Application of dendritic polypeptide in preparation of training immune agent

Abstract

The invention belongs to the field of biological medicine, and relates to application of dendritic polypeptide in preparation of a training immune agent. The technical problem to be solved by the invention is to stimulate and regulate an immune system by using a training immune agent so as to improve the resistance of a body to diseases. According to the scheme for solving the technical problem, the application of the dendritic DP7 polypeptide serving as a training immune agent to activate organism immunity is provided. The new application of the dendritic DP7 polypeptide can activate specific cells of the spleen to generate rapid immune response aiming at subsequent disease molecules, improves the resistance and treatment effect of the organism on diseases, is expected to bring important breakthrough to the fields of disease treatment and immune regulation, and improves the living quality and treatment effect of patients. Meanwhile, the invention also provides a powerful immunological research tool and method for researchers in related fields, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and in particular relates to the use of dendritic polypeptides in the preparation of trained immune agents. Background Art

[0002] In recent years, as our understanding of the function of the immune system has deepened, an immune response called "trained immunity" has attracted much attention and become a research hotspot due to its potential in fighting chronic diseases including cancer. Trained immunity, also known as "immune training," refers to the ability of the immune system to enhance its response to subsequent challenges through previous exposure to microorganisms or immune stimuli. Unlike traditional acquired immunity, trained immunity has long-term memory and can maintain enhanced immune responses without re-exposure to specific antigens. During trained immunity, the immune system "trains" immune cells by recognizing and remembering previous stimuli, such as pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), enabling them to respond quickly and effectively to similar stimuli in the future. This "training" not only improves the speed and intensity of immune cell immune responses, but also broadens the range of pathogens they recognize, enhancing the overall function of the immune system. The concept of trained immunity as a means of fighting infectious diseases and controlling tumor progression is emerging.

[0003] Many biologics, including β-glucans, have been reported to have the ability to induce trained immunity. β-glucans from Candida albicans and Versicolor fungi, as well as β-glucans from Saccharomyces cerevisiae, have been investigated for their ability to induce trained immunity. Although β-glucans have been used as immunomodulators for cancer, the use of β-glucans to induce trained immunity for the treatment of cancer is just beginning to emerge. β-glucan-mediated trained immunity can induce epigenetic reprogramming of myeloid cells, particularly monocytes and macrophages, thereby enhancing the inflammatory state and antimicrobial activity. Trained immunity can also protect dogs from rabies and protect mice from high doses of Candida albicans and Mycobacterium tuberculosis.

[0004] Because trained immunity can lead to nonspecific protection and prevent reinfection, it may provide a new tool for the prevention and treatment of chronic diseases, including cancer. Although the concept of trained immunity has been widely recognized and studied, there is a shortage of trained immune agents and their specific mechanisms of action are still not fully understood. Therefore, it is crucial to explore new trained immune agents and elucidate their mechanisms.

[0005] VQWRIRVAVIRK peptide is a cationic hydrophilic antimicrobial peptide. It is dendritic designed and synthesized to obtain dendritic DP7 peptide, which can be used as a carrier for drug delivery. Summary of the invention

[0006] The technical problem to be solved by the present invention is to stimulate and regulate the immune system, thereby improving the body's resistance to diseases and providing a new immune training agent.

[0007] The solution to the technical problem provided by the present invention is to provide the use of a dendritic polypeptide in the preparation of a trained immune agent. The dendritic polypeptide is a dendritic polypeptide formed by coupling 2 to 16 polypeptide molecules with the amino acid sequence of VQWRIRVAVIRK (SEQ ID No. 1) to each other.

[0008] The dendrimer polypeptide described in the above use is formed by coupling 2, 4 or 8 DP7 polypeptide molecules.

[0009] Wherein, the VQWRIRVAVIRK polypeptide molecules described in the above use are coupled by using lysine as a connecting medium.

[0010] Wherein, the sites of the VQWRIRVAVIRK polypeptide involved in the coupling are the amino acid residues at the carbon terminal and / or the nitrogen terminal.

[0011] Wherein, the structural formula of the dendritic polypeptide described in the above use is at least one of the following:

[0012]

[0013]

[0014] Furthermore, the administration route of the training immune agent in the above use is intravenous administration.

[0015] The present invention also provides the use of a dendritic polypeptide in the preparation of an anti-tumor drug or an anti-infective drug, wherein the dendritic polypeptide is a dendritic polypeptide formed by coupling 2 to 16 polypeptide molecules having a VQWRIRVAVIRK amino acid sequence with each other; the dendritic polypeptide is the main active ingredient in the anti-tumor drug.

[0016] Wherein, the dendritic polypeptide described in the above use is formed by coupling 2, 4, or 8 DP7 polypeptide molecules.

[0017] Wherein, the VQWRIRVAVIRK polypeptide molecules described in the above use are coupled by using lysine as a connecting medium.

[0018] Wherein, the sites of the VQWRIRVAVIRK polypeptide involved in the coupling are the amino acid residues at the carbon terminal and / or the nitrogen terminal.

[0019] Furthermore, the structural formula of the dendritic polypeptide described in the above use is at least one of the following:

[0020]

[0021]

[0022] Wherein, the dendritic polypeptide described in the above use is used alone as an anti-tumor active ingredient in an anti-tumor drug. Furthermore, the administration route of the anti-tumor drug is intravenous administration.

[0023] Wherein, the tumor described in the above use is at least one of melanoma, lung cancer, colon cancer, or lymphoma. Wherein, the infectious disease is a disease caused by bacterial infection or viral infection. Further, the disease caused by bacterial infection is a disease caused by Gram-negative bacterial infection.

[0024] The present invention also provides an anti-tumor drug, which includes an antibody of an immune checkpoint inhibitor and a dendritic polypeptide which are separately packaged; the dendritic polypeptide is a dendritic polypeptide formed by coupling 2 to 16 polypeptide molecules with the amino acid sequence of VQWRIRVAVIRK to each other; the dendritic polypeptide is the main active ingredient in the anti-tumor drug.

