Pharmaceutical compositions, methods of making and using the same

Abstract

The application relates to a method for expanding the antigen spectrum of a DC vaccine, initiating an adaptive immune response and enhancing the anti-tumor effect of the DC vaccine by adding allogeneic dendritic cells and / or viral antigen peptides into a traditional DC vaccine and by means of the exogenous DCs.

Description

[0001] Priority information

[0002] none Technical Field

[0003] This invention relates to the field of cell engineering, specifically to pharmaceutical compositions, their preparation methods and applications, and more specifically to pharmaceutical compositions, dendritic cells (DCs), methods for preparing pharmaceutical compositions or DCs, methods for treating cancer patients, and methods for enhancing the immune killing power of DCs. Background Technology

[0004] This invention relates to the field of cell immunotherapy, specifically to a dendritic cell (DC) composition or a method for preparing a dendritic cell vaccine formulation, which can be used for tumor immunotherapy or personalized precision immunotherapy.

[0005] Tumor immunotherapy is a current research hotspot in the medical field and has become another important cancer treatment method after surgery, radiotherapy, and chemotherapy. A growing body of research confirms that activating the body's immune system to produce a sustained or specific anti-tumor immune response can effectively promote the regression of cancer or tumor lesions and prevent tumor recurrence and metastasis. The body's immune system plays a crucial role in the occurrence and development of malignant tumors; therefore, effectively controlling the body's immune system can better prevent and control tumors.

[0006] Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) in the body, capable of taking up, processing, and presenting in vivo and in vitro antigens to CD4+. + and CD8 + T lymphocytes induce a specific immune response in the body. Due to the unique role of dendritic cells (DCs) in inducing the body's immune response, DC-based immunotherapy has received increasing attention in clinical applications in recent years and has become a research hotspot both domestically and internationally.

[0007] A growing body of research indicates that using tumor-associated antigens or cancer-testis antigens such as alpha-fetoprotein (AFP), melanoma antigen gene MAGE-A3, and tumor-testis antigen NY-ESO-1 as vaccines, or using DC cells loaded with these antigenic peptides as vaccines to activate the body's immune system, can effectively improve the prognosis of cancer patients. For example, Palmer et al. demonstrated that autologous DC vaccines loaded with hepatocellular carcinoma whole-cell antigens (i.e., tumor cell lysates) can treat patients with advanced primary liver cancer to some extent, with 28% of patients achieving partial remission or disease stabilization after treatment, and 23.5% of patients experiencing a significant decrease in serum AFP levels after treatment. Currently, three DC tumor vaccines have been approved for marketing globally, under the brand names sipuleucel-T (Dendreon, USA), CreaVaxRCC (CreaGene, South Korea), and Hybricell (Genoa Biotechnologia, Brazil), demonstrating the significant research value and broad clinical application prospects of tumor immunotherapy represented by DC vaccines.

[0008] DC vaccines loaded with tumor antigens are a safe and effective tumor immunotherapy. However, further analysis revealed that although DC vaccines can benefit some patients, their overall efficacy is limited and uncertain. Currently, the antigenic peptides used to prepare DC vaccines are mostly single antigens (such as MAGE, NY-ESO-1, GPC3, etc.), which have a single antitumor spectrum and limited antigenic immunogenicity. This means that the T lymphocytes activated after vaccination only have partial killing activity, which is insufficient to eliminate tumor cells with complex antigenic heterogeneity and to inhibit tumor growth. Secondly, during the occurrence and development of tumors, a complex immune escape mechanism is gradually established to resist the body's immune attack. For example, tumor antigen expression defects / antigen modulation, downregulation or absence of MHC-I molecules and adhesion / co-stimulatory molecules on the surface of tumor cells, etc., all prevent DC cells from effectively performing their antigen-presenting function in vivo and are insufficient to provide enough activation signals to mobilize the body's immune system.

[0009] Therefore, the function of traditional anti-tumor DC vaccines needs further improvement in order to maximize their effectiveness. Summary of the Invention

[0010] The present invention aims to solve at least one of the technical problems existing in the prior art.

[0011] The inventors of this application have achieved a more effective T-cell response and enhanced immune response to the DC vaccine by adding viral antigen peptides and / or low-dose allogeneic DC cells to traditional DC vaccine formulations as non-specific immune enhancers or adjuvants. This method leverages the function of viral antigens and exogenous DC cells to initiate the body's adaptive immune response. The method is highly efficient and simple, significantly improving the anti-tumor effect of DC vaccines and providing greater assurance for clinical treatment with DC vaccines.

