Tumor vaccine for multi-target immune checkpoint tumor antibody loaded immune cells, and use of tumor vaccine

Abstract

The present invention relates to the technical field of medicine, and provides a tumor vaccine for multi-target immune checkpoint tumor antibody loaded immune cells, and a use of the tumor vaccine. The tumor vaccine is obtained by gene transduction of a ZG16 recombinant protein or a LECTIN short peptide to DCs or in-vitro incubation of a ZG16 recombinant protein, a LECTIN short peptide, and DCs. According to the present invention, combination of ZG16 and DCs based on a ZG16 gene transduction method can up-regulate CD40 expression of the DCs, and promote maturation of the DCs. In addition, in-vitro incubation of the ZG16 recombinant protein, the LECTIN short peptide, and the DCs can up-regulate the expression of CD40 on the surface of the DCs by means of intervention and direct combination, and promote the function of the DCs, thereby effectively reducing immunosuppression in solid tumors.

Description

A tumor vaccine containing multi-target immune checkpoint tumor antibodies and immune cells and its application Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a tumor vaccine comprising multi-target immune checkpoint tumor antibody-loaded immune cells and its application. Background Art

[0002] Pancreatic ductal adenocarcinoma (PDAC) is one of the most common gastrointestinal malignancies in the world. Pancreatic cancer is currently the fourth leading cause of cancer-related death in the United States and the third in Europe. It is projected to become the second leading cause of cancer-related death by 2030. The prevalence, incidence, and mortality rates of pancreatic cancer in my country are lower than those in developed countries such as Europe, the United States, and Japan. However, the survival rate for pancreatic cancer in China is worse than in most other countries. The mortality-to-prevalence ratio for pancreatic cancer in China is 0.85, compared to 0.4-0.7 in other countries. Only 10%-20% of all pancreatic cancer patients are eligible for surgical resection, and the recurrence rate is very high. Coupled with the poor response rate to adjuvant chemotherapy, the cure rate for pancreatic cancer is extremely low. Currently, there is an urgent need for new and effective treatments to improve the treatment of pancreatic cancer worldwide.

[0003] Dendritic cells (DCs) are a type of cell with specific antigen-presenting capabilities. They are present in nearly all tissue types and are the most potent T cell activators of the immune system, playing a key role in the initiation and regulation of both innate and adaptive responses. Studies have shown that deficient resident dendritic cells (cDCs) function in early-stage pancreatic ductal adenocarcinoma (PDAC) can lead to dysfunctional immune surveillance and T cell inactivation in pancreatic cancer, a key factor in pancreatic cancer immune escape.

[0004] CD40-CD40L is a key signaling pathway in the immune system, supporting the maturation and function of B cells, monocytes, and especially DCs, as well as regulating cell-mediated immune responses. CD40 induces DC differentiation through the CD40L signal on the surface of MHC-restricted activated CD4+ T cells, providing DCs with effective maturation and anti-apoptotic signals, enhancing the killing effect of antigen-specific cytotoxic T lymphocytes (CTLs), inducing interleukin-12 (IL-12) production, promoting T helper 1 (Th1) cell proliferation, overcoming peripheral T cell tolerance, and strengthening tumor immunity.

[0005] Studies have demonstrated that combined therapy with DC vaccines and CD40 agonists elicits T cell-dependent antitumor immunity in pancreatic cancer mouse models and promotes apoptosis of PDAC tumor cells. CD40 agonistic antibodies have demonstrated promising results in modulating DCs and even the PDAC TME, promoting T cell infiltration, and regulating the fibrous stroma of cancerous tissue. Loading DCs with CD40 tumor antigens to stimulate maturation and function can overcome the defects of PDAC DCs and restore their function. DCs loaded with CD40 can successfully induce immune responses and clinical effects in various less immunogenic malignancies as vaccinations, showing great promise in PDAC immunotherapy. However, solid tumors contain a highly robust and immunosuppressive tumor microenvironment (TME), which limits the intratumoral delivery and function of immune vaccines such as DCs. Clinical efforts are also underway to explore novel antigens targeting the PDAC immune microenvironment and new approaches to reduce immunosuppression within solid tumors.

[0006] Immunotherapy has produced remarkable results in the treatment of many malignancies, but clinical responses in pancreatic cancer have been less than satisfactory. Among solid tumors, PDAC is one of the most immune-tolerant tumor types and an immunologically "cold" tumor characterized by an extreme lack of cytotoxic T cell infiltration, which allows it to evade immune surveillance and has a poor response rate to immune checkpoint antibodies. To date, single-agent immunomodulatory agents have been shown to be clinically ineffective in PDAC, and multimodal therapies targeting immunotherapy resistance mechanisms may be required.

[0007] Summary of the Invention

[0008] The present invention aims to develop a multi-target immune checkpoint tumor antibody-loaded immune cell tumor vaccine and its application. By combining ZG16 with DCs through transgenic ZG16, the vaccine can upregulate CD40 expression on DCs and promote their maturation. Furthermore, in vitro incubation of DCs with recombinant ZG16 protein and LECTIN peptides can intervene, directly bind to, and upregulate CD40 expression on the DC surface, promoting DC function and thereby effectively reducing immunosuppression within solid tumors.

[0009] The technical solution of the present invention is achieved as follows:

[0010] The present invention provides a tumor vaccine that loads immune cells with multi-target immune checkpoint tumor antibodies. The tumor vaccine is obtained by transducing DCs with ZG16 recombinant protein or LECTIN short peptide genes or by in vitro incubation of DCs with ZG16 recombinant protein and LECTIN short peptide.

