore, the preparation method also includes a maturation-promoting culture step for dendritic cells.

[0049] Furthermore, the dendritic cells are subjected to the blocking and / or activation treatment during or after the maturation culture process. Preferably, the blocking and / or activation treatment is performed 1 to 6 hours before the completion of the culture.

[0050] Furthermore, the preparation method also includes the step of loading dendritic cells with tumor neoantigens or tumor-associated antigens.

[0051] Furthermore, the dendritic cells are loaded with tumor neoantigens or tumor-associated antigens prior to the blocking and / or activation treatment.

[0052] Furthermore, the dendritic cells subjected to the blocking and / or activation treatment are PD-L1 negative and / or dendritic cells with CD40 agonists bound to their cell surface.

[0053] Furthermore, in the dendritic cell preparation prepared by the method described in this application, the proportion of PD-L1 negative cells to the total number of cells in the preparation is ≥80%, and / or the proportion of cells with CD40 agonists bound to their cell surfaces is ≥80% of the total number of cells in the preparation.

[0054] Furthermore, this application also provides the use of the aforementioned dendritic cell preparation in the prevention, treatment, and postoperative recurrence prevention of cancer.

[0055] Furthermore, the cancer includes solid tumor cancer or hematological cancer. Preferably, the solid tumor cancer includes breast cancer, ovarian cancer, pancreatic cancer, lung cancer, liver cancer, nasopharyngeal cancer, gastric cancer, colorectal cancer, kidney cancer, bladder cancer, prostate cancer, sarcoma, esophageal cancer, cervical cancer, gallbladder cancer, glioblastoma, or melanoma. The hematological cancer includes leukemia, lymphoma, and multiple myeloma.

[0056] Furthermore, in the applications of cancer prevention, treatment, and postoperative recurrence prevention, the dendritic cell preparation can be used in combination with radiotherapy, chemotherapy, or immunotherapy drugs.

[0057] More preferably, the dendritic cell preparation can be used in combination with temozolomide (TMZ) and / or immune checkpoint inhibitors.

[0058] Invention Effects

[0059] The technical solution provided in this application yields a novel dendritic cell preparation by co-incubating dendritic cells with a PD-L1 monoclonal antibody (or a multispecific antibody containing a PD-L1 antibody), a CD40 agonist monoclonal antibody (or a multispecific antibody containing a CD40 antibody or recombinant CD40L protein). On one hand, the PD-L1 antibody effectively blocks PD-L1 molecules on the surface of dendritic cells, thereby releasing the dendritic cell co-stimulatory molecule B7.1, significantly enhancing the ability of dendritic cells to stimulate CD28 molecules on T cells, thus activating T cells and preventing them from entering a state of functional exhaustion, thereby increasing the ability of dendritic cells to activate T cells. On the other hand, the CD40 agonist antibody can bind to CD40 on the surface of dendritic cells, inducing dendritic cells to express higher levels of co-stimulatory molecules and enhance factor secretion, promoting the maturation and activation of dendritic cells, while also enhancing the antigen cross-presentation capacity of dendritic cells.

[0060] The injection solution developed using the technical solution of this application, when administered to cancer patients, can stimulate the activation and expansion of antigen-specific T lymphocytes in the patient's body. Through the specific recognition and killing of cancer cells by the activated T lymphocytes, it can play a role in treating cancer or preventing postoperative recurrence of cancer. It has high clinical safety and significant anti-cancer and cancer prevention functions, and has broad application prospects in the field of cancer immunotherapy. Attached Figure Description

[0061] Figure 1 is a schematic diagram of a mature dendritic cell structure loaded with tumor neoantigens and bound to CD40 and PD-L1 monoclonal antibodies.

[0062] Figure 2 shows the morphology of dendritic cells from multiple batches of this application observed under a confocal microscope.

[0063] Figure 3 shows the detection results of dendritic cell-related molecular markers in this application by flow cytometry.

[0064] Figure 4 shows the changes in the PD-L1 positivity rate of dendritic cells after PD-L1 monoclonal antibody blocking treatment.

[0065] Figure 5 shows the detection results of the positive rate of CD40 antibody on the surface of dendritic cells treated with CD40 monoclonal antibody.

[0066] Figure 6 shows the change in the number of T cells secreting IFN-γ stimulated by dendritic cells activated by CD40 monoclonal antibody.

[0067] Figure 7 shows a comparison of the ability of dendritic cells to activate T cells and the proportion of PD-L1+ dendritic cells in the control group and the PD-L1 monoclonal antibody treatment group in Example 3.

[0068] Figure 8 shows the blocking effect of different concentrations of monoclonal antibodies on PD-L1 molecules on the surface of dendritic cells in Example 3.

[0069] Figure 9 is a statistical chart showing the detection results of the effects of different agonists on dendritic cells in Example 4.

[0070] Figure 10 shows the detection results of dendritic cells treated with different concentrations of CD40 monoclonal antibody in Example 4.

[0071] Figure 11 shows the results of dendritic cell detection in each treatment group in Example 5.

[0072] Figure 12 shows CD8+ T cells and P53 cells before culture. R175H Flow cytometry phenotypic analysis of T and control T cells.

[0073] Figure 13 shows the results of electrical impedance method for detecting the killing effect of CTL cells on tumor cells.

[0074] Figure 14 is a statistical graph showing the changes in tumor volume in the test mice during the drug efficacy experiment.

[0075] Figure 15 shows the tumor imaging results of the subjects in Example 8.

[0076] Figure 16 shows the tumor imaging results of a subject in Example 9.

[0077] Figure 17 shows the tumor imaging results of a subject in Example 9.

[0078] Figure 18 is a flowchart of a method for preparing dendritic cells provided in this application. Detailed Implementation

[0079] The following detailed embodiments illustrate and explain the implementation methods of this application, but the following content should not be construed as limiting this application in any way. The technical terms mentioned in this specification have the same meaning as commonly understood by those skilled in the art; in case of conflict, the definitions in this specification shall prevail. Those skilled in the art, under the guidance of this application, can make many modifications without departing from the spirit and scope of the claims, and all such modifications are within the protection scope of this application.

[0080] Secondly, the terms "embodiment" or "specific implementation" used below refer to a specific feature, structure, or characteristic that may be included in at least one implementation of this application. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that excludes other embodiments. The following descriptions are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims.

[0081] This application relates to a dendritic cell preparation comprising PD-L1 negative dendritic cells and / or dendritic cells with a CD40 agonist bound to their cell surface.

[0082] In specific embodiments, the proportion of PD-L1 negative cells in the formulation provided in this application is ≥80% of the total cells in the formulation, for example, the proportion of PD-L1 negative cells is ≥81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and / or the proportion of cells with CD40 agonists bound to their cell surfaces is ≥80% of the total cells in the formulation, for example, the proportion of cells with CD40 agonists bound to their cell surfaces is ≥81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0083] In a specific implementation, the PD-L1 negative dendritic cells are dendritic cells whose cell surface is bound to PD-L1 antibodies.

[0084] In a specific embodiment, the PD-L1 antibody is selected from PD-L1 monoclonal antibodies or multispecific antibodies containing PD-L1.

[0085] In some specific embodiments, the PD-L1 monoclonal antibody is selected from monoclonal antibodies Atezolizumab, Adebrelimab, Durvalumab, or Avelumab.

[0086] In a specific embodiment, the CD40 agonist is selected from CD40 agonist antibodies or CD40L ligands. The CD40 agonist antibodies include CD40 monoclonal antibodies and multispecific antibodies containing CD40, and the CD40L ligands include CD40L recombinant proteins and fusion proteins.

[0087] In some specific embodiments, the CD40 agonist is selected from CD40 monoclonal antibodies Mitazalimab, Cifurtilimab, Sotigalimab, or bispecific antibodies Tecaginlimab.

[0088] In a specific embodiment, the dendritic cell is a dendritic cell whose cell surface is simultaneously bound to a PD-L1 antibody and a CD40 agonist, or a dendritic cell bound to a PD-L1 and CD40 bispecific antibody, or a dendritic cell bound to a multispecific antibody containing PD-L1 and CD40.

[0089] In a specific implementation, the dendritic cells are derived from human peripheral blood mononuclear cells.

[0090] In one specific embodiment, in the formulation provided by this application, the proportion of CD80, CD86, HLA-DR, and CD83 positive cells accounts for 50% or more of the total cells in the formulation, preferably 80% or more.