[0012] In a first aspect, the present invention provides a pharmaceutical composition. According to embodiments of the present invention, the pharmaceutical composition comprises autologous dendritic cells (DCs) and allogeneic DCs. It should be noted that "autologous DCs" in this application refers to DCs derived from the individual to whom the pharmaceutical composition is to be administered; for example, if the pharmaceutical composition is to be administered to a cancer patient, then the autologous DCs in the pharmaceutical composition refer to DCs derived from that cancer patient. Correspondingly, "allogeneic DCs" in this application refers to DCs derived from an individual to whom the pharmaceutical composition is not administered. It should be noted that the "individual not to whom the composition is administered" and the "individual to whom the composition is to be administered" refer to the same allogeneic species; for example, "allogeneic DCs" are derived from a healthy individual. The pharmaceutical composition according to embodiments of the present invention significantly enhances the immune response in the body.

[0013] According to embodiments of the present invention, the above-described pharmaceutical composition may further include at least one of the following additional technical features:

[0014] According to embodiments of the present invention, the autologous dendritic cells are derived from tumor patients, and the allogeneic dendritic cells are derived from healthy individuals. The pharmaceutical composition according to embodiments of the present invention significantly enhances the immune-killing effect on tumor cells.

[0015] According to embodiments of the present invention, the ratio of autologous DC cells to allogeneic DC cells is 20:1 to 3:1. The inventors have found that within the above-mentioned ratio range, the enhancement effect of autologous DC cells and allogeneic DC cells on the body's immune response is more significant.

[0016] According to embodiments of the present invention, the autologous dendritic cells (DCs) are loaded with tumor antigen peptides (including patient-specific neoantigens, tumor-associated antigens (TAAs), and tumor-specific antigens (TSA)) and / or viral antigen peptides. DCs loaded with tumor antigen peptides can effectively activate and generate T lymphocytes, thereby killing tumor cells. Simultaneously, the inventors discovered that further loading viral antigen peptides onto DCs can more effectively induce T cell responses and enhance the body's immune response to the pharmaceutical composition.

[0017] According to embodiments of the present invention, the viral antigenic peptide includes at least one selected from Epstein-Barr virus (EBV) antigenic peptide and cytomegalovirus (CMV) antigenic peptide.

[0018] According to a specific embodiment of the present invention, the viral antigen peptide has an amino acid sequence shown in any one of SEQ ID NO: 1 to 4.

[0019] GLCTLVAML(SEQ ID NO:1);

[0020] IVTDFSVIK (SEQ ID NO:2);

[0021] ATIGTAMYK (SEQ ID NO:3);

[0022] NLVPMVATV(SEQ ID NO:4).

[0023] Among them, the viral antigen peptide with the amino acid sequence shown in SEQ ID NO:1 is called EBV_A2, the viral antigen peptide with the amino acid sequence shown in SEQ ID NO:2 is called EBV_A11-1, the viral antigen peptide with the amino acid sequence shown in SEQ ID NO:3 is called EBV_A11-2, and the viral antigen peptide with the amino acid sequence shown in SEQ ID NO:4 is called CMVpp65.

[0024] In a second aspect, the present invention provides a dendritic cell (DC). According to embodiments of the invention, the DC is loaded with tumor antigen peptides and viral antigen peptides. The DCs according to embodiments of the invention can more effectively induce T cell responses and enhance the body's immune response to DCs.

[0025] According to embodiments of the present invention, the DC cells described above may further include at least one of the following additional technical features:

[0026] According to an embodiment of the present invention, the DC cells are derived from tumor patients.

[0027] According to embodiments of the present invention, the viral antigenic peptide includes at least one selected from EBV antigenic peptide and CMV antigenic peptide.

[0028] In a third aspect, the present invention provides a pharmaceutical composition. According to embodiments of the invention, the pharmaceutical composition comprises the aforementioned dendritic cells (DCs). The pharmaceutical composition according to embodiments of the invention can activate the body's adaptive immune response by means of viral antigens, thereby inducing a more effective T-cell response and enhancing the body's immune response to DC vaccines.

[0029] According to embodiments of the present invention, the above-described pharmaceutical composition may further include at least one of the following additional technical features:

[0030] According to embodiments of the present invention, the pharmaceutical composition further comprises autologous dendritic cells (DCs) from a tumor patient and / or allogeneic DCs from a healthy individual, wherein the autologous DCs from the tumor patient are loaded with tumor antigen peptides. This pharmaceutical composition according to embodiments of the present invention can induce a more effective T-cell response.

[0031] In a fourth aspect, the present invention proposes the use of the aforementioned pharmaceutical composition or DC cells in the preparation of a medicament for the treatment or prevention of cancer. It should be noted that the term "medicament" in this application should be interpreted broadly; it can refer to both preventative vaccines and therapeutic drugs. For example, the pharmaceutical composition or DC cells according to embodiments of the present invention can be used to prepare both DC vaccines with cancer-preventive effects and drugs with cancer-treating effects.