[0011] As a further improvement of the present invention, the multi-target immune checkpoint is selected from at least one of PD-L1, CD40, PD1 and CTLA4.

[0012] The present invention further protects the use of the above-mentioned multi-target immune checkpoint tumor antibody-loaded immune cell tumor vaccine in pancreatic cancer vaccines.

[0013] As a further improvement of the present invention, the tumor vaccine is used in combination with ZG16 protein or LECTIN short peptide or chemotherapy drugs.

[0014] As a further improvement of the present invention, the chemotherapy drug is gemcitabine.

[0015] The present invention has the following beneficial effects: In the present invention, ZG16 enhances the expression of immune co-stimulatory molecules in DCs by acting on the CD40 protein at the post-transcriptional level, thereby promoting their maturation. It can be used as a new type of immune checkpoint antibody (instead of CD40 antibody) to load immune cells (DCs) with tumor immune molecule vaccines. ZG16 reduces the ubiquitination level of CD40 protein by binding to CD40 and NEDD4 (ubiquitin ligase). Co-IP experiments verifying the binding of ZG16 and CD40 demonstrate direct binding of ZG16 to CD40 in DCs and regulation of CD40 expression at the post-transcriptional level. ZG16 intervention can directly bind to and upregulate CD40 expression in DCs. It was found that ZG16 can regulate the maturation of DCs by upregulating CD40. Upregulating CD40 rather than downregulating CD40 is a completely different effect. That is, upregulating CD40 has a tumor immune stimulatory effect that can promote cancer cell apoptosis; downregulating CD40 has a tumor immune suppressive effect that inhibits cancer cell apoptosis.

[0016] Experiments on the effects of ZG16 on DC maturation and activity demonstrate that combining ZG16 with DCs via ZG16 transgenic methods can upregulate CD40 expression in DCs and promote DC maturation. Furthermore, in vitro incubation of DCs with ZG16 recombinant protein and LECTIN peptide (detailed methods are provided below) can intervene, directly bind to, and upregulate CD40 expression on the DC surface, thereby promoting DC function. This protein incubation method avoids the toxicity of gene transfection reagents and lentiviral gene vectors to DCs. Furthermore, systemic injection of ZG16 recombinant protein and LECTIN peptide into the body can maintain the survival and function of exogenous DCs.

[0017] This study, through animal experiments involving intratumoral injection of ZG16 gene-transduced DCs and combined systemic injection of ZG16 and LECTIN recombinant proteins, compared the in vivo efficacy of DCs transduced with either CD40 or ZG16 alone. The results demonstrate that repeated, low-volume systemic injections of ZG16 and LECTIN proteins can enhance the function of exogenous gene-transduced DCs in vivo. This reduces the toxic side effects of frequent gene transduction reagents and enhances the biological effects of exogenous DCs in suppressing pancreatic cancer in vivo.

[0018] The present invention also conducted animal experiments using systemic injection of ZG16 recombinant protein and combined chemotherapy drugs, and compared the in vivo efficacy with that of CD40 agonistic antibodies, proving that ZG16 and LECTIN proteins stimulate enhanced DCs function and, in combination with gemcitabine chemotherapy drugs, inhibit the biological effects of pancreatic cancer in vivo.

[0019] ZG16 has the potential to improve the TME and enhance the efficacy and reduce the toxicity of combined chemotherapy drugs in vivo. Based on the advantages of ZG16 systemic injection established in previous research, and the fact that ZG16 is a low-molecular-weight protein with a molecular weight twice that of CD40, this study further highlights the technical advantages of ZG16 in gene transfection. ZG16 has also been shown to further promote pancreatic cancer tumor immunity in vivo by directly upregulating CD40 expression on DCs in tumor tissue, potentially reducing the use of expensive and difficult-to-obtain exogenous dendritic cells (which require isolation of DCs from animals and humans). In contrast, the commercially available CD40 agonist antibody FGK45 does not directly upregulate CD40 on DCs when regulating tumor immunity in vivo. This demonstrates the distinct biological effects of ZG16 and commercially available CD40 antibodies. Furthermore, compared to chemotherapy, ZG16 protein has virtually no toxic side effects. Furthermore, it has been shown that loading a small dose of a chemotherapy drug (gemcitabine) with ZG16 protein has the greatest effect on CD40 proliferation. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 shows the results of CD40 protein ubiquitination in ZG16-overexpressing CDs; among them, a: ubiquitinated protein expression in DCs; b: ZG16 and CD40 ubiquitination E3 ligase NEDD4 binding protein expression in DCs; c: ZG16 and CD40 binding protein expression in DCs; d: ZG16 and CD40 RNA expression relationship in DCs.

[0022] Figure 2 shows the effects of ZG16 on DCs maturation and activity;

[0023] Figure 3 shows the number of CD40-positive DCs detected by flow cytometry 24 hours after mouse and human DCs (Immature) were infected with adenovirus or given ZG16 recombinant protein / LECTIN; a: CD40-positive DCs flow cytometric expression graph, hZG16 treatment group (DCs incubated with human recombinant protein, flow cytometry); b: CD40-positive DCs flow cytometric expression graph, human IgG protein isotype control incubated DCs, flow cytometric detection; c: CD40-positive DCs flow cytometric expression graph, LECTIN incubated DCs treatment Group, flow cytometry; d: Flow cytometric expression of CD40-positive DCs, IgG isotype control incubated DCs treatment group, flow cytometric analysis; e: Flow cytometric expression of CD40-positive DCs, ad-hZG16 (human ZG16 gene overexpressing DCs) treatment group, flow cytometric analysis; f: Flow cytometric expression of CD40-positive DCs, ad-Vector (empty control) transfected DCs treatment group, flow cytometric analysis; g: Flow cytometric expression of CD40-positive DCs, blank control (Control-1) DCs, flow cytometric analysis. h: CD40 protein expression on DCs, upregulation of CD40 protein expression on DCs treated with human ZG16 recombinant protein (Western blot method), hZG16: human recombinant protein.