[0091] In one specific embodiment, the proportion of CD14-positive cells in the formulation provided in this application is ≤20% of the total cells in the formulation, preferably ≤10%.

[0092] In one specific embodiment, in the formulation provided by this application, the sum of the proportions of CD3, CD19, and CD56 positive cells to the total number of cells in the formulation is ≤20%, preferably ≤10%, and more preferably ≤5%.

[0093] In a specific embodiment, the dendritic cells in the formulation may also be loaded with tumor neoantigens or tumor-associated antigens.

[0094] In one specific implementation, tumor antigens can be loaded during the maturation-promoting stage of monocyte culture. Methods of loading antigens include, but are not limited to, adding antigenic peptides or mRNAs that encode antigen information via electrotransfer.

[0095] In specific embodiments, the formulations of this application may also include cell cryopreservation solutions, cell protectants, or pharmaceutically acceptable carriers.

[0096] In this application, those skilled in the art will fully understand that any cell cryopreservation solution can be used to preserve the formulation of this application. Those skilled in the art can select from existing commercially available cell cryopreservation solutions, from literature reports, or formulate their own using commercially available cell cryopreservation solutions. Similarly, those skilled in the art will fully understand that any cell preservative can be used to preserve the formulation of this application. Those skilled in the art can select from existing commercially available cell preservatives, from literature reports, or formulate their own using commercially available cell preservatives.

[0097] In some specific embodiments, the formulation of this application comprises a cell cryopreservation solution and a cell protectant, wherein the cell cryopreservation solution and the cell protectant constitute 40-90% of the total composition. CS10 cryopreservation solution + 5-30% human serum albumin solution + 5-30% sodium chloride injection solution.

[0098] This application also relates to a method for preparing dendritic cells, comprising: blocking and / or activating dendritic cells with a treatment agent, said treatment agent comprising PD-L1 antibody and / or CD40 agonist.

[0099] In a specific embodiment, the PD-L1 antibody is selected from PD-L1 monoclonal antibodies or multispecific antibodies containing PD-L1.

[0100] In a specific embodiment, the CD40 agonist is selected from CD40 agonist antibodies or CD40L ligands. The CD40 agonist antibodies include CD40 monoclonal antibodies and multispecific antibodies containing CD40, and the CD40L ligands include CD40L recombinant proteins and fusion proteins.

[0101] In a specific embodiment, the treatment agent includes a PD-L1 antibody and a CD40 agonist. For example, the treatment agent can be any of the following combinations: a combination of a PD-L1 monoclonal antibody and a CD40 agonist monoclonal antibody / a bispecific or multispecific antibody containing CD40 / a recombinant CD40L protein; or a PD-L1+CD40 bispecific antibody; or a PD-L1+CD40+multispecific antibody; or a combination of a PD-L1-containing bispecific or multispecific antibody and a CD40 monoclonal antibody or a recombinant CD40L protein.

[0102] In some specific embodiments, the PD-L1 monoclonal antibody is selected from monoclonal antibodies Atezolizumab, Adebrelimab, Durvalumab, or Avelumab.

[0103] In some specific embodiments, the CD40 agonist is selected from CD40 monoclonal antibodies Mitazalimab, Cifurtilimab, Sotigalimab, or bispecific antibodies Tecaginlimab.

[0104] The PD-L1 antibody, CD40 agonist antibody, and CD40L ligand described in this application can be obtained by purchasing commercially available products (such as the monoclonal antibody Atezolizumab and the monoclonal antibody Mitazalimab listed above), or can be synthesized by those skilled in the art using conventional methods.

[0105] In a specific embodiment, the working concentration of the PD-L1 antibody is ≥1 μg / mL, preferably 1-50 μg / mL, more preferably 10-20 μg / mL, for example 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 μg / mL.

[0106] In a specific embodiment, the working concentration of the CD40 agonist is ≥0.1 μg / mL, preferably 0.1-10 μg / mL, more preferably 1-5 μg / mL, for example 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 μg / mL.

[0107] In a specific embodiment of this application, the blocking and / or activation treatment includes the step of adding the PD-L1 antibody and / or CD40 agonist, etc., to dendritic cells and co-incubating them.

[0108] In some specific embodiments, the co-incubation conditions are a temperature of 35-38°C and 5% carbon dioxide. In one specific embodiment, the co-incubation conditions are a temperature of 37°C and 5% carbon dioxide.

[0109] In embodiments of this application, the preparation method further includes a maturation-promoting culture step for dendritic cells. More preferably, the blocking and / or activation treatment is performed using mature dendritic cells before the culture is completed.

[0110] In a specific embodiment, mature dendritic cells can be obtained by the following methods for use in the dendritic cell preparation described in this application or for use in the preparation method described in this application:

[0111] Mature dendritic cells were obtained by culturing mononuclear cells from human peripheral blood or CD34+ precursor cells from human bone marrow or umbilical cord blood using serum-free culture medium and a combination of differentiation-promoting factors and maturation-promoting factors.

[0112] The serum-free culture medium used in this application is not limited to any specific brand or composition; any serum-free culture medium commonly used in the art can be used. Serum contains various plasma proteins, peptides, carbohydrates, growth factors, hormones, etc. Serum composition is complex, and there are differences between batches, making consistency impossible to guarantee. Furthermore, while serum contains many components beneficial to cell growth, it inevitably contains some components that can harm cells, such as complement, antibodies, and endotoxins. Therefore, cells cultured from high-concentration serum are unsuitable for clinical applications and increase the risk of clinical allergies. Serum-free culture medium, on the other hand, has no adverse effects on the growth, differentiation, morphology, and function of dendritic cells.

[0113] In one specific implementation, the selected serum-free culture medium is a culture medium with a clearly defined chemical composition and free of animal-derived components. The manufacturing process, component testing, and release of the product comply with GMP guidelines and can be used for the in vitro preparation of clinical-grade dendritic cell drugs.

[0114] After obtaining the precursor cells, the cells were suspended in serum-free medium to a concentration of 1-5 × 10⁻⁵. 6Cells / mL were added to a combination of differentiation-promoting cytokines rhGM-CSF (Recombinant human granulocyte-macrophage colony-stimulating factor) and rhIL-4 (Recombinant human interleukin 4). The specific composition can be found in reference 1 [Laura, Ridolfi, Francesco, de Rosa, Laura, Fiammenghi et al. Complementary vaccination protocol with dendritic cells pulsed with autologous tumour lysate in patients with resected stage III or IV melanoma: protocol for a phase II randomised trial (ACDC Adjuvant Trial). [J]. BMJ Open, 2018, 8:0.]. The cell suspension was transferred to a culture flask and cultured at 37°C in a 5% CO2 incubator for 2-3 days. After adding culture medium, the cells were cultured for another 2-3 days to obtain immature dendritic cells (imDCs). Subsequently, imDCs were induced into mature DCs using a combination of maturation-promoting cytokines (including, but not limited to, rhGM-CSF, rhIL-4, and rhTNF-α). In specific embodiments, the maturation-promoting cytokines combination may be the composition described in the aforementioned literature.

[0115] In a specific embodiment, the dendritic cells undergo the aforementioned blocking and / or activation treatment during or after the maturation culture process. In one specific embodiment, the treatment is performed 1–6 hours before the completion of the maturation culture.

[0116] In embodiments of this application, the preparation method further includes the step of loading dendritic cells with tumor neoantigens or tumor-associated antigens.

[0117] In one specific implementation, the antigen is loaded prior to the blocking and / or activation treatment (immature dendritic cell stage). Methods of loading the antigen include, but are not limited to, adding the antigenic peptide to the cell culture system or electroporating mRNA encoding antigen information into the dendritic cells.

[0118] In one specific implementation, tumor neoantigen peptides are dissolved according to their solubility properties and then added to imDC cell suspension for co-incubation and culture.

[0119] Figure 18 is a flowchart of a specific embodiment of the dendritic cell preparation method provided in this application. The main steps include:

[0120] 1. Using serum-free culture medium and a combination of differentiation-promoting factors and a combination of maturation-promoting factors to culture mononuclear cells from human peripheral blood or CD34+ precursor cells from human bone marrow or umbilical cord blood;

[0121] 2. Loading antigens into immature dendritic cells: This can be done by adding antigenic peptides to the cell culture system or by electroporating mRNA encoding antigen information into the dendritic cells.