[0032] In a fifth aspect, the present invention provides a method for preparing the aforementioned pharmaceutical composition. According to an embodiment of the present invention, the method comprises: (1) inducing differentiation culture of CD14-positive cells from tumor patients and healthy individuals respectively to obtain immature dendritic cells (DCs); (2) loading immature DCs from tumor patients with tumor antigen peptides and inducing maturation treatment to obtain mature DCs from tumor patients; (3) inducing maturation treatment of immature DCs from healthy individuals to obtain mature DCs from healthy individuals; and (4) mixing mature DCs from tumor patients and mature DCs from healthy individuals to obtain the pharmaceutical composition. The method for preparing the pharmaceutical composition according to the embodiments of the present invention is simple and efficient, and the obtained pharmaceutical composition can elicit a stronger immune response in the body.

[0033] According to embodiments of the present invention, the above method may further include at least one of the following additional technical features:

[0034] According to an embodiment of the present invention, the ratio of mature DC cells from tumor patients to mature DC cells from healthy individuals is 20:1 to 3:1.

[0035] According to an embodiment of the present invention, step (2) further includes loading mature DC cells from a tumor patient with viral antigen peptides.

[0036] According to embodiments of the present invention, the viral antigenic peptide includes at least one selected from EBV antigenic peptide and CMV antigenic peptide.

[0037] In a sixth aspect, the present invention provides a method for preparing the aforementioned DC cells or pharmaceutical compositions. According to embodiments of the present invention, the method comprises: (1) inducing differentiation culture of CD14-positive cells from tumor patients to obtain immature DC cells; (2) loading the immature DC cells from tumor patients with tumor antigen peptides and inducing maturation treatment to obtain mature DC cells from tumor patients; and (3) loading the DC cells from tumor patients with viral antigen peptides to obtain the DC cells or pharmaceutical compositions. The method for preparing pharmaceutical compositions according to embodiments of the present invention is simple and efficient, and the obtained DC cells or pharmaceutical compositions can elicit a stronger immune response in the body.

[0038] According to embodiments of the present invention, the above method may further include at least one of the following additional technical features:

[0039] According to an embodiment of the present invention, the viral antigen peptide loading treatment of mature DC cells from tumor patients is performed as follows: the mature DC cells are incubated with the viral antigen peptide at 37°C and 5% CO2 for 4-6 hours, the mature DC cells are suspended in serum-free culture medium, and the concentration of the mature DC cells in the serum-free culture medium is 1*10⁻⁶. 6 / mL, the concentration of the viral antigen peptide in the incubation system is 1 μM. According to the method described above in embodiments of the present invention, the loading rate of viral antigen peptide in mature DC cells can reach 80-95%.

[0040] In a seventh aspect, the present invention provides a method for treating cancer patients. According to embodiments of the invention, the method includes: obtaining PBMCs from cancer patients and / or healthy individuals, and sorting CD14-positive cells from the obtained PBMCs; preparing a pharmaceutical composition or DC cells from the CD14-positive cells according to the preceding method; and reinfusing the pharmaceutical composition or DC cells into the cancer patient. The method according to embodiments of the invention can persistently and effectively kill tumor cells, inhibit tumor cell growth, and achieve significant therapeutic effects.

[0041] According to embodiments of the present invention, the above method may further include at least one of the following additional technical features:

[0042] According to an embodiment of the present invention, before infusing the pharmaceutical composition or DC cells into the tumor patient, the method further includes: co-culturing the pharmaceutical composition or DC cells with the tumor patient's autologous T cells in vitro to obtain activated and expanded T cells, and then infusing the T cells into the tumor patient.

[0043] In an eighth aspect, the present invention provides a method for enhancing the immune killing power of autologous dendritic cells (DCs). According to an embodiment of the invention, the method includes mixing the autologous DCs with allogeneic DCs. The method according to the embodiment of the invention can significantly enhance the activation or stimulation of T cell proliferation by DCs and their ability to kill tumor cells.

[0044] According to embodiments of the present invention, the above method may further include at least one of the following additional technical features:

[0045] According to an embodiment of the present invention, the autologous DC cells are derived from a tumor patient, and the allogeneic DC cells are derived from a healthy individual.

[0046] According to an embodiment of the present invention, the ratio of autologous DC cells to allogeneic DC cells is 20:1 to 3:1.

[0047] According to an embodiment of the present invention, the autologous DC cells are loaded with tumor antigen peptides and / or viral antigen peptides.

[0048] According to embodiments of the present invention, the viral antigenic peptide includes at least one selected from EBV antigenic peptide and CMV antigenic peptide.

[0049] In a ninth aspect, the present invention provides a method for enhancing the tumor-killing immune capacity of dendritic cells (DCs). According to an embodiment of the invention, the method includes loading the DCs with tumor antigen peptides and viral antigen peptides. The method according to the embodiment of the invention can significantly enhance the ability of DCs to activate or stimulate T cell proliferation and their ability to kill tumor cells.

[0050] According to embodiments of the present invention, the viral antigenic peptide includes at least one selected from EBV antigenic peptide and CMV antigenic peptide.