[0025] Wherein, the dendritic polypeptide described in the above use is formed by coupling 2, 4, or 8 DP7 polypeptide molecules.

[0026] Wherein, the VQWRIRVAVIRK polypeptide molecules described in the above use are coupled by using lysine as a connecting medium.

[0027] Wherein, the sites of the VQWRIRVAVIRK polypeptide involved in the coupling are the amino acid residues at the carbon terminal and / or the nitrogen terminal.

[0028] Furthermore, the structural formula of the dendritic polypeptide described in the above use is at least one of the following:

[0029]

[0030]

[0031] Among them, the immune checkpoint inhibitor described in the above-mentioned use is at least one of CTLA-4, PD-1, PD-L1, LAG-3, TIM-3, TIGIT (T cell immunoglobulin and ITIM domain protein) or VISTA (V-DomainImmunoglobulin-Containing Suppressor of T Cell Activation).

[0032] Wherein, the administration route of the dendrimer polypeptide in the above use is intravenous administration.

[0033] The present invention also provides the use of a dendritic polypeptide in activating lymphocytes. The dendritic polypeptide is a dendritic polypeptide formed by coupling 2 to 16 polypeptide molecules with the amino acid sequence of VQWRIRVAVIRK.

[0034] Wherein, the lymphocytes described in the above use are CD11b + cell.

[0035] Wherein, the dendritic polypeptide described in the above use is formed by coupling 2, 4, or 8 DP7 polypeptide molecules.

[0036] Wherein, the VQWRIRVAVIRK polypeptide molecules described in the above use are coupled by using lysine as a connecting medium.

[0037] Wherein, the sites of the VQWRIRVAVIRK polypeptide involved in the coupling are the amino acid residues at the carbon terminal and / or the nitrogen terminal.

[0038] Furthermore, the structural formula of the dendritic polypeptide described in the above use is at least one of the following:

[0039]

[0040]

[0041] Wherein, the lymphocytes described in the above use are isolated lymphocytes.

[0042] The present invention further provides a dendritic polypeptide-activated lymphocyte, wherein the dendritic polypeptide is a dendritic polypeptide formed by coupling 2 to 16 polypeptide molecules with the amino acid sequence of VQWRIRVAVIRK.

[0043] Wherein, the lymphocytes in the above-mentioned dendritic polypeptide activated lymphocytes are CD11b + cell.

[0044] The dendritic polypeptide in the lymphocytes activated by the dendritic polypeptide is formed by coupling 2, 4 or 8 DP7 polypeptide molecules.

[0045] Wherein, the VQWRIRVAVIRK polypeptide molecules in the lymphocytes activated by the dendrimer polypeptide are coupled with each other by using lysine as a connecting medium.

[0046] Wherein, the sites of the VQWRIRVAVIRK polypeptide involved in the coupling in the lymphocytes activated by the dendritic polypeptide are the amino acid residues at the carbon terminal and / or the nitrogen terminal.

[0047] Furthermore, the structural formula of the dendritic polypeptide in the lymphocytes activated by the above dendritic polypeptide is at least one of the following:

[0048]

[0049]

[0050] Wherein, the lymphocytes in the above-mentioned dendritic polypeptide activated lymphocytes are isolated lymphocytes. Further, the lymphocytes are CD11b in the non-lymphocyte layer of the spleen. + cell.

[0051] Wherein, the activation method in the above-mentioned lymphocytes activated by dendritic polypeptide is to co-incubate the above-mentioned dendritic polypeptide with lymphocytes.

[0052] In addition, the activation method in the above-mentioned dendritic polypeptide-activated lymphocytes can be to intravenously inject the above-mentioned dendritic polypeptide into the body, and then separate the CD11b of the non-lymphocyte layer of the spleen of the target donor. + Cells lymphocytes.

[0053] The present invention also provides the use of the lymphocytes in preparing anti-tumor drugs or anti-infection drugs.

[0054] Wherein, the lymphocytes described in the above use are derived from the autologous lymphocytes of the patient to be used.

[0055] Wherein, the dendritic polypeptide described in the above use is formed by coupling 2, 4, or 8 DP7 polypeptide molecules.

[0056] Wherein, the VQWRIRVAVIRK polypeptide molecules described in the above use are coupled by using lysine as a connecting medium.

[0057] Wherein, the sites of the VQWRIRVAVIRK polypeptide involved in the coupling are the amino acid residues at the carbon terminal and / or the nitrogen terminal.

[0058] Wherein, the structural formula of the dendritic polypeptide described in the above use is at least one of the following:

[0059]

[0060]

[0061] The beneficial effects of the present invention are as follows: the present invention creatively discovered that the dendritic DP7 polypeptide has the excellent function of inducing trained immunity to help the body resist diseases. First, after intravenous administration, the dendritic DP7 polypeptide can enable the body to acquire the ability to resist tumor growth in subsequent tumor inoculation. Further studies have found that the mechanism is that the dendritic DP7 polypeptide, as a trained immune agent, significantly improves the body's resistance to diseases by stimulating and regulating the immune system. The results of further in vitro and in vivo experiments of the present invention also show that the dendritic DP7 polypeptide can activate CD11b in the non-lymphocyte layer of the spleen. + Cell groups promote the generation and regulation of immune responses, thereby enhancing the body's immune function, resulting in a significant phenomenon of trained immunity. This is of great significance for the subsequent use of it in the prevention and treatment of various infectious diseases and cancers, and is expected to improve the quality of life and treatment effects of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 : Tumor growth curves monitored after intravenous injection of DP7 in branches 1, 2, 4, and 8. ***P<0.001, ****P<0.0001.

[0063] Figure 2 :Tumor growth curves monitored after intravenous injection of different doses of KK2DP7 and inoculation of tumors. ***P<0.001, ****P<0.0001.