[0122] 3. Harvest the cells after co-incubating them with PD-L1 antibody (or multispecific antibody containing PD-L1) and CD40 agonist antibody (or multispecific antibody containing CD40) before the antigen-loaded dendritic cells have completed the maturation culture.

[0123] In a specific embodiment of this application, the dendritic cells subjected to the blocking and / or activation treatment are PD-L1 negative and / or dendritic cells with CD40 agonists bound to their cell surface.

[0124] In one specific embodiment, the dendritic cell preparation provided in this application is prepared by the following method:

[0125] Human monocytes were cultured using serum-free medium and a combination of differentiation-promoting and maturation-promoting factors to obtain dendritic cells with a mature phenotype;

[0126] Optionally, tumor neoantigens or tumor-associated antigens may be loaded during the maturation culture stage. The loading methods include, but are not limited to, adding antigenic peptides or electroporating mRNA encoding antigen information.

[0127] The mature dendritic cells loaded with antigens were co-incubated with CD40 agonist antibody and / or PD-L1 antibody (or a multispecific antibody containing at least one of PD-L1 and CD40).

[0128] Optionally, the treated cells can be washed, purified, and then suspended in a cell cryopreservation agent to obtain a dendritic cell preparation.

[0129] The expression levels of PD-L1, CD80, CD86, HLA-DR, CD83, CD14, CD3, CD19, and CD56, as well as the binding rate of CD40 agonists, described in this application can all be determined using conventional cellular and molecular biology detection methods in the art. For example, the expression levels of PD-L1, CD80, CD86, HLA-DR, CD83, CD14, CD3, CD19, and CD56 on the surface of dendritic cells, as well as the binding rate of CD40 agonists, can be determined using flow cytometry (FACS), immunofluorescence (IF), immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), and Western blotting. Those skilled in the art can also use other detection methods deemed appropriate. This application makes no limitation in this regard. Furthermore, the above detection methods can also be used to determine various functional indicators of dendritic cells.

[0130] For example, in this application, flow cytometry (FACS) can be used to detect the proportion of PD-L1 positive cells in the dendritic cell preparation of this application, and then the proportion of PD-L1 negative cells can be calculated by subtracting the number of PD-L1 positive cells from the total number of cells. Of course, those skilled in the art will understand that, depending on the method used, the proportion of PD-L1 negative cells in the cell preparation can also be detected directly.

[0131] In this application, flow cytometry (FACS) can be used to detect the number of cells with CD40 agonists bound to their surfaces in the dendritic cell formulation of this application, and further calculate their proportion in the cell formulation.

[0132] In one specific manner, immunofluorescence (IF) can be used to detect the number of PD-L1 positive cells in the formulation and the number of cells with CD40 agonists bound to their cell surfaces, and further calculate the proportion of PD-L1 negative cells to the total number of cells in the formulation and the proportion of cells with CD40 agonists bound to their cell surfaces to the total number of cells in the formulation.

[0133] In one specific manner, an enzyme-linked immunosorbent assay (ELISA) can be used to detect the number of PD-L1 positive cells in the formulation and the number of cells with CD40 agonists bound to their cell surfaces. The proportion of PD-L1 negative cells to the total number of cells in the formulation and the proportion of cells with CD40 agonists bound to their cell surfaces to the total number of cells in the formulation can be further calculated.

[0134] In a specific embodiment of this application, flow cytometry is used to detect the ratio of PD-L1 and CD40 molecules on the surface of dendritic cells to their corresponding antibodies. CD40 is detected directly by using fluorescently labeled anti-human IgG antibodies to detect the antibody binding level, while PD-L1 is detected indirectly by using fluorescently labeled PD-L1 antibodies to detect unblocked PD-L1 sites on the cell surface.

[0135] In one specific embodiment of this application, the flow cytometry detection method used is as follows:

[0136] After adding the cells to be tested to PBS, centrifuging and washing, and discarding the supernatant, the cells were resuspended in PBS to an appropriate cell concentration. After adding the live / dead cell dye, the mixture was mixed and incubated at room temperature in the dark for 10 minutes. After centrifugation and washing, the supernatant was discarded. Cell Staining Buffer was added to each tube to resuspend the cells. The corresponding flow cytometry antibody (as described in the method section of reference 2 [Sara, Nava, Marta, Dossena, Simona, Pogliani et al. An optimized method for manufacturing a clinical scale dendritic cell-based vaccine for the treatment of glioblastoma.[J]. PLoS One, 2013, 7:0.]) was added to each flow cytometry tube. After vortexing and mixing, the cells were incubated at room temperature in the dark for 30 minutes. After adding PBS to each tube, mixing, centrifuging, washing, and resuspending the cells, the samples were analyzed by flow cytometry to obtain the positive rates of various indicators such as CD80, CD86, HLA-DR, CD83, CD3 / CD56 / CD19, CD14, and PD-L1 on the cell surface, as well as the binding rate of cells to CD40 agonists. In this application, there are no specific limitations on the flow cytometer used.

[0137] Figure 1 is a schematic diagram of a mature dendritic cell structure loaded with tumor neoantigens and bound to CD40 and PD-L1 monoclonal antibodies, provided in a specific embodiment of this application. Figure 2 shows the microscopic morphology of dendritic cells in a specific dendritic cell injection solution provided in a specific embodiment of this application. These novel dendritic cells are stellate or polygonal in shape, with a diameter between 10-20 μm, and numerous spiky protrusions on their surface. Figure 3 shows the flow cytometry results of the cell preparation provided in a specific embodiment of this application, indicating that the positive rates of cell surface molecular markers such as CD80, CD86, HLA-DR, and CD83, which are maturation markers of dendritic cells, are all above 50%, preferably above 80%.

[0138] The dendritic cell preparation provided in this application, by flow cytometry detection of the expression level of the PD-L1 molecular marker on the surface of dendritic cells, preferably shows that the proportion of PD-L1-positive dendritic cells decreases to ≤20% (i.e., PD-L1 antibody blocking efficiency ≥80%). In one specific embodiment, the PD-L1 positivity rate of dendritic cells treated with PD-L1 monoclonal antibody decreased from 99.99% before treatment to 5.27% after treatment (as shown in Figure 4), indicating that the PD-L1 site was effectively blocked. Simultaneously, flow cytometry detection of the proportion of CD40 molecules binding to CD40 antibodies on the surface of dendritic cells shows that, preferably, the proportion of dendritic cells bound to CD40 monoclonal antibody or multispecific antibodies containing CD40 increases to ≥80%. In one specific embodiment, the positive rate of CD40 antibody on the surface of dendritic cells treated with CD40 monoclonal antibody significantly increased, from 0% before treatment to 99.88% after treatment (as shown in Figure 5).

[0139] In addition, the ability of dendritic cells to activate antigen-specific T cells was detected using the classic Elispot method (enzyme-linked immunospot assay). (T cells that secrete IFN-γ cytokines are considered to be antigen-specific T cells activated by dendritic cells). The number of activated T cells stimulated by dendritic cells activated with CD40 monoclonal antibody increased by 35.5% compared with the untreated group (as shown in Figure 6).

[0140] Compared to conventional dendritic cells, the innovation of this application lies in co-incubating mature dendritic cells loaded with antigens with a CD40 agonist and / or a PD-L1 antibody (or a multispecific antibody containing at least one of CD40 and PD-L1 antibodies), followed by washing, purification, and cryopreservation to obtain a dendritic cell injection solution with strong anti-cancer / tumor-preventive effects. Preferably, the cryopreservation protectant for the dendritic cells comprises 40-90% of the cell culture. CS10 cryopreservation solution + 5-30% human serum albumin solution + 5-30% sodium chloride injection. Preferably, the proportion of PD-L1 negative dendritic cells is ≥80%, and the proportion of dendritic cells bound to CD40 agonists is ≥80%.

[0141] Compared with conventional dendritic cell culture methods, this application innovatively adds PD-L1 antibody (or PD-L1-containing multispecific antibody) and CD40 agonist (including CD40 agonist monoclonal antibody or CD40-containing multispecific antibody or CD40L recombinant protein, etc.) to the dendritic cell preparation stage to enhance the function of dendritic cells. On the one hand, it blocks PD-L1 molecules on the surface of mature dendritic cells, enhancing the ability of dendritic cells to activate T cells; on the other hand, it activates CD40 receptors on the surface of dendritic cells, improving the expression of surface co-stimulatory molecules and the efficiency of antigen presentation, thereby enhancing their ability to stimulate and activate T cells.