[0051] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0052] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0053] Figure 1 This is a flowchart illustrating the preparation process of a novel personalized DC vaccine according to an embodiment of the present invention;

[0054] Figure 2 This is a graph showing the results of how the novel DC vaccine according to an embodiment of the present invention can stimulate T cells to secrete more IFN-γ.

[0055] Where T only indicates a T-cell control without DC vaccine,

[0056] DC+T indicates co-culturing of conventional DC vaccines with T cells.

[0057] DC+EBV+T indicates that a standard DC vaccine is supplemented with the viral peptide EBV and then co-cultured with T cells.

[0058] DC+alloDC+T indicates that a standard DC vaccine is co-cultured with T cells after adding a small amount of allogeneic DCs.

[0059] OKT3+T represents OKT3-stimulated cultured T cells, a positive control;

[0060] Figure 3 This is a graph showing the enhanced T cell proliferation capacity after stimulation with the novel DC vaccine according to an embodiment of the present invention.

[0061] Where T only indicates a T-cell control without DC vaccine,

[0062] DC+T indicates co-culturing of conventional DC vaccines with T cells.

[0063] DC+EBV+T indicates that a standard DC vaccine is supplemented with the viral peptide EBV and then co-cultured with T cells.

[0064] DC+alloDC+T means that a conventional DC vaccine is co-cultured with T cells after adding a small amount of allogeneic DCs.

[0065] Figure 4 This is a graph showing the enhanced tumor-killing ability of T cells stimulated by the novel DC vaccine according to an embodiment of the present invention.

[0066] T+T2 indicates co-culture of control T cells and T2 tumor cells.

[0067] DC+T+T2 indicates co-culture of T cells stimulated by conventional DC vaccine with T2 tumor cells.

[0068] DC+EBV+T+T2 indicates that T cells stimulated by the viral peptide EBV and T2 tumor cells are co-cultured with a standard DC vaccine.

[0069] DC+alloDC+T+T2 indicates that T cells stimulated by allogeneic DCs and T2 tumor cells are co-cultured with conventional DC vaccines.

[0070] Figure 5 The tumor growth curves of tumor-bearing mice in different groups according to embodiments of the present invention are shown below.

[0071] Pbs represents the blank control group, which received PBS infusion.

[0072] DC-T refers to the control DC-T cell group, whose T cells were stimulated by infusion of a conventional DC vaccine that does not contain peptides.

[0073] alloDC-T represents the control group of allogeneic DC-T cells, which are T cells stimulated by the infusion of allogeneic DC cells.

[0074] peptide-DC-T refers to the T-cell therapy group stimulated by conventional DC vaccines, which involves infusion of T cells stimulated by conventional DC vaccines loaded with peptides.

[0075] peptide-alloDC-DC-T refers to the T-cell therapy group that receives a conventional DC vaccine plus allogeneic DC stimulation, and is infused with T cells that have received a conventional DC vaccine loaded with peptides plus allogeneic DC stimulation.

[0076] Figure 6 This is a comparison of tumor inhibition rates in mice after infusion according to an embodiment of the present invention, wherein...

[0077] Pbs represents the blank control group, which received PBS infusion.

[0078] DC-T refers to the control DC-T cell group, whose T cells were stimulated by infusion of a conventional DC vaccine that does not contain peptides.

[0079] alloDC-T represents the control group of allogeneic DC-T cells, which are T cells stimulated by the infusion of allogeneic DC cells.

[0080] peptide-DC-T refers to the T-cell therapy group stimulated by conventional DC vaccines, which involves infusion of T cells stimulated by conventional DC vaccines loaded with peptides.

[0081] peptide-alloDC-DC-T refers to the T-cell therapy group receiving a conventional DC vaccine supplemented with allogeneic DC stimulation, which involves infusion of T cells stimulated with allogeneic DCs after receiving a conventional DC vaccine loaded with peptides; and

[0082] Figure 7 This is a flow cytometry diagram of peripheral blood T cells in mice after infusion, according to an embodiment of the present invention.

[0083] Detailed description of the invention

[0084] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0085] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0086] To address the limitations of existing DC vaccine preparation methods, which result in DC vaccines with a limited antitumor spectrum and limited antigenic immunogenicity, thus affecting their effectiveness and specificity, this invention provides a method to enhance the function of traditional DC vaccines by adding viral antigenic peptides and / or allogeneic DCs, thereby improving their activation of the body's immune response. The aim is to obtain a simple, efficient method for preparing DC vaccines that can elicit a stronger immune response from the body.