[0024] Figure 4 shows the expression of ZG16 in DCs detected by Western blot; a: ZG16 protein expression in DCs cells, Western blot detection of ZG16 expression in mouse DCs (mature and immature); b: ZG16 protein expression in DCs cells, Western blot detection of ZG16 expression in human DCs (mature and immature); c: ZG16 protein expression in DCs cells, Western blot detection of ZG16 expression in human DCs, respectively: blank control (Control, ad-Vector), ZG16 overexpressing (ad-mZG16) DCs, LPS-treated (LPS, LPS+ad-Vector) DCs, LPS+ad-mZG16 (LPS+ZG16 transgenic) treated DCs.

[0025] Figure 5 shows an experiment in which ZG16 exerts its effect through CD40;

[0026] Figure 6 shows the effects of intratumoral injection of ZG16 gene-transduced DCs and combined LECTIN short peptide / ZG16 protein on the in vivo growth of pancreatic cancer; a: (tumor image) confirms the effects of intratumoral injection of ZG16 gene-transduced DCs and combined in vivo injection of short peptide system on the in vivo growth of pancreatic cancer tumors, b: (body weight image) body weight growth graph of animals in the saline-treated control group, gene transfection (including CD40 and ZG16 overexpression) group, and chemotherapy drug (gemcitabine) application group.

[0027] Figure 7 shows the effect of systemic injection of ZG16 recombinant protein and combined chemotherapy drugs on the in vivo growth of pancreatic cancer; a (tumor growth graph) confirms the effect of systemic injection of ZG16 recombinant protein and combined chemotherapy drugs on the in vivo growth of pancreatic cancer tumors, b (weight growth graph) shows the effect of systemic injection of ZG16 recombinant protein and combined chemotherapy drugs on the weight growth of mice.

[0028] FIG8 shows that ZG16 protein directly upregulates CD40 protein expression in DCs in tumor tissues and compares it with other treatments and the commercially available CD40 agonist antibody FGK45;

[0029] FIG9 shows the killing rate of pancreatic cancer cells by ZG16-transduced DCs loaded with 5-FU and other chemotherapy drugs. DETAILED DESCRIPTION

[0030] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0031] Example 1 Co-IP verification of the binding of ZG16 to CD40

[0032] The experimental steps are:

[0033] 1. Co-IP verification of the binding of ZG16 to CD40

[0034] 1) Construct an overexpression vector and negative control based on human ZG16 (NM_152338.4, CDs region length 504 bp);

[0035] 2) Construct an overexpression vector and negative control based on human CD40 (NM_011611, CDs region length 870 bp);

[0036] 3) Normally culture human dendritic cells: 90% L-15 medium + 10% serum. When the cells are in good growth condition, select the above-constructed plasmid for transfection. The specific grouping and treatment methods are as follows:

[0037] 4) 48 hours after cell transfection, the expression level of ZG16 mRNA in cells was detected by real-time PCR;

[0038] 5) 48 hours after cell transfection, the expression level of CD40 mRNA in cells was detected by real-time PCR;

[0039] 6) 48 hours after cell transfection, the expression levels of ZG16 and CD40 fusion proteins in cells were detected by Western blot.

[0040] 7) Co-immunoprecipitation (Co-IP) experiments to verify the interaction between ZG16 and CD40;

[0041] 2. DCs were transfected with ZG16 and PD-L1 plasmids, respectively. 48 hours after transfection, cells were harvested for Co-IP experiments.

[0042] (1) Experimental setup:

[0043] DCs+CD40

[0044] DCs+ZG16

[0045] DCs+CD40+ZG16

[0046] (2) Group settings:

[0047] IP was performed with ZG16 antibody, and Western blot was performed with CD40 antibody;

[0048] IP was performed with ZG16 antibody, and Western blot was performed with ZG16 antibody;

[0049] IP was performed with CD40 antibody, and Western blot was performed with ZG16 antibody;

[0050] IP was performed with CD40 antibody, and Western blot was performed with CD40 antibody;

[0051] (Similar to the above steps to detect the co-precipitation binding of NEDD4 and CD40);

[0052] CD40 ubiquitination expression in ZG16-overexpressing CDs.

[0053] 3. CD40 overexpression plasmid was transfected into 293 cells to achieve high expression. Protein was extracted and NEDD4 and CD40 were co-incubated with UBE1, UbeH13-Uev 1a, and HA-ubiquitin. The degree of ubiquitination was detected by IP and WB.

[0054] Figure 1: a: Overexpression of ZG16 in DCs leads to decreased ubiquitination of CD40 protein; b: Co-IP validation of the binding of ZG16 and NEDD4 in mouse DCs; c: Co-IP validation of the binding of ZG16 and CD40 in mouse DCs; d: Overexpression of ZG16 has no effect on RNA expression in DCs. ****: P < 0.0001, ns: no difference.