[0142] To further demonstrate the ability of dendritic cells in this application to activate naïve antigen-specific T cells (CTL cells), and the ability of activated CTL cells to specifically recognize and kill tumor target cells, the following experiments were designed for verification:

[0143] To express p53 R175H The human endometrial cancer cell line KLE, expressing the neoantigen (HMTEVVRHC), was used as the target cell line, and the human pancreatic cancer cell line CFPAC-1, which does not express the neoantigen, was used as the negative control cell line (both cell lines were HLA-A*02:01 genotyped). Immunodeficiency dendritic cells (imDCs) were cultured from healthy donor monocytes with the same HLA genotype, and Neo-DCs were prepared (loaded with p53 during the maturation phase). R175H Neo-DCs (mature dendritic cells) were obtained by treating the neoantigen peptide with CD40 and PD-L1 antibodies, and Mock DCs (mature dendritic cells with the same loading but without PD-L1 and CD40 monoclonal antibody treatment served as controls). Neo-DCs and Mock DCs were used to stimulate CD8+ T cells from the same healthy donor in vitro for 10 days to harvest p53 cells. R175H T and control T cells. The composition of these CD8+ T cells obtained from in vitro stimulation was analyzed by flow cytometry, and their cytotoxic effects on target cells and negative control tumor cells were detected. The experimental results confirmed that Neo-DC cells had a better ability to induce CD8+ T cell activation than Mock DC cells, yielding antigen-specific T cells (CTL cells) containing a high proportion of effector memory T cell subsets. These CTL cells exhibited a strong ability to kill tumor cells.

[0144] To evaluate the antitumor effect of the dendritic cells described in this application in a tumor model of humanized huPBMC-NCG mice subcutaneously transplanted with human pancreatic cancer cells CFPAC-1, the following experiment was designed: Human pancreatic cancer cells CFPAC-1 in logarithmic growth phase were collected and subcutaneously inoculated into huPBMC-NCG mice. When the average tumor volume of the mice reached a certain level, they were randomly divided into three groups (G1-G3) based on body weight and tumor volume, with six mice in each group. Each mouse received an intraperitoneal injection of 100 μL of PBMCs with HLA-A*02:01 typing for humanized immune system reconstruction. The day of injection was recorded as D0. Simultaneously, Neo-DC cells (loaded with human pancreatic cancer cells KRAS) were prepared using monocytes from the same donor according to the cell preparation procedure described in this application. G12V Neo-antigen peptides generated by mutations (mature dendritic cells treated with CD40 and PD-L1 antibodies) and Mock DC cells (mature dendritic cells loaded with the same neo-antigen peptides but not treated with CD40 and PD-L1 antibodies) were used. Mice in the G1 group (immune reconstitution completed) were subcutaneously injected with the solvent as a control; mice in the G2 group were subcutaneously injected with Mock DC cells; and mice in the G3 group were subcutaneously injected with the same dose of Neo-DC cells. Administration was performed via two subcutaneous injections at the groin area of ​​the hind leg of mice, administered on days 14, 18, 22, and 30 after immune reconstitution, for a total of four administrations. The pharmacodynamics of the test substance in a humanized huPBMC-NCG mouse tumor model with subcutaneous transplantation of human pancreatic cancer cells CFPAC-1 were evaluated by combining observations of tumor volume changes, body weight, CBA analysis, GvHD score, and peripheral blood immune cell detection results during and after administration. The experimental results ultimately showed that repeated subcutaneous injections of Neo-DC cells and Mock DC cells into human PBMC-reconstructed immunodeficient mice (with subcutaneous tumor formation of human pancreatic cancer cells) were effective in inhibiting tumor growth, with statistically significant differences compared to the solvent control. Furthermore, Neo-DC cells showed a more significant effect in inhibiting tumor growth than Mock DC cells. This indicates that subcutaneous injection of Neo-DC cells has a stronger inhibitory and therapeutic effect on human pancreatic cancer lesions in humanized huPBMC-NCG mice.

[0145] Several human clinical trials of the Neo-DC cell injection solution of this application, conducted as a single agent or in combination with one or more of PD-1 antibodies, PD-L1 antibodies, temozolomide, etc., have shown that the Neo-DC cell injection solution has good clinical safety and has certain clinical efficacy for patients with pancreatic cancer, liver cancer, and glioblastoma, whether used as a single agent or in combination.

[0146] The above demonstrates that the Neo-DC cell injection of this application possesses good anti-cancer and cancer-preventive activity and clinical accessibility, showing broad application prospects in the field of immunotherapy for cancer and other diseases. It can be used alone or in combination with radiotherapy and chemotherapy drugs such as PD-1 antibodies, PD-L1 antibodies, and temozolomide for the clinical treatment of cancer or postoperative recurrence prevention. The cell-based drugs and / or combination drugs of this application are applicable to solid tumors such as breast cancer, ovarian cancer, pancreatic cancer, lung cancer, liver cancer, nasopharyngeal carcinoma, gastric cancer, colorectal cancer, kidney cancer, bladder cancer, prostate cancer, sarcoma, esophageal cancer, cervical cancer, gallbladder cancer, glioblastoma, or melanoma, as well as hematological cancers such as leukemia, lymphoma, and multiple myeloma. Compared with existing traditional dendritic cell culture methods, this application, by adding PD-L1 antibodies and / or CD40 agonists during dendritic cell culture, significantly improves the maturity, antigen-presenting ability, and ability to activate specific T cells of dendritic cells, thereby enhancing their clinical anti-cancer and cancer-preventive effects.

[0147] Example

[0148] This application provides a general and / or specific description of the materials and experimental methods used in the experiments. In the following examples, unless otherwise specified, % represents volume percentage. Reagents or instruments used, unless otherwise specified, are all commercially available conventional reagent products. imDC cells and mDC cells are immature dendritic cells and mature dendritic cells, respectively, cultured using the methods of Examples 1 and 2 of this application. The Neo-DC cells described in this application are mature dendritic cells loaded with the corresponding antigen, cultured using the method of Example 6 of this application.

[0149] Example 1: Methods for preparing mononuclear cells and culturing imDC (immature dendritic cells)

[0150] The imDC cell culture method includes four steps: obtaining blood from apheresis or peripheral blood, PBMC isolation and purification, mononuclear cell sorting and purification, and imDC cell culture.

[0151] 1. Blood collection or peripheral blood acquisition

[0152] On the day of collection or the day before, the donor undergoes routine blood tests, coagulation tests, and infectious disease screening to confirm that the patient is free of active hepatitis B, hepatitis C, syphilis, HIV, and other infectious diseases. Peripheral blood is collected via apheresis or direct venous collection using the white blood cell mode of a blood cell separator. After the collected blood samples are transported to the laboratory, blood samples that pass the initial quality confirmation proceed to the next cell sorting step, with strict aseptic technique followed.

[0153] 2. PBMC Isolation and Purification

[0154] The day blood samples were first processed was recorded as Day 0 of culture. Plasma was removed from the blood samples. An appropriate amount of PBS solution was added to the blood cell sample using a pipette. The diluted blood was slowly loaded onto the surface of the Ficoll solution and centrifuged for 30 minutes. After centrifugation, the white membrane layer was carefully transferred to a new centrifuge tube using a pipette. PBS was added to the tube for dilution and mixing, followed by centrifugation. After washing again, the cell pellet was resuspended in sorting buffer to form a PBMC cell suspension.

[0155] 3. Mononuclear cell sorting and purification

[0156] The MACS technology was used to sort mononuclear cells, and the specific purification procedure was as follows:

[0157] Based on the PBMC cell counting results from the previous step, adjust the cell density to a suitable level using sorting buffer, add CD14 magnetic beads, mix well, and incubate at 2-8℃ for 15 minutes. Then, add 1×10⁻⁶ cells per cell. 8 Add 10-20 mL of buffer to each cell, mix well, centrifuge, discard the supernatant, resuspend the cells in buffer, adjust the volume, centrifuge again, discard the supernatant, and resuspend the cells in buffer. Place the cell sorting column in a magnetic sorting rack, rinse the column with buffer, and add the cell suspension evenly to the sorting column. After all the cell suspension has flowed down, wash the column with buffer and remove the magnetic rack. Collect CD14+ cells at the bottom of the sorting column, centrifuge, and resuspend in serum-free medium to obtain a CD14+ mononuclear cell suspension. Count the cell concentration and viability.