[0087] To achieve the above objectives, the technical solution adopted by this invention is a method that expands the antigenic spectrum and immune response of traditional DC vaccines by adding allogeneic dendritic cells and / or viral antigen peptides to traditional DC vaccines, thereby initiating an adaptive immune response and enhancing the anti-tumor effect of DC vaccines, specifically including the following steps:

[0088] (1) Obtaining autologous and allogeneic CD14-positive cells: CD14-positive cells from PBMCs of patients and healthy volunteers were sorted using magnetic beads. After sorting, the cells were cultured in DC serum-free medium. Resuspend and count the cells in GMP DC Medium (20801-0500) and adjust the cell density to 5 x 10^5 / ml.

[0089] (2) Induction and culture of autologous and allogeneic immature DCs: CD14-positive cells from patients and volunteers were seeded at a density of 5 x 10^5 / ml, and GM-CSF (800 U / ml) and IL-4 (1000 U / ml) were added to the culture medium. The cells were cultured at 37°C in a 5% CO2 incubator for 5 days, with half of the medium changed every other day. When changing the medium, 1 / 2 (or 1 / 3) of the old culture medium was carefully aspirated from the culture dish or flask and transferred to a new 15 ml centrifuge tube, which was centrifuged at 400 g for 5 minutes. After centrifugation, the supernatant was discarded, the cell pellet was collected, and an equal volume of fresh culture medium was added. After mixing by pipetting, the pellet was added back to the original culture dish, and an equal amount of cytokines GM-CSF (800 U / ml) and IL-4 (1000 U / ml) were added.

[0090] (3) Personalized neoantigen long peptide loaded DC and DC maturation induction in tumor patients: On the 5th day of induction culture of autologous immature DCs, 10 μM long peptide (27-30 aa) was added to the culture medium after changing the medium, allowing the immature DCs to phagocytose. The cells were then cultured in a 37℃, 5% CO2 incubator for 16 h. After loading DCs with long peptide, CD4 and CD8 positive T cells could be activated simultaneously. On the 6th day, maturation factors TNF-α (40 ng / ml) + IL-6 (20 ng / ml) + IL-1β (20 ng / ml) + PGE2 (100 ng / ml) + PolyIC (5 ug / ml) were added to the immature DC culture medium to induce DC cell maturation.

[0091] (4) Personalized neoantigen short peptide-loaded DCs for cancer patients: On day 7 of culture, DC cells matured, and most cells were observed to be in suspension with obvious small synapses under a microscope. At this time, the suspended DC cells were collected, centrifuged at 400g for 5 minutes, the supernatant was discarded, and the cells were resuspended in culture medium. The cells were counted, and the cell density was adjusted to 1×10^6 / ml. Then, 1μM of antigen short peptide was added (this step can be omitted if the DC vaccine treatment regimen uses only long peptide-loaded DCs). The cells were incubated at 37℃ in a 5% CO2 incubator for 4 hours. The short peptide-loaded DC cells can be used to activate CD8 positive T cells. After incubation, the peptide-loaded DC cells were washed with culture medium, centrifuged at 400g for 5 minutes, and the cell pellet was collected and resuspended in culture medium for later use. The DC cells obtained at this time can be used directly as material for preparing novel DC vaccines, or co-cultured in vitro with autologous T lymphocytes for subsequent functional analysis and flow cytometry identification.

[0092] (5) Flow cytometry detection of DC cell phenotype: The DC cells obtained in the previous step were stained with the following antibodies: CD80 (PE-Cy7), CD83 (APC), CD86 (PE), HLA-DR (PerCP-Cy5.5), CD14 (APC-Cy7), and then analyzed by flow cytometry to detect the expression of each antibody and the maturation of DC.

[0093] (6) DC harvesting and preparation of novel DC vaccine final products: Two days after DC cell maturation culture, DC cell suspension is collected and tested for microorganisms, endotoxins and mycoplasma. The qualified sterile, mycoplasma-free and endotoxin-free autologous DC cells are mixed with allogeneic DCs. The mixing ratio can be any ratio between 20:1, 10:1, 5:1, 3:1 or 20:1 to 3:1 to obtain a novel personalized DC vaccine preparation containing allogeneic DCs. If, after DC maturation, autologous DC cells are loaded with relevant viral antigen peptides such as EBV antigen peptides, CMV antigen peptides, etc., and then mixed with allogeneic DCs, a novel personalized DC vaccine containing allogeneic DCs and viral antigen peptides is obtained. Similarly, a novel personalized DC vaccine loaded only with viral antigen peptides without allogeneic DCs can also be obtained.

[0094] (7) In vitro functional testing of novel DC vaccine: The prepared novel DC vaccine is co-cultured with T cells. After one week, its ability to activate T cells and promote T cell proliferation can be detected in vitro. DC-T cells are co-cultured with corresponding tumor cells, and their tumor killing effect and IFN-γ secretion can be detected in vitro.

[0095] For ease of understanding, the applicant represents the preparation process of the aforementioned novel personalized DC vaccine as follows: Figure 1 .