[0064] Figure 3 : Tumor growth curves monitored after intravenous injection of KK2DP7 at different frequencies. ****P<0.0001. Figure 4 :Tumor growth curves monitored after intravenous injection of KK2DP7 followed by tumor inoculation, or after intravenous injection of KK2DP7 followed by tumor inoculation. a) EG7 model. b) CT26 model. c) LL2 model. ***P<0.001, ****P<0.0001.

[0065] Figure 5 : After intravenous injection of KK2DP7, the secretion levels of TNF-α, IL-6, IL-1β, and IFN-γ in the a) spleen, b) bone marrow, and c) PBMC of mice after LPS stimulation. **P<0.01, ***P<0.001, ****P<0.0001.

[0066] Figure 6: After intravenous injection of KK2DP7, the secretion levels of TNF-α, IL-6, IL-1β, and IFN-γ in a) lymphocyte layer cells and b) non-lymphocyte layer cells of mouse spleen after LPS stimulation. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0067] Figure 7 :a) CD11b in the non-lymphocyte layer of mouse spleen after intravenous injection of KK2DP7 + cells, b) CD11b - The secretion levels of TNF-α, IL-6, IL-1β, and IFN-γ in cells after LPS stimulation. ****P<0.0001.

[0068] Figure 8 :After intravenous injection of KK2DP7, CD11b in the non-lymphocyte layer of mouse spleen + The secretion levels of a) TNF-α, b) IL-6, c) IL-1β, and d) IFN-γ after the cells were stimulated by normal cells and different tumor cells. ****P<0.0001.

[0069] Fig. 9 : CD11b in non-lymphocyte layers of spleens trained and untrained by intravenous injection of KK2DP7 + Anti-tumor effect of cells. ****P<0.0001.

[0070] Fig.10 :KK2DP7 trains CD11b in the non-lymphocyte layer of spleen in vitro + Cells can induce cytokine secretion in response to secondary stimulation. a) TNF-α, b) IL-6, c) IL-1β, d) IFN-γ secretion levels. **P<0.01, ****P<0.0001. Fig.11 : Antitumor effect of intravenous injection of KK2DP7 combined with anti-PD-1 antibody in B16 model. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. DETAILED DESCRIPTION

[0071] When studying the function of dendritic DP7 polypeptide, the present invention accidentally discovered that the dendritic DP7 polypeptide, which was originally used as a polypeptide and siRNA carrier for subcutaneous injection and as a vaccine delivery system for lymph node targeting, also has a significant anti-tumor effect after intravenous infusion.

[0072] On this basis, further research was conducted and it was found that the dendritic DP7 peptide can be very effective in producing anti-tumor immune effects if it is injected intravenously before tumor cells come into contact with the body. On this basis, lipopolysaccharide (LPS) was used to simulate the source of infection of infectious diseases, verifying that the dendritic DP7 peptide has the excellent function of being a trained immune agent, inducing trained immunity to help the body resist diseases.

[0073] Lipopolysaccharide (LPS) is a unique component in the cell wall of Gram-negative bacteria, also known as endotoxin. LPS activates the expression of inflammatory cytokine genes and causes inflammatory response by binding to host cell surface receptors, thus initiating the pathogenic process of bacterial infection. LPS can induce the host's response through signal transduction pathways, stimulate immune cells to produce a large number of inflammatory cytokines with pyrogenic effects, and cause overactivation of the immune system. In practice, lipopolysaccharide can be used to establish animal models of acute lung injury, acute liver failure, acute pancreatitis, etc.

[0074] The present invention found that after the dendritic DP7 polypeptide training and LPS stimulation, the secretion of TNF-α, IL-6, IL-1β, and IFN-γ in spleen cells increased significantly, but no significant difference in the secretion of the above cytokines was found in the bone marrow and PBMC. This shows that the cells that produce immune responses to secondary stimuli such as LPS after dendritic DP7 polypeptide training are located in the spleen. Further experiments found that the cells that produce immune responses to secondary stimuli after dendritic DP7 polypeptide training are CD11b in the non-lymphocyte layer of the spleen. + cell.

[0075] In this regard, the present invention provides a technical solution for the use of dendritic DP7 polypeptides in the preparation of trained immune agents.

[0076] The trained immune agent described in the present invention refers to an agent that can enable the innate immune system to produce a memory function when encountering the stimulation of the agent, so that the body can produce a faster or stronger immune response to the same or different subsequent stimuli. Trained immunity (also known as immune training) refers to the ability of the immune system to enhance its response to subsequent challenges through previous exposure to microorganisms or immune stimuli. Trained immunity has long-term memory and can maintain an enhanced immune response without re-exposure to specific antigens. This "training" not only improves the speed and intensity of the immune response of immune cells, but also broadens their range of pathogen recognition and enhances the overall function of the immune system. A class of biological agents including β-glucan has been reported to have the ability to induce trained immunity, and there have been many reports on the use of β-glucan to induce trained immunity to treat cancer and enhance antibacterial activity. Trained immunity is being studied and applied more as a new means to resist infectious diseases and control tumor progression.

[0077] The dendritic polypeptide described in each technical solution of the present invention, also known as the dendritic DP7 polypeptide, is a dendritic polypeptide formed by coupling 2 to 16 DP7 (amino acid sequence VQWRIRVAVIRK, SEQ ID No. 1) polypeptide molecules to each other. Further, the dendritic polypeptide is formed by coupling 2, 4 or 8 DP7 polypeptide molecules. In the dendritic polypeptide used in the examples of the present invention, these VQWRIRVAVIRK polypeptide molecules are coupled by lysine as a connecting medium to complete the coupling.

[0078] Furthermore, the sites of the VQWRIRVAVIRK polypeptide involved in coupling are the amino acid residues at the carbon terminal and / or the nitrogen terminal.

[0079] The structural formula of the dendritic polypeptide is at least one of the following:

[0080]

[0081]

[0082] The above-mentioned dendritic DP7 polypeptides are named KDP7, KK2DP7, and KK2K4DP7 respectively.