[0158] 4. Immediately synthesized dendritic cells (imDCs) were obtained from mononuclear cell differentiation culture.

[0159] Adjust the mononuclear cell suspension obtained in the above steps to a concentration of 1-5 × 10⁻⁵ using serum-free culture medium. 6 Cells / mL were added to a combination of differentiation-promoting cytokines rhGM-CSF and rhIL-4, and the cell suspension was transferred to a culture flask. The flask was then incubated at 37°C in a 5% CO2 incubator for 2-3 days. After adding the culture medium, the cells were cultured for another 2-3 days to obtain immature dendritic cells (imDCs).

[0160] Example 2: mDC (mature dendritic cell) culture, antigen loading, and treatment methods

[0161] imDC cells were collected and cultured for maturation. The required amount of cytokines was calculated based on the volume of the maturation-promoting medium. Various maturation-promoting cytokines were added to the serum-free medium (the combination of maturation-promoting cytokines includes, but is not limited to, rhGM-CSF, rhIL-4, rhTNF-α, etc., for example, the maturation-promoting factor composition described in Reference 1). Antigen peptides (specific selections are given in the examples below) were dissolved according to their solubility properties. The peptide solutions were filtered through a membrane and added to the imDC cell suspension. The imDC cells were then seeded into culture flasks and placed back into a 37°C, 5% CO2 incubator for further culture.

[0162] On days 6 or 7 (approximately one day before the end of maturation) of dendritic cells undergoing accelerated maturation culture, a working concentration of PD-L1 monoclonal antibody and a DC cell agonist (specific selections are described in the examples below) are added to the culture medium. The cells are then incubated at 37°C in a 5% CO2 incubator for 1–6 hours. Afterward, the cells are removed and their morphological characteristics are observed and recorded under an inverted microscope. The cells are collected in centrifuge tubes, centrifuged, and samples are retained for quality testing. The cell pellet is resuspended in washing buffer and counted. Based on the counting results, an appropriate amount of cell suspension is retained for testing of viable cell concentration, cell viability, and cell surface molecular markers such as CD80 / CD86 / HLA-DR / CD83 / PD-L1. After sampling, the obtained cells are centrifuged again, the supernatant is discarded, and the cells are used for subsequent assays.

[0163] Furthermore, cryoprotectants (prescription ingredients 40-90%) can be formulated according to the concentration standards for clinical use, based on cell count results. The cell pellet was resuspended in CS10 cryopreservation solution (5-30% human serum albumin solution + 5-30% sodium chloride injection), and an appropriate sample was taken for safety testing. Then, the cell suspension resuspended in the cryopreservation agent was precisely measured using a pipette and filled and frozen according to established standards.

[0164] Example 3: Determination of the effect of PD-L1 monoclonal antibody treatment on enhancing mDC cell function

[0165] This study used two marketed humanized monoclonal antibodies, adebrelimab and atezolizumab, for experimental purposes. The aim was to first determine whether co-incubation of PD-L1 monoclonal antibodies with mDC cells could effectively reduce the proportion of PD-L1-expressing mDC cells, and whether it could subsequently enhance the ability of mDC cells to activate T cells.

[0166] Experimental Methods: Following the methods described in Examples 1 and 2, HLA-A*02:01 genotyped healthy donor mononuclear cells were cultured in vitro to obtain imDCs (imDCs). These cells were then loaded with cytomegalovirus (CMV) peptides and cultured to promote maturation. Subsequently, different PD-L1 antibody treatments were performed according to the following experimental groups: A. Conventional control group (no PD-L1 antibody added to the culture system); B. Adebrelimab group; C. Atezolizumab group. Both antibody treatments were added to the culture system one day before the completion of mDC cell culture and incubated at 37°C for 3 hours. After incubation, the mDCs were harvested and washed, followed by cell counting, viability testing, and flow cytometry phenotyping to examine the blocking effect of PD-L1 molecules on the DC surface. The Elispot method was used to detect the ability of mDCs to activate T cells. The results of the three batches of experiments are shown in Table 1 and Figures 7(a) and (b).

[0167] Table 1. Statistical analysis of mDC cell detection data in the control group and PD-L1 monoclonal antibody treatment group.

[0168] Experimental results showed that when the concentration of PD-L1 monoclonal antibody was 2 μg / mL, there were significant differences in the activation capacity of mDC cells for T cells between the control group, the Adebrelimab group, and the Atezolizumab group, but there was no significant difference between the two antibody groups. Analysis of the number of T cells secreting IFN-γ after activation by mDC cells using the Elispot assay (Figure 7(a)) showed that the mean number in the control group was 1817, the mean number in the Adebrelimab group was 2261 (an increase of 24.4%), and the mean number in the Atezolizumab group was 2253 (an increase of 24.0%). This indicates that the addition of a PD-L1 inhibitor (PD-L1 antibody) binds to PD-L1 on the surface of DCs, enabling DC cells to better activate T cells. Regarding the PD-L1 blocking effect of monoclonal antibodies (Figure 7(b)), both antibody treatment groups achieved over 95% blocking effect on PD-L1 on the DC surface (the mean number of DC cells with PD-L1+ ratio was 99.9% in the control group, 1.3% in the adebrelimab group, and 1.4% in the atezolizumab group, respectively). Based on the analysis of the above indicators, it is confirmed that adding PD-L1 antibody blocking treatment before mDC cell harvesting can effectively block PD-L1 and significantly enhance the functional activity of DC cells.

[0169] Further, the working concentration of Atezolizumab monoclonal antibody was explored and optimized, and the blocking effect of different gradient concentrations of monoclonal antibody on PD-L1 molecules on the DC surface was detected. The results are shown in Table 2 and Figure 8 below.

[0170] Table 2. Results of PD-L1+ ratio detection in mDC cells treated with different concentrations of PD-L1 monoclonal antibody

[0171] The results showed that as the concentration of PD-L1 monoclonal antibody treatment increased, the proportion of PD-L1+ in mDC cells decreased, exhibiting an S-shaped curve. When the concentration of Atezolizumab monoclonal antibody was ≥1 μg / mL, it could block more than 98% of PD-L1 molecules on the surface of mDC cells.

[0172] Example 4: Determination of the effect of DC cell agonist treatment on enhancing mDC cell function

[0173] In this embodiment, five DC cell agonists were selected for experiments: agonist A (OK432), agonist B (PolyI:C), agonist C (CD40 monoclonal antibody Cifurtilimab), agonist D (CD40 monoclonal antibody Mitazalimab), and agonist E (CD40L recombinant protein) to verify the effects of different agonists on the quality and function of mDCs.

[0174] Experimental Methods: Following the methods described in Examples 1 and 2, HLA-A*02:01 genotyped healthy donor mononuclear cells were cultured in vitro to obtain imDCs (mDCs). These imDCs were then loaded with cytomegalovirus (CMV) peptides and cultured to promote maturation. On the last day of the dendritic cell maturation culture, different agonists (working concentration 2 μg / mL) were added to the culture medium according to the settings of the five experimental groups described above. A control group (mDC cultured using conventional methods) was also included. After culture, the mDCs were harvested and washed. Subsequently, mDC cell counting and viability were performed, along with flow cytometry phenotyping and Elispot assays to determine the ability of DCs to activate T cells. Differences between groups were analyzed, and yield, viability, factor secretion, maturity, and antigen presentation were compared to determine the effects of different agonists on the quality and function of mDCs. The experimental results are shown in Table 3 and Figure 9.

[0175] Table 3. Statistical analysis of mDC cell detection data in different agonist treatment groups

[0176] Experimental results showed that, compared with the control group, all five agonists could enhance the maturation of dendritic cells (DCs), factor secretion, and T cell activation to varying degrees. However, the treatment effects of CD40 monoclonal antibody and recombinant CD40L protein were significantly better than those of OK432 and PolyI:C. The Cifurtilimab and Mitazalimab groups showed the most significant enhancement in T cell activation by mDCs, with no significant difference between the two antibody groups. The enhancement effect of CD40L was second only to Cifurtilimab. Subsequently, the working concentration of Cifurtilimab was explored and optimized. The effects of different concentrations of the monoclonal antibody on the maturation phenotype of DCs (CD83 molecules), IL-12 factor secretion, and T cell activation were detected. The results are shown in Table 4 and Figure 10.

[0177] Table 4. Statistical analysis of mDC cell detection data after different CD40 monoclonal antibody concentrations.