[0055] The experimental results showed that ZG16 binds to CD40 in DCs and regulates its expression at the post-transcriptional level, providing direct evidence for the mechanism by which ZG16 acts on DCs.

[0056] Example 2 Experiment on the Effect of ZG16 on DCs Maturation and Activity

[0057] The experimental steps are:

[0058] 1) Commercially available human DCs were cultured in 1640 complete medium supplemented with IL-2 and activated with LPS.

[0059] 2) The groups were as follows: A. Negative control group DCs cells (Control, blank vector ad-Vector), B. LECTIN short peptide group (35 μg / ml low dose) treated DCs cells, C. Human recombinant ZG16 short peptide group (hZG16, 35 μg / ml low dose) treated DCs cells, D. ZG16 gene transfection group (ad-hZG16), E. LECTIN short peptide group (35 μg / ml low dose) incubated with ZG16 gene transfected DCs cells.

[0060] 3) After DCs were activated for 48 hours, the activated DCs were collected into a 50 ml centrifuge tube and centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded and the cells were resuspended in 1640 complete medium and counted. The resuspended DCs were then incubated with the above-mentioned grouping components and adjusted concentrations.

[0061] Gene transfection steps:

[0062] (1) Experimental method of lentiviral transfection of DCs cells

[0063] 1. 18-24 hours before lentiviral transfection, DCs were cultured at a density of 1×10 5 / well in a 24-well plate. The number of cells at the time of lentiviral transfection is 2×10 5 / hole around.

[0064] 2. On the second day, replace the original culture medium with 2 ml of fresh culture medium containing 6 μg / ml polybrene and add an appropriate amount of virus suspension. Incubate at 37°C.

[0065] After 3 or 4 hours, add 2 ml of fresh culture medium to dilute the polybrene.

[0066] 4. Continue culturing for 24 hours and replace the virus-containing medium with fresh medium.

[0067] 5. Incubate for 24 hours. After 48 hours, collect the DCs cells suspended in the culture medium into a 1.5 ml EP tube and place it on ice. Stimulate the DCs cells 6 hours before collecting the cells by adding PMA and ionomycin to stimulate the production of intrinsic factor, 1 ul each per well. After incubation for 1 hour, add BFA, 1 ul per well, and incubate for 5 hours to block the secretion of intrinsic factor to the extracellular space.

[0068] 6. Mouse dendritic cells (Immature) were infected with a ZG16-overexpressing adenovirus or / and treated with hZG16 and LECTIN peptide (35 μg / ml). 24 hours later, the following assay was performed: IL-1β, IL-6, TNF-α, and IL-12 / p70 expression in the cell supernatant was measured using kits. ELISA kits were used to quantify the concentrations of these markers in DC culture supernatants (using ad-ZG16 / ZG16 and Lectin as culture templates). The ELISA kit steps are as follows:

[0069] 1. Solution preparation: Coating solution: 50mM Na2CO3 (pH9.6) Blocking solution: 5% BSA

[0070] 2. Washing solution: 0.02-0.05% Tween-20 in 0.1M phosphate buffer (pH 7.4)

[0071] 3. Dissolve the antigen in the coating solution at an appropriate concentration.

[0072] 4. Add 100 μl of antigen to the corresponding wells and incubate at room temperature for 2 h.

[0073] 5. Empty the liquid and pat dry the remaining liquid, then wash twice with 300μl washing solution.

[0074] 6. Add 300 μl of blocking solution to each well and incubate for 1 hour.

[0075] 7. Empty the liquid and pat dry the remaining liquid, then wash twice with 300μl washing solution.

[0076] 8. Add 100 μl of primary antibody to each well and incubate at 37°C for 1 hour or at room temperature for 3 hours.

[0077] 9. Empty the liquid and pat dry the remaining liquid, fill each well with washing solution, empty the liquid and pat dry the remaining liquid, repeat 3 times.

[0078] 10. Add 100 μl of secondary antibody to each well and incubate at room temperature for 1 hour.

[0079] 11. Empty the liquid and pat dry any remaining liquid. Fill each well with wash solution, empty the liquid, and pat dry any remaining liquid. Repeat three times. Patting dry in this step means turning the container upside down on the bench and tapping it to drain the liquid and dry it. The same applies below.

[0080] 12. Soak in detergent for 5 minutes and pat dry any remaining liquid.

[0081] 13. Fill each well with washing solution, empty the liquid and pat dry the remaining liquid, repeat 5 times.

[0082] 14. Add 100 μl of substrate to each well, develop color for 30 minutes and read immediately at 405-410 nm.

[0083] The results are shown in Figure 2, which shows the effects of ZG16 on DC maturation and activity. The results include (mouse) Control (blank), ad-Vector (blank control), ad-hZG16 (human DCs overexpressing ZG16 via lentivirus), hZG16 (DCs incubated with human recombinant ZG16 protein), LECTIN (short peptide), and LECTIN + ad-hZG16 (short peptide incubation + DCs overexpressing ZG16 gene transfection). The experimental results in Figure 2 demonstrate that ZG16 promotes the activity and function of DCs, providing in vitro experimental evidence for the fundamental scientific problem addressed by the present invention: enhancing DC cellular function.

[0084] Example 3 ZG16 / LECTIN intervention (transgenic and direct incubation methods) can upregulate CD40 expression in DCs

[0085] The experimental steps are:

[0086] 1) Commercially available human DCs were cultured in 1640 complete medium supplemented with IL-2 and activated with LPS.