[0096] The novel DC vaccine prepared by the above method can increase the activation / stimulation of T cell proliferation by 5-50 times, expand T cells by 200-500 times, and increase the effective T cell tumor killing ability by 3-6 times compared with traditional DC vaccines.

[0097] The DC vaccine obtained by the above-mentioned method can improve the ability of T cells to be activated / stimulated and the in vitro tumor killing ability by 3 to 6 times compared with the original technology, and the effective T cell expansion can be increased by 5 to 50 times. In vivo mouse experiments show that the tumor inhibition rate of DC-T cells obtained by this method can reach 80%, which is significantly higher than that of the traditional DC-T cell therapy group. This indicates that the effectiveness and specificity of DC vaccine have been greatly improved.

[0098] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in the art (e.g., refer to J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Science Press, translated by Huang Peitang et al.) or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products, such as those purchased from Miltenyi Biotec.

[0099] Example 1: Sorting of CD14-positive cells

[0100] PBMCs (HLA 0201) from colorectal cancer patients and healthy volunteers were obtained using the Ficoll lymphocyte separation method (HLA typing not limited). CD14-positive cells were then isolated using magnetic beads. The specific procedures are as follows: 1) Transfer the blood sample to a 50ml centrifuge tube, add 1 volume of DPBS to dilute the blood sample, and gently mix by pipetting; 2) Transfer 18mL of Ficoll separation solution to a 50ml centrifuge tube for later use; 3) Slowly add 20mL of diluted blood sample to the centrifuge tube containing Ficoll separation solution, spreading it evenly on the surface of the Ficoll separation solution; 4) Centrifuge at 800g for 25 minutes. After centrifugation, carefully aspirate the white membrane layer mononuclear cells and transfer them to a sterile 50mL centrifuge tube; 5) Add 3 volumes of DPBS to the centrifuge tube containing mononuclear cells, gently mix by pipetting several times, and centrifuge at 400g for 10 minutes; 6) After centrifugation, discard the supernatant, gently tap the cell pellet at the bottom of the centrifuge tube, and add 1mL of DPBS. Resuspend in T009 serum-free medium and centrifuge again at 400g for 10 minutes; 7) PBMC counting: add 20μL LCD14 sorting beads and 80μL LMACS buffer per 1X10^7 cells, gently mix the beads with the cell mixture, and incubate at 4°C in the dark for 15 minutes; 8) After incubation, add 1-2mL buffer / 10 7 9) Wash cells with MACS buffer and centrifuge at 300g for 10 minutes; 10) Resuspend the cell pellet in 1mL MACS buffer, place it on a magnetic sorting rack for sorting, collect the unlabeled effluent cell suspension and the labeled cells in the sorting column, count them and set aside.

[0101] Example 2 DC cell culture

[0102] 1) CD14-positive cell differentiation stimulation: Sorted CD14-positive cells from colorectal cancer patients and healthy volunteers were seeded at a density of 5 x 10^5 / mL, and GM-CSF (800 U / mL) and IL-4 (1000 U / mL) were added to the culture medium. Cells were cultured at 37°C in a 5% CO2 incubator for 5 days, with half the medium changed every other day. During medium change, 1 / 2 (or 1 / 3) of the old culture medium was carefully aspirated from the culture dish or flask and transferred to a new 15 mL centrifuge tube. The tube was centrifuged at 400 g for 5 minutes. After centrifugation, the supernatant was discarded, the cell pellet was collected, and an equal volume of fresh culture medium was added. The pellet was then mixed thoroughly and added back to the original culture dish, along with equal amounts of GM-CSF (800 U / mL) and IL-4 (1000 U / mL).

[0103] 2) DC cell maturation culture and peptide loading: On day 5 of autologous immature DC induction culture, after changing the medium, 10 μM of colorectal cancer patient-specific neoantigen long peptides IC-1, IC-2, or IC-3 were added to the culture medium (wherein, the amino acid sequences of IC-1, IC-2, or IC-3 are as follows: IC-1: WPLLVFLLPACLYLFASCCAHTFSSMS; IC-2: KSLRVQKIRPSILDCNILRVEYSLLIY; IC-3: LVIPLVELSAKQVTFHIPFEVVEKVYP), allowing immature DCs to phagocytose, and were cultured in a 37℃, 5% CO2 incubator for 16 h; on day 6, maturation factors TNF-α (40 ng / mL) + IL-6 (20 ng / mL) + IL-1β (20 ng / mL) + PGE2 (100 ng / mL) + PolyIC (5 μg / mL) were added to the immature DC culture medium to induce DC cell maturation. Healthy volunteer DC cells do not require peptide loading. They can be directly added on day 6 with maturation factors TNF-α (40 ng / mL) + IL-6 (20 ng / mL) + IL-1β (20 ng / mL) + PGE2 (100 ng / mL) + PolyIC (5 μg / mL) to stimulate maturation and prepare for use.