[0178] The results showed that with increasing CD40 monoclonal antibody concentration, the secretion of CD83 molecules and IL-12 factor in mDC cells and their ability to activate T cells all increased, exhibiting an S-shaped curve. When the concentration of Cifurtilimab monoclonal antibody was ≥0.1 μg / mL, it could significantly enhance various functional and activity indicators of mDC cells.

[0179] Example 5: Determination of the effect of combined treatment with PD-L1 monoclonal antibody and DC cell agonist on enhancing mDC function

[0180] This embodiment uses PD-L1 monoclonal antibody (Atezolizumab), a combination of PD-L1 monoclonal antibody (Atezolizumab) and CD40 monoclonal antibody (Cifurtilimab), and PD-L1 and CD40 bispecific antibodies for experiments (CD40-PD-L1-IgG4 bispecific antibody is a bispecific antibody containing two parts, Fab and Fc, synthesized by Sinopharm Biotechnology Co., Ltd. on behalf of the applicant. It contains four polypeptide chains (i.e., two light chains and a heavy chain). The two branches of Fab bind to different antigens (i.e., CD40 and PD-L1). It adopts CrossMab design, the constant region remains unchanged, and the variable region of the anti-PD-L1 light chain is exchanged and linked with the variable region of the PD-L1 heavy chain, so that the light chain of anti-CD40 is paired with the heavy chain of anti-CD40, and the light chain of anti-PD-L1 is paired with the heavy chain of anti-PD-L1. The sequences of CD40_L, CD40_H, PD-L1_L and PD-L1_H used are shown as SEQ ID No. 1-4, respectively).

[0181] Experimental Methods: Following the methods described in Examples 1 and 2, HLA-A*02:01 genotyped healthy donor mononuclear cells were cultured in vitro to obtain imDC cells. These cells were then loaded with cytomegalovirus (CMV) peptides and subjected to imDC cell maturation-promoting culture. The following experimental groups were established: A. Control group (conventional DC culture maturation-promoting factor combination plus PD-L1 monoclonal antibody treatment); B. Monoclonal antibody combination (conventional DC culture maturation-promoting factor combination plus PD-L1 and CD40 monoclonal antibody treatment); C. Bispecific antibody group (conventional DC culture maturation-promoting factor combination plus PD-L1×CD40 bispecific antibody). Antibody treatment involved adding the corresponding antibody to the culture system before the completion of mDC cell culture and incubating at 37°C for 3 hours. After incubation, mDCs were harvested and washed, followed by mDC counting and viability detection, flow cytometry phenotyping, and Elispot assay to determine the ability of DCs to activate T cells. Differences between groups were analyzed, and yield, viability, factor secretion and maturation, as well as antigen presentation were compared to determine the effects of monoclonal antibodies and / or bispecific antibodies on mDC quality and function. The experimental results are shown in Table 5 and Figure 11.

[0182] Table 5. Statistics of mDC cell detection data for each treatment group

[0183] The experimental results showed that there were no significant differences in mDC cell yield and viability between the monoclonal antibody combination group, the bispecific antibody group, and the control group (mean yields of the control group, monoclonal antibody combination group, and bispecific antibody group were 28.8%, 28.5%, and 27.3%, respectively; mean viability was 87.5%, 87.5%, and 86.3%, respectively). Regarding DC maturity (represented by the proportion of CD83+ cells), the mean CD83+ proportions in the three groups were 80.35%, 95.72%, and 95.63%, respectively, indicating that adding either of the two monoclonal antibody combinations or the bispecific antibody treatment significantly improved DC cell maturity, with CD83 positivity rates increasing by 19.1% and 19.0%, respectively. Regarding the IL-12 cytokine secretion index, the mean values ​​for the three groups were 4.9, 746.3, and 704.1 pg / mL, respectively, indicating that the addition of two monoclonal antibodies or bispecific antibodies significantly enhanced the ability of mDC cells to secrete cytokines. For the IFN-γ secreting cell count, which reflects the antigen-presenting capacity of mDCs, there were also significant differences among the three groups, with mean values ​​of 1936, 2359, and 2413, respectively. The monoclonal antibody combination and the bispecific antibody group showed increases of 21.8% and 24.6% respectively compared to the control group, indicating that the addition of two monoclonal antibody combinations or bispecific antibodies significantly enhanced the ability of DC cells to activate T cells, resulting in a significant increase in the number of T cells secreting IFN-γ. Based on the above analysis, it was determined that co-incubation treatment with PD-L1 monoclonal antibody and CD40 monoclonal antibody or PD-L1×CD40 bispecific antibody before DC harvest can promote DC maturation, cytokine secretion, and enhance the ability to activate T cells without significantly affecting the yield and viability of mDC cells.

[0184] Example 6: Dendritic cell-induced activation of cytotoxic T lymphocytes (CTLs) and in vitro killing of tumor cells.

[0185] To further demonstrate the ability of dendritic cells presenting tumor neoantigens to activate naïve antigen-specific T cells (CTL cells), and the ability of activated CTL cells to specifically recognize and kill tumor target cells, the following experiment was designed:

[0186] To express p53 R175H The human endometrial cancer cell line KLE, which expresses the neoantigen (HMTEVVRHC), was used as the target cell line, and the human pancreatic cancer cell line CFPAC-1, which does not express this neoantigen, was used as the negative control cell line (both cell lines were HLA-A*02:01 genotyped). Immunodeficiency dendritic cells (imDCs) were cultured from healthy donor monocytes with the same HLA genotype and loaded with the antigen (p53 in this example) during the maturation phase. R175HNeo-DC cells were mature dendritic cells (hereinafter referred to as Neo-DC cells) loaded with the corresponding antigen and treated with CD40 antibody (Cifurtilimab, 2 μg / mL) and PD-L1 antibody (Atezolizumab, 2 μg / mL). Mature dendritic cells of the same origin but not treated with PD-L1 and CD40 monoclonal antibodies were used as control cells (hereinafter referred to as Mock DC). The specific culture, loading, and antibody treatment methods are as described in Examples 1 and 2. Neo-DC and Mock DC cells were used to stimulate CD8+ T cells from the same healthy donor in vitro for 10 days to obtain p53 cells. R175H T cells and control T cells. The composition of these CD8+ T cells obtained from in vitro stimulation was analyzed by flow cytometry, and the killing effect of these cells on target cells and negative control tumor cells was detected. The experimental results are shown in Figure 12.

[0187] Results and Analysis: Cell surface glycoprotein CD69 and chemokine receptor CXCR3 are important biomarkers of effector T cell activation. Memory is one of the important biological characteristics of effector T cells, and T cell memory is formed during the proliferation and differentiation of antigen-specific T cell clones. Naïve T cells ( Activated T cells (CCR7+CD45RA+) can differentiate into central memory T cells (TCM, CCR7+CD45RA-), effector memory T cells (TEM, CCR7-CD45RA-), and terminally differentiated effector memory T cells (Teff, CCR7-CD45RA+). Reactive memory of T cells is mediated by TCM, which proliferates and differentiates into effector cells in response to antigen stimulation. Protective memory is mediated by TEM, which migrates to peripheral tissues to exert immediate effector functions when needed. Flow cytometry analysis of CD8+ T cells derived from the same healthy donor after 10 days of stimulation of Neo-DC and Mock DC cells, using pre-stimulation CD8+ T cells as the baseline sample, showed that the expression level of CXCR3 on the surface of CD8+ T cells before stimulation was 0.55%, and after stimulation, p53... R175H The expression levels of CXCR3 on the surface of T cells and control T cells reached 78.2% and 73.9%, respectively, both significantly higher than before culture. This indicates that both Neo-DC cells and Mock DC cells can effectively activate CD8+ T cells. The experiment also found that p53... R175HThe proportions of TCM cell subsets in T and control T cells were 7.88% and 5.59%, respectively, both significantly higher than the 1.84% before culture; the proportions of TEM cell subsets were 54.8% and 28.9%, respectively, both significantly higher than the 7.19% before culture. This indicates that both Neo-DC cells and Mock DCs can significantly induce CT8+ T cells to differentiate into central memory T cells and effector memory T cell subsets, but Neo-DC cells have a stronger induction function, with the proportions of central memory T cells and effector memory T cell subsets induced being 1.4 times and 1.9 times that of control T cells, respectively.