[0087] 2) The groups were as follows: negative control group (IgG antibody, blank vector ad-Vector), LECTIN short peptide group (35 μg / ml small dose) treated DCs cells, human recombinant ZG16 short peptide group (35 μg / ml small dose) treated DCs cells, ad-hZG16 (DCs with high expression of human ZG16 gene).

[0088] 3) After 48 hours of DCs activation, the activated DCs were collected into a 50ml centrifuge tube and centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded and the cells were resuspended in 1640 complete medium and counted. The resuspended DCs were incubated with the cells from step 2) (negative control group (IgG antibody, blank vector ad-Vector), DCs treated with LECTIN short peptide group (35μg / ml low dose), DCs treated with human recombinant ZG16 short peptide group (35μg / ml low dose), and ad-hZG16 gene transfection). After 48 hours, the DCs suspended in the culture medium were collected into a 1.5ml EP tube and placed on ice. Six hours before cell collection, the DCs were stimulated by adding PMA and ionomycin to stimulate the production of intrinsic factor, each at 1ul / well. After incubation for 1 hour, BFA was added at 1ul / well and incubated for 5 hours to block the secretion of intrinsic factor to the extracellular space.

[0089] 4) Flow cytometry and western blot were used to detect the expression of CD40 antigen, a cell surface molecule of the TNFR superfamily, in DCs, demonstrating that ZG16 binds to and regulates CD40 protein in DCs.

[0090] Streaming operation steps:

[0091] 1. Store the antibody at 2-8°C in the dark.

[0092] 2. Quickly centrifuge the antibody to recover the maximum volume.

[0093] The operation should be carried out on ice or at 2-8°C and protected from light as much as possible.

[0094] 3. Mix 100 μL sample with 100 μL IC fixative (eBioscience) TM (1.4% paraformaldehyde fixative). Before staining, cells were pre-incubated with 20 μL purified human Fc receptor binding inhibitor / 100 μL at 2-25°C for 10-20 min.

[0095] 4. Add equal amount of 50 μL cell suspension (10 5 -10 8 cells).

[0096] 5. Add the primary antibody to the cells, so that the final volume is 100 μL (i.e., 50 μL cell sample + 50 μL antibody mixture). Gently pulse-vortex to mix.

[0097] 6. Incubate at 2-8°C or on ice for at least 30 minutes. Protect from light.

[0098] 7. Wash the cells by adding flow cytometry staining solution. For microtiter plates, use 2 mL / tube or 200 μL / well. Centrifuge at 400-600 x g for 5 minutes at room temperature. Discard the supernatant.

[0099] 8. Repeat the previous step (step 7).

[0100] 9. Dilute an appropriate amount of fluorescent dye-labeled secondary reagent in 100 μL of flow cytometry staining buffer and add to the cells. Incubate at 2-8°C or on ice for at least 30 minutes. Protect from light.

[0101] 10. Analyze samples by flow cytometry.

[0102] The results are shown in Figure 3. Mouse and human DCs (Immature) were infected with adenovirus or administered with ZG16 recombinant protein / LECTIN. 24 hours later, the number of CD40-positive cells was determined by flow cytometry. a: hZG16 (DCs incubated with human recombinant protein); b: human IgG protein isotype control incubation; c: LECTIN; d: IgG isotype control; e: ad-hZG16 (DCs overexpressing human ZG16); f: ad-Vector (empty control); g: blank control (Control-1). h: Upregulation of CD40 protein expression in DCs incubated with human ZG16 recombinant protein (Western blot). hZG16: human recombinant protein.

[0103] The experimental results showed that direct intervention of ZG16 protein can bind to and upregulate the expression of CD40 in DCs, providing direct evidence that ZG16 regulates the maturation of DCs by upregulating CD40.

[0104] Experimental Example 4 Western blot detection of ZG16 expression in mouse DCs and human DCs

[0105] The experimental steps are:

[0106] 1) Commercially available human DCs were cultured in 1640 complete medium supplemented with IL-2 and activated with LPS.

[0107] 2) The groups were as follows: blank control (Control, ad-Vector), ZG16 overexpression (ad-mZG16), LPS treatment (LPS, LPS+ad-Vector), LPS+ad-mZG16 (LPS+ZG16 transgenic).

[0108] 3) After 48 hours of DCs activation, collect the activated DCs into a 50ml centrifuge tube and centrifuge at 1500 rpm for 5 minutes. Discard the supernatant, resuspend in 1640 complete medium, and count. Resuspended DCs were grouped as described above. After 48 hours, collect the DCs suspended in the medium into a 1.5ml EP tube and place on ice. Six hours before harvesting, stimulate the DCs with 1µl of PMA and ionomycin to stimulate intrinsic factor production. After incubation for 1 hour, add 1µl of BFA per well and incubate for 5 hours to block the secretion of intrinsic factor.

[0109] 4) Western blot was used to detect the expression of ZG16 antigen in DCs protein samples, demonstrating that ZG16 protein is significantly upregulated during DCs maturation.

[0110] The results are shown in Figure 4. Mouse DCs (Immature) derived from bone marrow of C57BL / 6 mice and purchased human DCs (Immature) were induced to differentiate into mature DCs. Figure (a): Western blot analysis of ZG16 expression in mouse DCs; b: Western blot analysis of ZG16 expression in human DCs; c: ZG16 expression in DCs: blank control (control, ad-vector), ZG16 overexpression (ad-mZG16), LPS treatment (LPS, LPS + ad-vector), and LPS + ad-mZG16 (LPS + ZG16 transgenic).