[0083] When the dendritic DP7 polypeptide of the present invention is used as a training immune agent, the administration route is intravenous administration.

[0084] At the same time, the present invention also provides the use of dendritic DP7 polypeptides in the preparation of anti-tumor drugs or anti-infective drugs. The anti-tumor drugs or anti-infective drugs prepared by the dendritic DP7 polypeptides are administered before or when the tumor occurs, and the body's resistance to the generation or further deterioration of the tumor is enhanced in a training immune manner. The anti-infective drugs prepared by the dendritic DP7 polypeptides can be administered before or after the infection occurs, so that the body can resist diseases caused by various sources of infection. The tumor can be various common solid tumors or blood tumors, such as melanoma, lung cancer, colon cancer, or lymphoma, etc. The infectious disease is a disease caused by bacterial infection or viral infection. Preferably, the infectious disease is a disease caused by various Gram-negative bacterial infections.

[0085] Further, the anti-tumor drug refers to an anti-tumor drug with dendritic DP7 polypeptide as the main active ingredient, rather than using the dendritic DP7 polypeptide as a carrier to load and deliver other anti-tumor drug components into the body to help other anti-tumor drug components reach tumor tissues and better exert their effects. Alternatively, the anti-tumor drug is an anti-tumor drug with only the dendritic DP7 polypeptide as an active ingredient. Further, the administration route of the anti-tumor drug is intravenous administration.

[0086] Furthermore, in the above use, when the dendritic DP7 polypeptide is administered intravenously, the optional intravenous dosage range is 0.05-4 mg / kg.

[0087] The present invention also found that the combination of dendritic DP7 polypeptide and immune checkpoint inhibitor can produce a synergistic effect. Therefore, the present invention also provides an anti-tumor drug, which includes an antibody of immune checkpoint inhibitor and a dendritic DP7 polypeptide, which are separately packaged.

[0088] The immune checkpoint inhibitor may be at least one selected from CTLA-4, PD-1, PD-L1, LAG-3, TIM-3, TIGIT (Tcell immunoglobulin and ITIM domain protein) or VISTA (V-DomainImmunoglobulin-Containing Suppressor of T Cell Activation).

[0089] Wherein, the administration route of the dendritic polypeptide in the above use is intravenous administration. In one example of the present invention, it was demonstrated that the tumor growth of mice treated with KK2DP7 combined with anti-PD-1 was significantly slower than that of the single KK2DP7 group and anti-PD-1 group.

[0090] When used in combination, the dendritic DP7 polypeptide and the immune checkpoint inhibitor can be injected at different time points.

[0091] On the basis of the above technical solution, the present invention also provides the use of dendritic DP7 polypeptide in activating lymphocytes. It is understood that the lymphocytes activated by the dendritic DP7 polypeptide are in vitro lymphocytes. Further, the lymphocytes are CD11b +- Preferably, the lymphocytes are CD11b cells in the non-lymphocyte layer of the spleen. + cell.

[0092] The activation mentioned in the present invention refers to enabling lymphocytes to rapidly secrete cytokines to kill pathogens after receiving secondary pathogen stimulation.

[0093] The present invention also provides the above lymphocytes activated by dendritic DP7 polypeptides.

[0094] In the examples of the present invention, a typical activation method is provided, which is to co-incubate the dendritic DP7 polypeptide with lymphocytes in vitro.

[0095] Of course, another common activation method is to inject the dendritic DP7 peptide intravenously into the body, and then separate the CD11b of the non-lymphocyte layer of the spleen of the target donor. + Cells lymphocytes.

[0096] The present invention also provides the use of lymphocytes activated by the above-mentioned dendritic DP7 polypeptide in the preparation of anti-tumor drugs or anti-infective drugs.

[0097] Those skilled in the art will appreciate that it is a better choice that the lymphocytes are derived from the patient's autologous lymphocytes.

[0098] The technical solution of the present invention is further described in detail below through specific descriptions of embodiments.

[0099] The main experimental materials and equipment used in the examples are as follows:

[0100] 1. Experimental cell lines and experimental animals

[0101] LL2, EG7, B16, and CT26 cell lines were purchased from the American Type Culture Collection (ATCC). They were cultured in RPMI-1640 (Gibco) medium containing 10% fetal bovine serum (FBS, Gibco). 6-8 week old C57 / BL6J female mice were purchased from Beijing Weitong Lihua Experimental Animal Co., Ltd. and raised in an SPF environment.

[0102] 2. Main reagent materials and kits

[0103] Experimental cell culture medium: RPMI-1640 culture medium and fetal bovine serum (FBS) were purchased from Gibco, USA.

[0104] The DP7 polypeptide and various dendritic DP7 polypeptides used in the experiment were synthesized by Shanghai Chupeptide Biotechnology Co., Ltd. Example 1 Tumor inhibition effect of DP7 polypeptide and dendritic polypeptides KDP7, KK2DP7, and KK2K4DP7 after intravenous injection

[0105] 6-8 week old C57BL / 6J female mice were randomly divided into 5 groups (PBS group, DP7 group, KDP7 group, KK2DP7 group, KK2K4DP7 group), with 6 mice in each group.

[0106] On days -6 and -3, mice were intravenously injected with DP7, KDP7, KK2DP7, and KK2K4DP7 (1 mg / kg, 100 μl), respectively. On day 0, each mouse was subcutaneously inoculated with 1×10 6 EG7-OVA tumor cells were collected and the tumor size of the mice was continuously measured. The tumor volume was calculated as 0.52×length×width 2 , and statistical analysis was performed.

[0107] The results showed that the tumor growth of each group injected with DP7, KDP7, KK2DP7, and KK2K4DP7 before inoculation of tumor cells was significantly slowed down, and there was a significant difference compared with the PBS group ( Figure 1 ).

[0108] Example 2 Tumor inhibition effect of different doses of dendrimer peptide KK2DP7 after intravenous injection

[0109] 6-8 week old C57BL / 6J female mice were randomly divided into 5 groups (PBS group, 0.25 mg / kg KK2DP7 group, 0.5 mg / kg KK2DP7 group, 1 mg / kg KK2DP7 group, and 2 mg / kg KK2DP7 group), with 6 mice in each group.