[0188] To detect p53 R175H The ability of effector T cells in T and control T to recognize and kill tumor cells will be enhanced by the expression of p53. R175H Neoantigen-bearing tumor cells KLE (abbreviated as KLE) mut As target cells, tumor cells expressing wild-type p53, CFPAC-1, served as negative control cells (abbreviated as CFPAC-1). wt ), and the p53 obtained in the aforementioned experiment R175H T cells or control T cells were co-cultured with two different cell types at different effector-to-target ratios of 2.5:1, 5:1, and 10:1. Target cell death was monitored using an RTCA cell analyzer, and the cytotoxic activity of T cells against tumor cells was calculated. The cytotoxic effect of CTL cells against tumor cells was detected using the analyzer's impedance method. 1 x 10⁻⁶ cells per well. 4 A density of cells per 100 μL will reduce tumor cells (KLE) mut Or CFPAC-1 wt The cells were seeded into a 96-well microplate for electrical resistance detection, and effector cells (p53) were added in different proportions after 24 hours. R175H The system resistance change was detected using either T or control T, and the cytotoxicity of effector cells over 6 hours was calculated as follows: Cytolysis = (CI of cells without T cells - CI of experimental cells) / CI of cells without T cells × 100%. The experimental results are shown in Figure 13, where A: effector-to-target ratio was 2.5:1; B: effector-to-target ratio was 5:1; C: effector-to-target ratio was 10:1; D: co-incubation for 4 hours. All data are expressed as mean ± standard deviation, and the differences between the means of different groups were assessed using the Student's t-test (no statistical difference: ns; P < 0.05: *, P < 0.01: **; P < 0.001: ***; P < 0.0001: ****).

[0189] Results and Analysis: Under three different effect-to-target ratios, p53 R175H Compared to the control T, both T and p53 can more significantly kill the expression. R175HKLE antigen-targeted tumor cells mut For those who do not express p53 R175H CFPAC-1 antigen wt The cell-killing effect was not significant. Statistical analysis was performed on the killing efficiency at 4-hour co-incubation time points between effector and target cells. Under three different effector-to-target ratios (2.5:1, 5:1, and 10:1), p53... R175H The killing efficiency of T cells against target cells was approximately 3, 2, and 1.5 times that of control T cells, respectively, showing significant differences; p53 R175H T vs KLE mut The killing efficiency against target cells differed significantly from that against the negative control CFPAC-1 cells, with the former being approximately 12 times, 3 times, and 2.5 times higher, respectively. (Note p53) R175H T exhibits significant specific recognition and killing expression (p53). R175H The ability of antigens to target cells.

[0190] The above experimental results confirm that Neo-DC cells have a better ability to induce CD8+ T cell activation than Mock DC cells, and can obtain antigen-specific T cells (CTL cells) containing a high proportion of effector memory T cell subsets. These CTL cells have a strong ability to kill tumor cells.

[0191] Example 7 Pharmacodynamic evaluation of Neo-DC cells in a huPBMC-NCG mouse model of subcutaneous transplantation of CFPAC-1 cells

[0192] Human pancreatic cancer cells CFPAC-1 in the logarithmic growth phase were collected and resuspended in DPBS to a concentration of 1×10⁻⁶. 7 A suspension of cells / mL was subcutaneously injected into 18 huPBMC-NCG mice (200 μL / mouse). When the average tumor volume of the mice reached the preset volume, they were randomly divided into 3 groups (G1-G3) with 6 mice in each group according to their body weight and tumor volume. Each mouse received an intraperitoneal injection of 100 μL of PBMCs with HLA-A*02:01 typing for humanized immune system reconstruction. The day of injection was recorded as D0. At the same time, Neo-DC cells (in this example, the antigen loaded is human pancreatic cancer cell KRAS) were prepared using mononuclear cells from the same donor according to the cell preparation procedure in Example 6. G12VNeo-antigen peptides generated by mutations (mature dendritic cells treated with CD40 and PD-L1 antibodies) and Mock DC cells (mature dendritic cells loaded with the same neo-antigen peptides but not treated with CD40 and PD-L1 antibodies) were used. G1 mice (immunely reconstituted) were subcutaneously injected with the solvent as a control; G2 mice were subcutaneously injected with Mock DC cells (100 μL / mouse); and G3 mice were subcutaneously injected with the same dose of Neo-DC cells. Administration was performed via two subcutaneous injections at the groin area of ​​the hind leg, on days 14, 18, 22, and 30 after immune reconstitution, for a total of four administrations. The pharmacodynamics of the test substance in a humanized huPBMC-NCG mouse tumor model with subcutaneous transplantation of human pancreatic cancer cells CFPAC-1 were evaluated by combining observations of tumor volume changes, body weight, CBA analysis, GvHD score, and peripheral blood immune cell detection results during and after administration.

[0193] Results and Analysis: Flow cytometry analysis of peripheral blood in mice during the experiment showed that more than half of the mice in each group had hCD45+ immune cells accounting for more than 10% of the peripheral blood. Among the hCD45+ immune cells, T cells accounted for more than 99%, indicating that the human PBMC immune reconstitution model was successfully established. Figure 14 also shows that after the third administration, the average tumor volume increase in the G3 group injected with Neo-DC peaked on day 28 and then began to decline, from 379 mm on day 28. 3 The temperature dropped to 327 mm on day 35. 3 Until day 42, 83mm 3 The average tumor volume in the G2 group mice injected with Mock-DC also peaked on day 28, and then began to decline, reaching 429 mm on day 28. 3 The diameter of day D35 was 376 mm. 3 The diameter of day D42 was 173 mm. 3 In the G1 group, the average tumor volume in the solvent control group mice peaked on day 35, and a slow decline was subsequently observed, reaching 512 mm on day 35. 3 The diameter of day D38 was 458 mm. 3 The diameter of day D42 is 282mm. 3 It is speculated that this may be related to the stability of the reconstructed human immune system in the later stages of the experiment.

[0194] The results of this experiment indicate that in immunodeficient mice reconstituted with human PBMCs (with subcutaneous tumor formation of human pancreatic cancer cells), repeated subcutaneous injections of Neo-DC cells and Mock DC cells were effective in inhibiting tumor growth, showing significant statistical differences compared to the solvent control group. Furthermore, Neo-DC cells demonstrated a more significant effect in inhibiting tumor growth than Mock DC cells. This suggests that subcutaneous injection of this dose of Neo-DC cells has a stronger inhibitory and therapeutic effect on human pancreatic cancer lesions in humanized huPBMC-NCG mice.

[0195] Example 8: Clinical study on the safety and efficacy of Neo-DC cell therapy for advanced malignant solid tumors.

[0196] In a single-center, single-arm, prospective phase I clinical trial, the participants were patients with advanced malignant solid tumors. Patients received either Neo-DC cell therapy (loaded with the patient's tumor antigen) as monotherapy or in combination with a PD-1 monoclonal antibody after surgery. The regimen consisted of five subcutaneous injections of 1.0 mL each, with the first three injections spaced two weeks apart and the last two three weeks apart. If combined with PD-1, the PD-1 monoclonal antibody was administered subcutaneously every three weeks at the clinically recommended dose. Three patients with advanced malignant solid tumors completed the treatment efficacy evaluation. Safety evaluation results: The only drug-related adverse reaction was transient fever following cell injection, which disappeared within 1-2 days without treatment. No other unexpected adverse events, serious adverse events, or dose-limiting toxicities occurred in any of the participants. Efficacy evaluation results: All three participants developed a strong anti-tumor T-cell immune response. Among the patients treated, one patient with advanced pancreatic cancer (Case P1) with systemic metastases experienced a 64% reduction in lesions after 3 months of Neo-DC cell injection combined with PD-1 monoclonal antibody treatment, achieving a partial response (PR) as assessed by RECIST, and was followed up for 28 months. Another patient with laryngeal cancer and lung metastases (Case P2) experienced a 46% reduction in lesions after 3 months of Neo-DC cell injection monotherapy, achieving a partial response (PR) as assessed by RECIST, and was followed up for 36 months. A third patient with advanced multiple hepatocellular carcinoma (Case P3) experienced a 13% reduction in lesions after 8 months of Neo-DC cell injection monotherapy combined with PD-1 monoclonal antibody treatment, achieving a stable disease (SD) as assessed by RECIST, and was followed up for 32 months. The experimental results are shown in Figure 15. These preliminary clinical data demonstrate the safety and clinical efficacy of Neo-DC cell injection monotherapy or its combination with PD-1 monoclonal antibody treatment.