[0111] The results in Figure 4 showed that ZG16 protein was significantly upregulated during DCs maturation, which demonstrated the role of ZG16 in DCs maturation.

[0112] Example 5 ZG16 acts through CD40

[0113] The experimental steps are:

[0114] 1) To isolate primary mouse DCs from bone marrow, use strict aseptic techniques to dissect the femur and humerus, flush out the bone marrow cells, treat with erythrocyte lysis buffer, treat with macrophage colony-stimulating factor, and incubate with cytokines to isolate mouse DCs. Culture in 1640 complete medium supplemented with IL-2 and activate with LPS.

[0115] 2) The groups were divided into the following groups: ZG16 gene transfection group was loaded with CD40 knockout vector (ad-mZG16+ad-shRNA-mCD40), and ZG16 gene transfection group was loaded with CD40 knockout blank control vector (ad-mZG16+ad-shNC).

[0116] Gene transfection steps:

[0117] (1) Experimental method of lentiviral transfection of DCs cells

[0118] 1. 18-24 hours before lentiviral transfection, DCs were cultured at a density of 1×10 5 / well in a 24-well plate. The number of cells at the time of lentiviral transfection is 2×10 5 / hole around.

[0119] 2. On the second day, replace the original culture medium with 2 ml of fresh culture medium containing 6 μg / ml polybrene and add an appropriate amount of virus suspension. Incubate at 37°C.

[0120] After 3 or 4 hours, add 2 ml of fresh culture medium to dilute the polybrene.

[0121] 4. Continue culturing for 24 hours and replace the virus-containing medium with fresh medium.

[0122] 5. After incubation for 24 hours and 48 hours, collect the DCs cells suspended in the culture medium into a 1.5 ml EP tube and place it on ice; stimulate the DCs cells 6 hours before cell collection by adding PMA and ionomycin to stimulate the production of intrinsic factor, 1 ul each per well. After incubation for 1 hour, add BFA, 1 ul per well, and incubate for 5 hours to block the secretion of intrinsic factor to the extracellular space.

[0123] 6. Use ELISA kits to quantitatively determine the concentrations of IL-1β, IL-6, TNF-α, IL-12 / p70 in the DCs culture supernatant (using ad-ZG16 / ZG16 and Lectin as culture templates). For the ELISA kit steps, refer to the experimental steps in the section on the effects of ZG16 on DCs maturation and activity.

[0124] The results, shown in Figure 5, show that mouse dendritic cells (Immature) were infected with a CD40-downregulating adenovirus and co-infected with the ZG16 gene. 48 hours later, assays using a kit detected a significant downregulation of IL-1β, IL-6, TNF-α, and IL-12 / p70 in the cell supernatant. This "reverse" experiment, by downregulating CD40, validated whether ZG16 exerts its effects through CD40 and clarified the preliminary research foundation, further solidifying the theoretical basis of this invention.

[0125] Example 6 Animal Experiment Effects of Intratumoral Injection of ZG16 Gene-Transduced DCs and Combined LECTIN Peptide / ZG16 Protein on Pancreatic Cancer Growth in Vivo

[0126] Experimental procedures: Mouse-derived PANC02 pancreatic cancer cells (5×10 5) were injected subcutaneously into 6-week-old female C57BL / 6 mice when the tumor volume approached 30 mm. 3 At about 14 h, adenovirus-infected DCs (5×10 5 Once a week) and / or LECTIN (200 μg / animal; intraperitoneal injection; twice a week) were administered, and the tumor volume (mm) was measured every two days. 3 ) and body weight (g). Monitor for about 20 days.

[0127] PANC02: murine pancreatic cancer cells, PANC02+DCs: untreated DCs injection group, PANC02+ad-ZG16-DCs: DCs transfected with ZG16 gene and treated with the gene, PANC02+GEM: gemcitabine-treated group, PANC02+ad-ZG16-DCs+LECTIN: DCs transfected with the ZG16 gene and intraperitoneally injected with LECTIN peptide, PANC02+ad-CD40-DCs: DCs transfected with the gene and treated with the gene. The results are shown in Figure 6.

[0128] Figure 6a (tumor image) demonstrates the effects of intratumoral injection of ZG16 gene-transduced DCs and combined short peptide systemic injection on pancreatic cancer growth in vivo. It was observed that DCs loaded with the novel ZG16 protein antigen significantly inhibited pancreatic cancer cell growth in vivo, and the effect was significantly greater than that of gemcitabine alone. Furthermore, ZG16 gene-transduced DCs combined with low-dose LECTIN also maximally inhibited pancreatic cancer growth in vivo. Volume unit: mm 3 ,****:P<0.0001,***P=0.0003, ns:P=0.2360.

[0129] Figure 6b (Weight Graph) shows no significant differences in weight gain between the saline-treated control group, the gene transfection (including CD40 and ZG16 overexpression) group, or the chemotherapy (gemcitabine) group. However, there was a difference in weight gain compared to the untreated DCs group, with the DCs-only group showing a more pronounced weight gain. The weight gain curves show that both the lentiviral vector, gene transfection reagent, and chemotherapy drugs all affect mouse weight gain, with chemotherapy drugs having the most significant impact. This means that while chemotherapy drugs have some effect, they also cause greater overall harm to the subjects. Notably, weight gain in the gene transfection groups (ZG16 and CD40) was faster than in the chemotherapy groups (chemotherapy alone or in combination with transgenic DCs), but there was no difference in weight gain between the two transgenic groups (CD40 vs. ZG16). Weight is measured in g.