[0104] 3) Viral antigen peptide-loaded autologous DC cells (optional): On day 7 of culture, when DC cells mature, collect the suspended DC cells, centrifuge at 400g for 5 minutes, discard the supernatant, resuspend in serum-free DC medium, count the cells, adjust the cell density to 1×10^6 / mL, and then add 1 μM of viral antigen peptide EBV_A2 or CMVpp65 (wherein, the amino acid sequence of EBV_A2 or CMVpp65 is as follows: EBV_A2: GLCTLVAML, CMVpp65: NLVPMVATV), and incubate at 37℃ in a 5% CO2 incubator for 4 hours. After incubation, wash the peptide-loaded DC cells with medium, centrifuge at 400g for 5 minutes, collect the cell pellet, and resuspend in medium for later use.

[0105] Example 3: DC cell phenotype detection

[0106] The DC cells obtained in the previous step were stained with the following antibodies: CD80 (PE-Cy7), CD83 (APC), CD86 (PE), HLA-DR (PerCP-Cy5.5), and CD14 (APC-Cy7). Then, the expression of each antibody and the maturation of DCs were detected by flow cytometry.

[0107] Example 4: Preparation of DC vaccine final product

[0108] Two days after DC cell maturation culture, DC cell suspensions were collected and tested for microorganisms, endotoxins, and mycoplasma. Qualified, sterile, mycoplasma-free, and endotoxin-free autologous colorectal cancer patient DC cells were mixed with allogeneic DC cells from healthy volunteers at ratios of 20:1, 10:1, and 3:1, thus obtaining a novel personalized DC vaccine for colorectal cancer patients containing allogeneic DC cells. If the autologous colorectal cancer patient DC cells loaded with relevant viral antigen peptides (EBV, CMV, etc.) from Example 2 above are mixed with allogeneic DC cells from healthy volunteers, a novel personalized DC vaccine containing both allogeneic DC cells and viral antigen peptides can be obtained. Similarly, a novel personalized DC vaccine loaded only with viral antigen peptides without allogeneic DC cells can also be obtained. The marker expression of different batches of novel DC vaccine cells is shown in Table 1.

[0109] Table 1: Cell Marker Expression in Different Batches of Novel DC Vaccine

[0110] NO.1 99.7％ 98.1％ 90.3％ 67.1％ NO.2 99.8％ 99.2％ 97.1％ 73.8％ NO.3 99.9％ 99.8％ 98.4％ 75.3％ NO.4 99.9％ 99.4％ 98.0％ 74.9％

[0111] Example 5: In Vitro Functional Testing

[0112] The novel DC vaccine prepared above was co-cultured with autologous T lymphocytes in vitro, and its ability to activate T cells, promote T cell proliferation, kill tumor cells, and secrete cytokines was tested. Results are as follows: Figures 2-4 As shown in the figure. The results indicate that the novel DC vaccine can stimulate T cells to secrete more IFN-γ, the T cells stimulated by the novel DC vaccine have stronger proliferative capacity, and the T cells stimulated by the novel DC vaccine have stronger in vitro tumor-killing ability.

[0113] Example 6: In vivo mouse experiment

[0114] 1) Construction of mouse tumor models containing individualized mutations from colorectal cancer patients

[0115] First, a stable K562 cell line containing the colorectal cancer patient-specific antigenic peptide mine-gene was constructed. This cell line was constructed by Wuhan Winosai Biotechnology Co., Ltd., commissioned by our company. Then, the cell line was expanded and cultured in vitro to a suitable quantity using RPMI 1640 medium containing 10% FBS. The cells were then subcutaneously infused into 6-week-old NSG immunodeficient mice, with each mouse receiving 1 x 10^7 mine-gene-K562 cells. Tumor growth was observed periodically after infusion, approximately 5 days later, when the tumor volume reached 50-100 mm². 3 For future use.

[0116] 2) Grouping and cell infusion

[0117] The NSG mice that were successfully modeled were randomly divided into the following 5 groups, 5 mice per group, and cells were infused into each group sequentially according to the experimental protocol:

[0118] a. Blank control group: Each mouse was injected with 100 μL of PBS via the tail vein;

[0119] b. Control DC-T cell group: Each mouse was injected with 2 x 10^7 DC-T cells stimulated by autologous DCs from a colorectal cancer patient via the tail vein and suspended in 100 μL PBS.

[0120] c. Control allogeneic DC-T cell group: Each mouse was injected with 2 x 10^7 alloDC-T cells stimulated by allogeneic DCs from colorectal cancer patients via the tail vein and suspended in 100 μL PBS.

[0121] d.peptide-DC-T cell group: Each mouse was injected via tail vein with 2 x 10^7 autologous DCs stimulated by individualized antigen peptides from colorectal cancer patients, i.e., peptide-DC-T cells, and suspended in 100 μL PBS.