[0110] On days -6 and -3, mice were intravenously injected with the above doses of KK2DP7 (100 μl), and on day 0, each mouse was subcutaneously inoculated with 1×10 6 EG7-OVA tumor cells were collected and the tumor size of the mice was continuously measured. The tumor volume was calculated as 0.52×length×width 2 , and statistical analysis was performed.

[0111] The results showed that the tumor growth of the KK2DP7 group injected intravenously at all doses was significantly slowed down, and there was a significant difference compared with the PBS group ( Figure 2 The tumor growth curve of the 1mg / kg group slowed down most significantly, and there was no significant difference with the 2mg / kg group ( Figure 2 ), it may be that the mouse immune system has been fully activated at a dose of about 1 mg / kg.

[0112] Example 3 Tumor inhibition effect after intravenous injection of 1 mg / kg of dendrimer peptide KK2DP7 at different frequencies

[0113] 6-8 week old C57BL / 6J female mice were randomly divided into 5 groups (PBS group, KK2DP7 single administration group, KK2DP7 twice administration group, KK2DP7 three times administration group, β-glucan group), with 6 mice in each group.

[0114] On day -6, day -6, -3, day -6, -4, and day -2, mice were intravenously injected with 1 mg / kg KK2DP7 (100 μl). On day -7, mice were injected with 1 mg of β-glucan. On day 0, each mouse was subcutaneously inoculated with 1×10 6 EG7-OVA tumor cells were collected and the tumor size of the mice was continuously measured. The tumor volume was calculated as 0.52×length×width 2 , and statistical analysis was performed.

[0115] The results showed that the tumor growth of the groups that were intravenously injected with KK2DP7 once, twice or three times before tumor cell inoculation was significantly slowed down, and there was a significant difference with the PBS group, and there was no significant difference with the positive control β-glucan group ( Figure 3 Among them, the tumor growth was the slowest after 1 mg / kg was intravenously administered three times ( Figure 3 ), and this dose and administration frequency were used in subsequent experiments.

[0116] Example 4 Antitumor effects of pre-administration and post-administration of KK2DP7 in EG7, CT26 and LL2 tumor models

[0117] 6-8 week old C57BL / 6J female mice were randomly divided into 3 groups (PBS group, KK2DP7 pre-administration group, KK2DP7 post-administration group), with 6 mice in each group.

[0118] In the preventive tumor model, mice were intravenously injected with 1 mg / kg KK2DP7 (100 μl) on days -6, -4, and -2, respectively. On day 0, each mouse was subcutaneously inoculated with 1×10 6 EG7-OVA tumor cells, or 1×10 6 CT26 tumor cells, or 1×10 6 LL2 tumor cells were collected and the tumor size of the mice was continuously measured. The tumor volume was calculated as 0.52×length×width. 2 , and statistical analysis was performed.

[0119] In the therapeutic tumor model, each mouse was subcutaneously inoculated with 1 × 10 6 EG7-OVA tumor cells, or 1×10 6 CT26 tumor cells, or 1×10 6 LL2 tumor cells were added. On days 2, 4, and 6, mice were intravenously injected with 1 mg / kg of KK2DP7 (100 μl). The size of the tumor was continuously measured, and the tumor volume was calculated as 0.52×length×width. 2 , and statistical analysis was performed.

[0120] The results showed that in the EG7 tumor model, both preventive and therapeutic administration of KK2DP7 could significantly inhibit tumor growth in mice ( Figure 4 a). In the CT26 tumor model, both preventive and therapeutic administration of KK2DP7 can significantly inhibit tumor growth in mice ( Figure 4 b). In the LL2 tumor model, both preventive and therapeutic administration of KK2DP7 can significantly inhibit tumor growth in mice ( Figure 4 c).

[0121] The results of this experiment can fully demonstrate that KK2DP7 can play a role in immune training, enabling the body to improve its immunity to tumor cells. In the future, we will continue to study the immune training function of dendritic peptides through experiments.

[0122] Example 5: Isolation of spleen, bone marrow and peripheral blood mononuclear cells from mice after intravenous administration of KK2DP7 to detect secretion levels of immune-training characteristic cytokines

[0123] On days -6, -4, and -2, mice were intravenously injected with 1 mg / kg KK2DP7 (100 μl) or PBS, and on day 0, the spleen, bone marrow, and PBMC of the mice were isolated.

[0124] The method for isolating spleen single cells is as follows: place the spleen on a 70 μm mesh, add culture medium and grind with a syringe to obtain a spleen cell suspension. After centrifugation and adding red blood cell lysis buffer, resuspend it into a single cell suspension with 1640 double culture medium.

[0125] The bone marrow cell extraction method is as follows: remove the mouse hind leg bone, blow the cells therein into the culture medium with a syringe, and obtain a bone marrow cell suspension. After centrifugation and adding red blood cell lysis buffer, resuspend it into a single cell suspension with 1640 double culture medium.

[0126] The PBMC isolation protocol is as follows: blood is collected from the mouse orbit and placed in an anticoagulant tube, PBMC isolation reagent is added, and then centrifugation is performed according to the protocol in the instruction manual, and single cells in the middle layer are collected for later use.

[0127] The spleen, bone marrow and PBMC obtained above were plated in 24-well plates, with 5×10 5 cells. LPS (100 ng / ml) was added for 24 h. The culture supernatant was collected and the secretion levels of TNF-α, IL-6, IL-1β, and IFN-γ were detected using ELISA kits (Elabscience, China) according to the instructions.