[0197] Example 9: Safety and preliminary clinical efficacy evaluation of adjuvant TMZ and Neo-DC cell injection in patients with glioblastoma after surgery.

[0198] In a single-center, single-arm, prospective phase I clinical trial, several subjects aged 18–75 years with newly diagnosed primary glioblastoma (GBM) confirmed by histopathology were enrolled. The histopathologically confirmed new-onset glioblastoma was WHO grade IV, with molecular pathological diagnosis confirming wild-type IDH1 and IDH2 genes, and greater than 90% resection of the enhancing lesions. After surgical resection, subjects received standard system (SoC) therapy, alternating with 5–6 subcutaneous injections of Neo-DC cells (loaded with the patient's tumor antigen) during routine adjuvant TMZ chemotherapy. If progression was confirmed after injection therapy, continued treatment with PD-1 monoclonal antibody or bevacizumab was possible. A total of 10 subjects completed the full cycle of treatment. Safety evaluation results: The only drug-related adverse reaction was transient fever after cell injection, which disappeared within 1–2 days without treatment; no other unexpected adverse events, serious adverse events, or dose-limiting toxicities occurred in any subject. The clinical efficacy evaluation results as of April 1, 2024, were as follows: 8 subjects achieved stable disease (SD), with no change in target lesions during the evaluation period and a disease control rate (DCR) of 80%; one subject maintained long-term relapse-free survival for 23 months (as shown in Figure 16); one subject, after 10.4 months of follow-up and radiographic progression, achieved advanced remission after 4 months of PD-1 monoclonal antibody therapy, achieving a CR on the iRANO assessment, currently at 13 months (as shown in Figure 17). The subjects' survival was significantly better than the previously reported median overall survival of 14.6 months and median progression-free survival of 6.9 months after standard treatment for newly diagnosed primary GBM patients. These results preliminarily demonstrate the safety and clinical efficacy of postoperative adjuvant TMZ and Neo-DC cell injection therapy.

[0199] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application, without departing from the scope of the technical solution of this application, shall still fall within the protection scope of this application.

Claims

1. A novel dendritic cell preparation comprising PD-L1 negative dendritic cells and / or dendritic cells with a CD40 agonist bound to their cell surface.

2. The dendritic cell preparation according to claim 1, wherein, The proportion of PD-L1 negative cells to the total number of cells in the formulation is ≥80%, and / or the proportion of cells with CD40 agonists bound to their cell surfaces is ≥80% of the total number of cells in the formulation.

3. The novel dendritic cell preparation according to claim 1 or 2, wherein, In the formulation, dendritic cells with a mature cell phenotype account for ≥50% of the total cells in the formulation, preferably ≥80%.

4. The novel dendritic cell preparation according to any one of claims 1 to 3, wherein, In the formulation, the proportion of CD80, CD86, HLA-DR, and CD83 positive cells to the total cells of the formulation is ≥50%, preferably ≥80%.

5. The novel dendritic cell preparation according to any one of claims 1 to 4, wherein, The proportion of CD14-positive cells in the formulation is ≤20% of the total cells in the formulation, preferably ≤10%.

6. The novel dendritic cell preparation according to any one of claims 1 to 5, wherein, The sum of the proportions of CD3, CD19, and CD56 positive cells in the total cells of the formulation is ≤20%, preferably ≤10%, and more preferably ≤5%.

7. The novel dendritic cell preparation according to any one of claims 1 to 6, wherein, The formulation contains PD-L1 antibodies bound to the surface of PD-L1-negative dendritic cells.

8. The novel dendritic cell preparation according to claim 7, wherein, The PD-L1 antibody is selected from PD-L1 monoclonal antibodies or multispecific antibodies containing PD-L1. Preferably, the PD-L1 antibody is selected from monoclonal antibodies Atezolizumab, Adebrelimab, Durvalumab, or Avelumab.

9. The novel dendritic cell preparation according to any one of claims 1 to 6, wherein, The CD40 agonist is selected from CD40 agonist antibodies or CD40L ligands. The CD40 agonist antibodies include CD40 monoclonal antibodies and multispecific antibodies containing CD40. The CD40L ligands include CD40L recombinant proteins and fusion proteins. Preferably, the CD40 agonist is selected from monoclonal antibodies Mitazalimab, Cifurtilimab, Sotigalimab, or bispecific antibodies Tecaginlimab.

10. The novel dendritic cell preparation according to any one of claims 1 to 9, wherein, The dendritic cells are derived from human peripheral blood mononuclear cells.

11. The novel dendritic cell preparation according to any one of claims 1 to 10, wherein, The dendritic cells in the formulation are also loaded with tumor neoantigens or tumor-associated antigens.

12. The novel dendritic cell preparation according to any one of claims 1 to 11, wherein, The formulation may also include cell cryopreservation solutions, cell protectants, or pharmaceutically acceptable carriers.

13. A method for preparing novel dendritic cells, comprising: Dendritic cells are blocked and / or activated by treatment agents, said treatment agents including PD-L1 antibodies and / or CD40 agonists.

14. The preparation method according to claim 13, wherein, The PD-L1 antibody is selected from PD-L1 monoclonal antibodies or multispecific antibodies containing PD-L1. Preferably, the PD-L1 antibody is selected from monoclonal antibodies Atezolizumab, Adebrelimab, Durvalumab, or Avelumab.

15. The preparation method according to claim 13, wherein, The CD40 agonist is selected from CD40 agonist antibodies or CD40L ligands. The CD40 agonist antibodies include CD40 monoclonal antibodies and multispecific antibodies containing CD40. The CD40L ligands include CD40L recombinant proteins and fusion proteins. Preferably, the CD40 agonist is selected from monoclonal antibodies Mitazalimab, Cifurtilimab, Sotigalimab, or bispecific antibodies Tecaginlimab.

16. The preparation method according to claim 13, wherein, The working concentration of the PD-L1 antibody is 1-50 μg / mL, and / or the working concentration of the CD40 agonist is 0.1-10 μg / mL.

17. The preparation method according to claim 16, wherein, The working concentration of the PD-L1 antibody is 10-20 μg / mL, and / or the working concentration of the CD40 agonist is 1-5 μg / mL.

18. The preparation method according to claim 13, wherein, The blocking and / or activation treatment includes the step of adding the treatment agent to the dendritic cells and co-incubating, preferably, the co-incubation conditions are: temperature 35-38°C, 5% carbon dioxide.

19. The preparation method according to claim 13, wherein, It also includes a maturation-promoting culture step for dendritic cells.

20. The preparation method according to claim 19, wherein, Dendritic cells are subjected to the blocking and / or activation treatment during or after the maturation culture process, preferably 1 to 6 hours before the completion of the culture.

21. The preparation method according to claim 13, wherein, It also includes the step of loading dendritic cells with tumor neoantigens or tumor-associated antigens.

22. The preparation method according to claim 21, wherein, The dendritic cells are loaded with tumor neoantigens or tumor-associated antigens prior to the blocking and / or activation treatment.

23. The preparation method according to any one of claims 13 to 22, wherein, The dendritic cells treated with the aforementioned blocking and / or activation methods are PD-L1 negative and / or have CD40 agonists bound to their cell surface.

24. The dendritic cell preparation obtained by the preparation method according to claim 23, wherein, The proportion of PD-L1 negative cells in the formulation is ≥80% of the total cells in the formulation, and / or the proportion of cells with CD40 agonists bound to their cell surfaces is ≥80% of the total cells in the formulation.

25. The novel dendritic cell preparation according to any one of claims 1 to 12, or its use in the prevention, treatment, and postoperative recurrence prevention of cancer.

26. The use according to claim 25, wherein, The cancers mentioned include solid tumor cancers or hematological cancers. Preferably, the solid tumor cancers include breast cancer, ovarian cancer, pancreatic cancer, lung cancer, liver cancer, nasopharyngeal cancer, gastric cancer, colorectal cancer, kidney cancer, bladder cancer, prostate cancer, sarcoma, esophageal cancer, cervical cancer, gallbladder cancer, glioblastoma, or melanoma. The hematological cancers include leukemia, lymphoma, and multiple myeloma.

27. The use according to claim 25 or 26, wherein, The novel dendritic cell preparation can be used in combination with radiotherapy, chemotherapy, or immunotherapy drugs. Preferably, the dendritic cell preparation can be used in combination with temozolomide (TMZ) and / or immune checkpoint inhibitors.

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