[0130] Combining the tumor volume and body weight data, we found that dendritic cells loaded with the ZG16 and CD40 genes had a more potent inhibitory effect on pancreatic cancer cells in vivo than lower-dose chemotherapy drugs, with less toxicity than chemotherapy drugs. ZG16-loaded DCs combined with LECTIN systemic injection demonstrated significantly better efficacy than the chemotherapy group (p<0.0001) and less toxicity than chemotherapy drugs. Furthermore, ZG16-loaded DCs combined with low-dose LECTIN systemic injection reduced toxicity and enhanced efficacy (the effect was significantly greater than that of dendritic cells loaded with the ZG16 and CD40 genes). In vivo injection of LECTIN peptides enhanced the in vivo effects of exogenous dendritic cells, reduced the use of expensive and difficult-to-obtain human peripheral blood dendritic cells, and theoretically prolonged the lifespan of DCs.

[0131] Therefore, this study conducted animal experiments using intratumoral injection of ZG16 gene-transduced DCs and combined with LECTIN, and compared the in vivo efficacy with that of CD40 gene transduction, providing in vivo re-evaluation data for ZG16-enhanced DCs function.

[0132] Based on the advantages of ZG16 systemic injection in previous research, this experiment also highlights the technical advantages of ZG16 small molecular weight protein (its molecular weight is 2 times smaller than that of CD40 protein) and gene transfection.

[0133] Example 7 Effects of Systemic Injection of ZG16 Recombinant Protein and Combination Chemotherapy on Pancreatic Cancer Growth in Vivo

[0134] Experimental procedures: Mouse-derived PANC02 pancreatic cancer cells (5×10 5 ) were injected subcutaneously into 6-week-old female C57BL / 6 mice when the tumor volume approached 30 mm. 3 Around 20 days, mice were given ZG16 recombinant protein (200 μg / per mouse, intraperitoneal injection, twice a week), low-dose GEM (15 mg / kg, intraperitoneal injection, twice a week), high-dose GEM (60 mg / kg, intraperitoneal injection, twice a week), and CD40 monoclonal antibody (12.5 μg / per mouse, twice a week). Tumor volume and mouse body weight were measured every two days for about 20 days.

[0135] PANC02: murine pancreatic cancer cells, PANC02 + GEM (15 mg / kg): low-dose gemcitabine treatment group, PANC02 + mZG16: recombinant protein injection group, PANC02 + mZG16 + GEM: recombinant protein plus gemcitabine treatment group, PANC02 + FGK45: CD40 agonist monoclonal antibody systemic injection treatment group, PANC02 + GEM (60 mg / kg): high-dose gemcitabine treatment group. The results are shown in Figure 7.

[0136] Figure 7a (tumor image) confirms the effect of systemic injection of ZG16 recombinant protein and combined chemotherapy drugs on the growth of pancreatic cancer in vivo. It was observed that the recombinant protein ZG16 protein can significantly inhibit the growth of pancreatic cancer cells in vivo, and its effect is significantly greater than the application of low-dose gemcitabine chemotherapy alone, P<0.001. In addition, the combination of low-dose ZG16 recombinant protein and low-dose chemotherapy drugs also plays a role in maximally inhibiting the growth of pancreatic cancer in vivo, achieving the effect of ultra-high-dose chemotherapy (4 times the dose) drug injection in vivo.

[0137] Figure 7b (Weight Chart) shows that chemotherapy drugs significantly affect mouse weight gain. While chemotherapy drugs have some effects, they also cause significant overall harm to the subjects. High-dose chemotherapy drugs, in particular, have a particularly significant impact on body weight (P < 0.001). Notably, the weight gain of animals in the recombinant ZG16 and CD40 monoclonal antibody groups was faster than that in the chemotherapy drug groups (low-dose and high-dose), P = 0.0021 and P < 0.001, respectively. Furthermore, the ZG16 recombinant protein group exhibited the fastest weight gain.

[0138] Combining the tumor volume and body weight data reveals that recombinant ZG16 and CD40 monoclonal antibodies inhibit pancreatic cancer cells in vivo more effectively than lower-dose chemotherapy drugs, without toxicity. The combination of recombinant ZG16 and low-dose chemotherapy drugs was significantly more effective than chemotherapy alone (p<0.001) and exhibited less toxicity than chemotherapy alone. This suggests that the combination of recombinant ZG16 and low-dose chemotherapy drugs reduces toxicity and enhances efficacy.

[0139] This study demonstrated that ZG16 further promotes pancreatic cancer tumor immunity in vivo by directly upregulating CD40. In contrast, the commercially available CD40 agonist antibody FGK45 does not directly upregulate CD40 in vivo. Furthermore, ZG16 has virtually no toxic side effects compared to chemotherapy.

[0140] Example 8 ZG16 protein directly upregulates CD40 protein expression in DCs in tumor tissues and compares it with other treatment methods and the commercially available CD40 agonist antibody FGK45

[0141] Experimental procedures: Mouse-derived PANC02 pancreatic cancer cells (5×10 5 ) were injected subcutaneously into 6-week-old female C57BL / 6 mice when the tumor volume approached 30 mm. 3 Around 20 days, mice were given ZG16 recombinant protein (200 μg / per mouse, intraperitoneal injection, twice a week), low-dose GEM (15 mg / kg, intraperitoneal injection, twice a week), high-dose GEM (60 mg / kg, intraperitoneal injection, twice a week), and CD40 monoclonal antibody (12.5 μg / per mouse, twice a week). Tumor volume and mouse body weight were measured every two days for about 20 days.