[0128] The results showed that in spleen cells, after KK2DP7 training and LPS stimulation, the secretion of TNF-α, IL-6, IL-1β, and IFN-γ by spleen cells was significantly higher than that of the group without KK2DP7 training, and higher than that of the group without KK2DP7 training but with LPS stimulation, and significantly higher than that of the group with KK2DP7 training but without LPS stimulation ( Figure 5 a). However, no significant difference in the secretion of the above cytokines was observed between the KK2DP7 trained and untrained groups after LPS stimulation in the bone marrow and PBMC ( Figure 5 b-5c). This indicates that the cells that produce immune responses to secondary stimulation after KK2DP7 training are located in the spleen of mice.

[0129] Example 6 After intravenous administration of KK2DP7, the spleen cells of mice were separated and further divided into lymphocyte layer cells and non-lymphocyte layer cells, and the secretion levels of training immune characteristic cytokines were detected

[0130] On days -6, -4, and -2, mice were intravenously injected with 1 mg / kg KK2DP7 (100 μl) or PBS, and the spleens of the mice were removed on day 0.

[0131] The method for separating spleen lymphocyte layer cells and non-lymphocyte layer cells is as follows: the spleen is placed on a 70 μm mesh, lymphocyte separation solution is added and the spleen is ground with a syringe, followed by density gradient centrifugation to separate spleen cells into lymphocyte layer cells and non-lymphocyte layer cells.

[0132] The lymphocyte layer cells and non-lymphocyte layer cells obtained above were plated in a 24-well plate, with 5×10 5 cells. LPS (100 ng / ml) was added for 24 h. The culture supernatant was collected and the secretion levels of TNF-α, IL-6, IL-1β, and IFN-γ were detected using ELISA kits (Elabscience, China) according to the instructions.

[0133] The results showed that in the spleen lymphocyte layer cells, there was no significant difference in the secretion of the above cytokines between the KK2DP7 trained group and the untrained group after LPS stimulation ( Figure 6 a). However, in the non-lymphocyte layer cells of the spleen, after KK2DP7 training and LPS stimulation, the secretion of TNF-α, IL-6, IL-1β, and IFN-γ by spleen cells was significantly higher than that of the group not trained with KK2DP7, and higher than that of the group not trained with KK2DP7 but stimulated with LPS, and also significantly higher than that of the group trained with KK2DP7 but not stimulated with LPS ( Figure 6b) The results showed that the cells that produced immune responses to secondary stimulation after KK2DP7 training were located in the non-lymphocyte layer of the mouse spleen.

[0134] Example 7: After intravenous administration of KK2DP7, the non-lymphocyte layer cells of the mouse spleen were separated and further divided into CD11b + Cell populations and CD11b - Cell populations, detection of secretion levels of immune-characteristic cytokines after LPS stimulation

[0135] On days -6, -4, and -2, mice were intravenously injected with 1 mg / kg KK2DP7 (100 μl) or PBS, and the spleens of the mice were removed on day 0.

[0136] The cells of the non-lymphocyte layer of the spleen were separated as follows: the spleen was placed on a 70 μm mesh, lymphocyte separation solution was added and the spleen was ground with a syringe, followed by density gradient centrifugation to separate the spleen cells into lymphocyte layer cells and non-lymphocyte layer cells.

[0137] The non-lymphocyte layer cells obtained above were incubated with CD11b magnetic beads at 4°C for 10 min, and then magnetic bead sorting was performed. + and CD11b - These cells were plated in 24-well plates, with 1×10 5 cells. LPS (100 ng / ml) was added for 24 h. The culture supernatant was collected and the secretion levels of TNF-α, IL-6, IL-1β, and IFN-γ were detected using ELISA kits (Elabscience, China) according to the instructions.

[0138] The results showed that CD11b in the non-lymphocyte layer of the spleen + In the cells, after KK2DP7 training and LPS stimulation, the secretion of TNF-α, IL-6, IL-1β, and IFN-γ by spleen cells was significantly higher than that of the group without KK2DP7 training, and higher than that of the group without KK2DP7 training but stimulated by LPS, and significantly higher than that of the group trained by KK2DP7 but not stimulated by LPS ( Figure 7 a). But in the spleen lymphocyte layer CD11b - In cells, no significant difference in the secretion of the above cytokines was observed between the KK2DP7 trained and untrained groups after LPS stimulation ( Figure 7 b) The experimental results show that the cells that produce immune responses to secondary stimulation after KK2DP7 training are CD11b in the non-lymphocyte layer of mouse spleen + cell.

[0139] Example 8 Isolation of CD11b from the non-lymphocyte layer of mouse spleen after intravenous administration of KK2DP7 + Cell populations, detection of secretion levels of immune-specific cytokines after stimulation of different tumor cells

[0140] On days -6, -4, and -2, mice were intravenously injected with 1 mg / kg KK2DP7 (100 μl) or PBS, and the spleens of the mice were removed on day 0.

[0141] The cells of the non-lymphocyte layer of the spleen were separated as follows: the spleen was placed on a 70 μm mesh, lymphocyte separation solution was added and the spleen was ground with a syringe, followed by density gradient centrifugation to separate the spleen cells into lymphocyte layer cells and non-lymphocyte layer cells.

[0142] The non-lymphocyte layer cells obtained above were incubated with CD11b magnetic beads at 4°C for 10 min, and then magnetic bead sorting was performed. + and CD11b - There are two groups of cells. + The cells were plated in 24-well plates, with 1×10 5 cells. 40% EG7, CT26 and LL2 cells were added and co-cultured for 24 h. The culture supernatant was collected and the secretion levels of TNF-α, IL-6, IL-1β and IFN-γ were detected using ELISA kits (Elabscience, China) according to the instructions.

[0143] The results showed that CD11b in the non-lymphocyte layer of the spleen + In the cells, after KK2DP7 immune training and stimulation with multiple tumor cells, the secretion of TNF-α, IL-6, IL-1β, and IFN-γ by spleen cells was significantly higher than that of the group without KK2DP7 training, and higher than that of the group without KK2DP7 training but stimulated with multiple tumor cells, and significantly higher than that of the group trained with KK2DP7 but not stimulated with multiple tumor cells ( Figure 8 a-8d). The experimental results prove that the cells that produce immune responses to multiple secondary stimuli after KK2DP7 training are indeed CD11b in the non-lymphocyte layer of mouse spleen + cell.