[0122] e.peptide-alloDC-DC-T cell group: Each mouse was injected with 2 x 10^7 T cells stimulated by a personalized antigen peptide-loaded DC vaccine containing allogeneic DC from a colorectal cancer patient via the tail vein. These were the novel DC vaccine-stimulated T cells prepared by this method, named peptide-alloDC-DC-T cells, and suspended in 100 μL PBS.

[0123] 3) Post-infusion testing and result analysis

[0124] Tumor diameter and body weight: Mice were weighed and tumor diameter was measured every 2 days after infusion. The tumor diameter was measured when the average tumor tissue of the control group mice reached 2500 mm. 3 The observation ended at that time; during the last observation, the mice in each group were photographed, and the tumors were removed and weighed. The results are as follows: Figure 5 and Figure 6 As shown in the figure. The results showed that T cells stimulated by the novel DC vaccine had stronger tumor-suppressing ability in vivo, and the growth of tumor tissue in mice was significantly slowed down. The tumor inhibition rate of the T cell group stimulated by the novel DC vaccine infusion reached 80%.

[0125] Peripheral blood T cell analysis: Two mice were randomly selected from each group one week after infusion and at the end of the observation period. 20 μL of blood was collected from the tail vein. Flow cytometry analysis was performed on the proportion of human CD3-positive T cells in the peripheral blood of the mice. The results are shown in Table 2 and 3. Figure 7 As shown in the figure. The results showed that T cells could still be detected in the peripheral blood of mice in the treatment group one week or nine days after infusion.

[0126] Table 2: Proportion of CD3-positive human T cells detected in peripheral blood of mice in each group 9 days after infusion.

[0127]

[0128] Pathological observation: Subcutaneous tumor tissues of mice in the control group and treatment group were stained with HE and TUNEL to detect apoptosis in the tumor tissues of each group.

[0129] Based on the above test results, the tumor inhibition effect was comprehensively evaluated from the perspectives of tumor growth curve, tumor inhibition rate, peripheral blood human T cell distribution, and tumor tissue apoptosis in each group. The method proposed in this invention, which involves adding a small amount of allogeneic dendritic cells or viral antigen peptides to a traditional DC vaccine, can effectively improve the ability of the DC vaccine to activate T cells, promote the effective proliferation of naïve T cells, and thus enhance the anti-tumor effect of the DC vaccine and DC-CTL.

[0130] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of this invention. The full scope of this invention is given by the appended claims and any equivalents thereof.

[0131] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

Claims

1. A pharmaceutical composition, characterized in that, include: Allogeneic DC cells that do not require peptide loading; as well as Autologous dendritic cells loaded with tumor antigen peptides and viral antigen peptides; The autologous DC cells are derived from colorectal cancer patients, while the allogeneic DC cells are derived from healthy individuals. The pharmaceutical composition is prepared by the following method: (1) CD14-positive cells from colorectal cancer patients and healthy individuals were induced to differentiate and cultured to obtain immature DC cells; (2) Immature DC cells from colorectal cancer patients were loaded with tumor antigen peptides and induced to mature in order to obtain mature DC cells from colorectal cancer patients, and the mature DC cells from colorectal cancer patients were loaded with viral antigen peptides. (3) Immature DC cells from healthy individuals are induced to mature in order to obtain mature DC cells from healthy individuals; (4) The mature DC cells from colorectal cancer patients loaded with tumor antigen peptides and viral antigen peptides and the mature DC cells from healthy individuals are mixed to obtain the DC drug composition. The ratio of mature dendritic cells (DCs) from cancer patients loaded with tumor antigen peptides and viral antigen peptides to mature DCs from healthy individuals is 20:1 to 3:

1. The tumor antigen peptide is IC-1, IC-2 or IC-3, wherein the amino acid sequence of IC-1 is WPLLVFLLPACLYLFASCCAHTFSSMS, the amino acid sequence of IC-2 is KSLRVQKIRPSILDCNILRVEYSLLIY, and the amino acid sequence of IC-3 is LVIPLVELSAKQVTFHIPFEVVEKVYP. The viral antigenic peptide is either an EBV antigenic peptide or a CMV antigenic peptide, wherein the amino acid sequence of the EBV antigenic peptide is GLCTLVAML, and the amino acid sequence of the CMV antigenic peptide is NLVPMVATV. The DC cells are CD14. + DC cells.

2. The pharmaceutical composition according to claim 1, characterized in that, The mature DC cells derived from tumor patients were subjected to viral antigen peptide loading treatment in the following manner: The mature DC cells were incubated with the viral antigen peptide at 37°C and 5% CO2 for 4-6 hours. The mature DC cells were then suspended in serum-free culture medium at a concentration of 1. 10 6 / mL, the concentration of the viral antigen peptide in the incubation system is 1μM.

3. Use of the pharmaceutical composition of claim 1 or 2 in the preparation of a medicament for the treatment of colorectal cancer.

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