[0142] PANC02: murine pancreatic cancer cells, PANC02 + GEM (15 mg / kg): low-dose gemcitabine treatment group, PANC02 + mZG16: recombinant protein injection group, PANC02 + mZG16 + GEM: recombinant protein plus gemcitabine treatment group, PANC02 + FGK45: CD40 agonist monoclonal antibody systemic injection treatment group, PANC02 + GEM (60 mg / kg): high-dose gemcitabine treatment group. The results are shown in Figure 8.

[0143] Preparation of mononuclear cell suspension from tumor tissue: Mince the tissue until it becomes viscous, place it on a filter as described above, rinse 3-4 times to obtain a cell suspension, centrifuge, collect the precipitate, add red blood cell lysis buffer, let it stand, centrifuge again, and wash to obtain mononuclear cells. Immobilized DC (mDC) cell culture: Resuspend the mononuclear cells obtained in the previous step in culture medium, inoculate and culture, centrifuge, and culture in culture medium supplemented with growth factors for 7 consecutive days to obtain immobilized DC (mDC). Induction culture: Add cell culture medium containing maturation-promoting factors to immobilized DC cells for 3 days.

[0144] Western blot analysis was performed to examine CD40 antigen expression in DCs isolated from tumor tissue in each group. This demonstrated that ZG16 directly upregulates CD40 to further promote pancreatic cancer tumor immunity in vivo. In contrast, the commercially available CD40 agonist antibody FGK45 does not directly upregulate CD40 in vivo, demonstrating distinct biological effects. This further demonstrated that ZG16 protein loading with a low-dose chemotherapy drug (gemcitabine) significantly stimulated CD40 proliferation.

[0145] The results in Figure 8 show that only the ZG16 protein-treated group showed a significant upregulation of CD40 expression in DCs from tumor tissue, while the chemotherapy and FGK45 antibody groups showed no direct CD40 upregulation. This suggests that ZG16 protein's unique tumor immunomodulatory effect of upregulating CD40 can reduce the use of expensive and difficult-to-obtain exogenous dendritic cells (which require isolation from animals and humans) and has virtually no toxic side effects compared to the chemotherapy ZG16. This further demonstrates that ZG16 protein loading with a low-dose chemotherapy drug (gemcitabine) has the greatest effect on CD40 proliferation.

[0146] Example 9 Experimental study on the killing rate of pancreatic cancer cells by ZG16-transduced DCs loaded with other chemotherapy drugs

[0147] Experimental steps: ZG16-transduced DCs were loaded with chemotherapy drugs and incubated with pancreatic cancer cells PANC1. In the control setting, chemotherapy drugs were used alone. PANC1 cells were cultured at 5×10 4Cells were seeded per well of a 96-well plate and cultured overnight. Mitoxantrone concentrations ranged from 1.5 μg / ml to 4.0 μg / ml, decitabine concentrations ranged from 1 μg / ml to 5.0 μg / ml, doxorubicin concentrations ranged from 1 μg / ml to 4.0 μg / ml, 5-FU concentrations ranged from 32 μg / ml to 64 μg / ml, 125 μg / ml to 256 μg / ml, and vinorelbine concentrations ranged from 0.5 μg / ml to 1.0 μg / ml, 5 μg / ml to 10 μg / ml. At the 48-hour incubation time point, cells were treated with 10 μL of CCK-8 reagent (Dojindo Molecular Technologies, Inc., Shanghai, China) and incubated at 37°C for approximately 1 hour. Cell viability was assessed by measuring absorbance at 450 nm. A control group was treated with culture medium alone.

[0148] Inhibition (killing rate) % = [(OD value of blank control group - OD value of treatment group) / OD value of blank control group] × 100%.

[0149] ZG16-transduced DCs loading increased the killing rate of 5-fluorouracil (5FU, 32-256μM) and doxorubicin (1μM, 4μM) by nearly 1-10 times. Furthermore, 5-fluorouracil, doxorubicin, and vinorelbine demonstrated a strong synergistic killing effect at certain low-dose chemotherapy doses. The loading effect of ZG16-transduced DCs on decitabine (1μM, 4μM) and mitoxantrone (1.5μM, 4μM) was no different from that of the chemotherapy drugs alone (possibly due to the synergistic effects of the chemotherapy drugs on the immune microenvironment).

[0150] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A tumor vaccine with multi-target immune checkpoint tumor antibody loading immune cells, characterized in that: The tumor vaccine is obtained by transducing DCs with ZG16 recombinant protein or LECTIN short peptide gene or by incubating DCs with ZG16 recombinant protein and LECTIN short peptide in vitro.

2. The tumor vaccine of multi-target immune checkpoint tumor antibody-loaded immune cells according to claim 1, characterized in that: The multi-target immune checkpoint is selected from at least one of PD-L1, CD40, PD1 and CTLA4.

3. A tumor vaccine containing multi-target immune checkpoint tumor antibodies and immune cells as described in claim 1 or 2 is used in pancreatic cancer vaccines.

4. The use according to claim 3, characterized in that: The tumor vaccine is used in combination with ZG16 protein or LECTIN short peptide or chemotherapy drugs.

5. The use according to claim 5, characterized in that: The chemotherapy drug is gemcitabine.

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