[0144] Example 9 Intravenous reinfusion of CD11b from non-lymphocyte layers of KK2DP7 trained and untrained spleen + Anti-tumor effect of cells

[0145] On days -6, -4, and -2, mice were intravenously injected with 1 mg / kg KK2DP7 (100 μl) or PBS, and the spleens of the mice were removed on day 0.

[0146] The spleen non-lymphocyte layer cells were separated as follows: the spleen was placed on a 70 μm mesh, lymphocyte separation solution was added and the spleen was ground with a syringe, followed by density gradient centrifugation to separate the spleen cells into lymphocyte layer cells and non-lymphocyte layer cells.

[0147] The non-lymphocyte layer cells obtained above were incubated with CD11b magnetic beads at 4°C for 10 min, and then magnetic bead sorting was performed. + and CD11b - Two groups of cells.

[0148] 6-8 week old C57BL / 6J female mice were randomly divided into 3 groups (PBS group, KK2DP7 group, CD11b + Cell group, CD11b sorted without KK2DP7 training + Cell group), with 6 mice in each group.

[0149] On day -2, mice were intravenously injected with 2 × 10 6 CD11b in the non-lymphocyte layer of the spleen of KK2DP7-trained or untrained mice + On day 0, 1×10 cells were inoculated subcutaneously on the right back of each mouse. 6 EG7-OVA tumor cells were collected and the tumor size of the mice was continuously measured. The tumor volume was calculated as 0.52×length×width. 2 , and statistical analysis was performed.

[0150] The results showed that CD11b in the non-lymphocyte layer of the spleen of mice trained by intravenous injection of KK2DP7 + The cells could significantly inhibit the growth of mouse tumors and had significant differences compared with the PBS group ( Fig. 9 ). However, the CD11b in the non-lymphocyte layer of the spleen of mice that were not trained with KK2DP7 was + cells were unable to slow tumor growth ( Fig. 9 The experimental results showed that CD11b in the non-lymphocyte layer of the spleen after intravenous infusion of KK2DP7 training + Cells can be used as a new means of anti-tumor or anti-infection treatment.

[0151] Example 10 KK2DP7 trains CD11b in the non-lymphocyte layer of the spleen in vitro + Cells can be induced to secrete cytokines in response to secondary stimulation

[0152] On day 0, the spleen of the mouse was removed, and the cells of the non-lymphocyte layer of the mouse spleen were separated as follows: the spleen was placed on a 70 μm mesh, lymphocyte separation solution was added, and the spleen cells were ground with a syringe. Subsequently, density gradient centrifugation was performed to separate the spleen cells into lymphocyte layer cells and non-lymphocyte layer cells.

[0153] The non-lymphocyte layer cells obtained above were incubated with CD11b magnetic beads at 4°C for 10 min, and then magnetic bead sorting was performed. + and CD11b - There are two groups of cells. + The cells were plated in 24-well plates, with 1×10 5 cells. KK2DP7 (1 μg / ml) was added to the cells on days 0, 2, and 4, and LPS (100 ng / ml) was added on day 6 for 24 h. The culture supernatant was collected and the secretion levels of TNF-α, IL-6, IL-1β, and IFN-γ were detected using an ELISA kit (Elabscience, China) according to the protocol in the manual.

[0154] The results showed that after in vitro training with KK2DP7, the secretion of TNF-α, IL-6, IL-1β, and IFN-γ by spleen cells after secondary stimulation with LPS was significantly higher than that of the group not trained with KK2DP7, and higher than that of the group not trained with KK2DP7 but stimulated with LPS, and significantly higher than that of the group trained with KK2DP7 but not stimulated with LPS ( Fig.10 a-10d). This indicates that KK2DP7 can train CD11b in the non-lymphocyte layer of the spleen in vitro + Cells mount an immune response to LPS stimulation.

[0155] Example 11 Anti-tumor effect of intravenous injection of KK2DP7 combined with immune checkpoint inhibitor antibody anti-PD-1 in the B16 model

[0156] 6-8 week old C57BL / 6J female mice were randomly divided into 4 groups (PBS group, KK2DP7 group, anti-PD-1 group, and KK2DP7+anti-PD-1 group), with 6 mice in each group.

[0157] On day 0, each mouse was subcutaneously inoculated with 5 × 10 5B16-F10 tumor cells were added to the mice in the KK2DP7 group. KK2DP7 (1 mg / kg, 100 μl) was injected intravenously into the mice in the KK2DP7 group on days 2, 4, and 6, respectively. 200 μg of antibody (200 μl) was injected intraperitoneally into the mice in the anti-PD-1 group and the KK2DP7+anti-PD-1 group on days 3, 6, 10, and 13, respectively. The tumor size of the mice was continuously measured. The tumor volume was calculated using the formula 0.52×length×width 2 , and statistical analysis was performed.

[0158] The results showed that the tumor growth of the intravenous injection of KK2DP7 group was significantly slower than that of the PBS group, and the tumor growth rate of the KK2DP7 group was also significantly slower than that of the anti-PD-1 group. The tumor growth of mice in the KK2DP7 combined with anti-PD-1 group was significantly slower than that of the KK2DP7 group and the anti-PD-1 group ( Fig.11 ).

[0159] In the above examples of the present invention, it was found through experiments that intravenous administration of dendritic peptides KDP7, KK2DP7 and KK2K4DP7 can inhibit tumor growth in mice. Taking KK2DP7 as an example, after exploring the dosage and frequency of administration, it was verified that KK2DP7 can inhibit tumor growth in mice by activating CD11b in the non-lymphocyte layer of the spleen. + The cells secrete cytokines with trained immunity characteristics and inhibit the growth of various tumors. These results show that the dendritic peptides KDP7, KK2DP7, and KK2K4DP7 can be used as trained immune agents to stimulate the body's trained immune response. This is of great significance for the subsequent use of them in the prevention and treatment of various infectious diseases and cancers.

Claims

1. Use of a dendritic polypeptide in the preparation of a trained immune agent, wherein the dendritic polypeptide is a dendritic polypeptide formed by coupling 2 to 16 polypeptide molecules with the amino acid sequence of VQWRIRVAVIRK to each other.

2. The use according to claim 1, characterized in that: The dendritic polypeptide is formed by coupling 2, 4 or 8 VQWRIRVAVIRK polypeptide molecules.

3. The use according to any one of claims 1 or 2, characterized in that The administration route of the training immune agent is intravenous administration.

4. Use of a dendritic polypeptide in the preparation of an anti-tumor drug or an anti-infective drug, wherein the dendritic polypeptide is a dendritic polypeptide formed by coupling 2 to 16 polypeptide molecules having a sequence of VQWRIRVAVIRK with each other; the dendritic polypeptide is the main active ingredient in the anti-tumor drug.

5. The use according to claim 4, characterized in that: The dendritic polypeptide is formed by coupling 2, 4 or 8 VQWRIRVAVIRK polypeptide molecules.

6. The method according to claim 4 or 5, characterized in that The dendrimer polypeptide is used alone as an anti-tumor active ingredient in an anti-tumor drug; further, the anti-tumor drug is administered intravenously.

7. The use according to any one of claims 4 to 6, characterized in that The tumor is at least one of melanoma, lung cancer, colon cancer or lymphoma; the infectious disease is a disease caused by bacterial infection or viral infection.

8. An anti-tumor drug, comprising an immune checkpoint inhibitor antibody and a dendritic polypeptide which are separately packaged; the dendritic polypeptide is a dendritic polypeptide formed by coupling 2 to 16 polypeptide molecules having a sequence of VQWRIRVAVIRK; the dendritic polypeptide is the main active ingredient in the anti-tumor drug.

9. The anti-tumor drug according to claim 8, characterized in that: The dendritic polypeptide is formed by coupling 2, 4 or 8 VQWRIRVAVIRK polypeptide molecules.

10. The antitumor drug according to any one of claims 8 or 9, characterized in that The immune checkpoint inhibitor is at least one of CTLA-4, PD-1, PD-L1, LAG-3, TIM-3, TIGIT (T cell immunoglobulin and ITIM domain protein) or VISTA (V-Domain Immunoglobulin-Containing Suppressor of T Cell Activation); or, the administration route of the dendritic polypeptide is intravenous administration.

11. Use of a dendritic polypeptide in activating lymphocytes, wherein the dendritic polypeptide is formed by coupling 2 to 16 polypeptide molecules having the amino acid sequence of VQWRIRVAVIRK to each other.

12. The use according to claim 11, characterized in that: The lymphocytes are CD11b + Cells; further, the lymphocytes are isolated lymphocytes.

13. The use according to claim 11, characterized in that: The dendritic polypeptide is formed by coupling 2, 4 or 8 VQWRIRVAVIRK polypeptide molecules.

14. A dendritic polypeptide activated lymphocyte, characterized in that: The dendritic polypeptide is formed by coupling 2 to 16 polypeptide molecules with the amino acid sequence of VQWRIRVAVIRK.

15. The lymphocyte according to claim 14, characterized in that: The lymphocytes are CD11b + cell.

16. The lymphocyte according to any one of claims 14 or 15, characterized in that: The lymphocytes are isolated lymphocytes; further, the lymphocytes are CD11b in the non-lymphocyte layer of the spleen. + cell.

17. The lymphocyte according to any one of claims 13 to 15, characterized in that: The lymphocyte activation method is to co-incubate the dendritic polypeptide with lymphocytes.

18. The lymphocyte according to any one of claims 13 to 16, characterized in that: The lymphocyte activation method is to intravenously inject the dendritic polypeptide into the body, and then separate the CD11b of the non-lymphocyte layer of the spleen of the target donor. + Cells lymphocytes.

19. The lymphocyte according to any one of claims 16 to 17, characterized in that: The dendritic polypeptide is formed by coupling 2, 4 or 8 VQWRIRVAVIRK polypeptide molecules.

20. Use of the lymphocyte according to any one of claims 13 to 18 in the preparation of anti-tumor drugs or anti-infective drugs.

21. Use of the dendritic polypeptide according to any one of claims 1 to 3 in the preparation of a trained immune agent, use of the dendritic polypeptide according to any one of claims 4 to 7 in the preparation of an anti-tumor drug or an anti-infective drug, the anti-tumor drug according to any one of claims 8 to 10, use of the dendritic polypeptide according to any one of claims 11 to 13 in activating lymphocytes, the lymphocyte according to any one of claims 14 to 19, characterized in that: The VQWRIRVAVIRK polypeptide molecules are coupled with each other using lysine as a connecting medium.

22. Use of the dendritic polypeptide according to any one of claims 1 to 3 in the preparation of a trained immune agent, use of the dendritic polypeptide according to any one of claims 4 to 7 in the preparation of an anti-tumor drug or an anti-infective drug, the anti-tumor drug according to any one of claims 8 to 10, use of the dendritic polypeptide according to any one of claims 11 to 13 in activating lymphocytes, the lymphocyte according to any one of claims 14 to 19, characterized in that: The sites of the VQWRIRVAVIRK polypeptide involved in coupling are the amino acid residues at the carbon terminal and / or the nitrogen terminal.

23. Use of the dendritic polypeptide according to any one of claims 1 to 3 in the preparation of a trained immune agent, use of the dendritic polypeptide according to any one of claims 4 to 7 in the preparation of an anti-tumor drug or an anti-infective drug, the anti-tumor drug according to any one of claims 8 to 10, use of the dendritic polypeptide according to any one of claims 11 to 13 in activating lymphocytes, the lymphocyte according to any one of claims 14 to 19, characterized in that: The structural formula of the dendritic polypeptide is at least one of the following:

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