Activated dendritic cells and their use in the treatment of cancer

Abstract

The present invention relates to activated dendritic cells and their use in the treatment of cancer. In particular, the present invention relates to a placental derived heat shock protein gp96-antigen complex, dendritic cells activated by said complex, and pharmaceutical compositions, vaccine adjuvants, immunomodulators and vaccine formulations containing said complex or dendritic cells and their use in anti-tumor.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to activated dendritic cells and their application in the treatment of cancer. Background Technology

[0002] Cellular immunotherapy is a technique that involves collecting the body's own immune cells, culturing them in vitro to increase their number thousands of times and enhance their targeted killing function, and then reinfusing them into the body to kill pathogens, cancer cells, or mutated cells in the blood and tissues.

[0003] Cell therapy using activated dendritic cells (DCs) is one of the promising approaches to cancer prevention and treatment. However, while existing immune cell nutrient solutions and culture media reagents can maintain cell growth and proliferation in vitro, improving their survival rate, cytotoxic activity, and expansion, the resulting cell efficacy remains unsatisfactory and lacks specificity. In particular, activated DCs cannot effectively stimulate the production of T cells that specifically recognize and kill tumors, significantly hindering their application and development. Therefore, there is an urgent need in this field for novel immune cell culture technologies to enhance the function and efficacy of related immune cell cultures in vitro. Summary of the Invention

[0004] To overcome the aforementioned problems, after extensive scientific research and validation, the inventors developed a dendritic cell (DC) immunotherapy targeting cancers such as glioma and cholangiocarcinoma. The DCs are obtained through activation of a placental-derived heat shock protein gp96-antigen complex. Using peptide microarray enrichment technology combined with mass spectrometry, the complex was identified as containing at least 50 potential tumor antigens, demonstrating its broad-spectrum anti-tumor ability. Following subcutaneous immunization, the bound antigens are effectively presented to DCs, simultaneously acting as a natural adjuvant to activate DCs, promoting DC maturation and the secretion of related cytokines. This effectively stimulates tumor-specific CTLs and enhances the body's non-specific immune response. The specific CTL response and the enhanced non-specific immune response synergistically mediate the immune response against the tumor.

[0005] Heat shock protein gp96-antigen complex

[0006] In one aspect, the present invention provides a heat shock protein gp96-antigen complex, which can be prepared by the following method:

[0007] The complex was obtained by sequentially separating the homogenate of isolated animal tissue by ConA-Sepharose gel column chromatography, HiTrap Q ion exchange chromatography, and CHT ion exchange chromatography.

[0008] In some embodiments, the ex vivo animal tissue is placental tissue or tumor tissue of a human or non-human mammal.

[0009] In some embodiments, the homogenate of the isolated animal tissue is prepared by the following method: the isolated animal tissue is added to a 10-50 mM NaHCO3 solution with a pH of 6-8 at a mass (g)-volume (ml) ratio of 1:4 to 1:8 and then ground to obtain the tissue homogenate.

[0010] In some embodiments, the concentration of the NaHCO3 solution is, for example, 10-20 mM, 10-30 mM, 10-40 mM, 20-30 mM, 20-40 mM, 20-50 mM, 30-40 mM, 30-50 mM, or 40-50 mM, preferably 30 mM. In some embodiments, the pH of the NaHCO3 solution is, for example, 6.2–8, 6.4–8, 6.6–8, 6.8–8, 7.0–8, 7.2–8, 7.4–8, 7.6–8, 7.8–8, 6.2–7.8, 6.4–7.8, 6.6–7.8, 6.8–7.8, 7.0–7.8, 7.2–7.8, 7.4–7.8, 7.6–7.8, 6.2–7.6, 6.4–7.6, 6.6–7.6, 6.8–7.6, 7.0–7.6, 7… .2~7.6, 7.4~7.6, 6.2~7.4, 6.4~7.4, 6.6~7.4, 6.8~7.4, 7.0~7.4, 7.2~7.4, 6.2~7.2, 6.4~7.2, 6.6~7.2, 6.8~7.2, 7.0~7.2, 6.2~7.0, 6.4~7.0, 6.6~7.0, 6.8~7.0, 6.2~6.8, 6.4~6.8, 6.6~6.8, 6.2~6.6, 6.4~6.6, or 6.2~6.4.

[0011] In some embodiments, prior to the above separation, the tissue homogenate is further purified by the following steps:

[0012] Step 1-1: Centrifuge the tissue homogenate at low temperature (e.g., 2-6°C) and collect the supernatant;

[0013] Step 1-2: Using ammonium sulfate fractionation precipitation, ammonium sulfate is added to the supernatant obtained in Step 1-1 under low temperature conditions (e.g., 2-6°C), and the precipitate obtained when the ammonium sulfate concentration is 50%-70% is collected to obtain the initial extract. In some embodiments, in Step 1-1, the tissue homogenate is centrifuged at 1000 rpm to 10000 rpm for 0.5 h to 1 h at 2-6°C, and the supernatant is collected. Optionally, the collected supernatant is centrifuged again at 1000 rpm to 10000 rpm for 0.5 h to 1 h at 2-6°C, and the supernatant is collected.

[0014] In some embodiments, in steps 1-2, the precipitate obtained when the ammonium sulfate concentration is greater than 50%, for example 52%–70%, 54%–70%, 56%–70%, 58%–70%, 60%–70%, 62%–70%, 64%–70%, 66%–70%, or 68%–70%.

[0015] In some embodiments, in steps 1-2, the precipitate is dissolved in a 5-50 mM Tris-HCl solution containing 50-200 mM NaCl at a mass (g) to volume (ml) ratio of 1:(2-20).

[0016] In some embodiments, in steps 1-2, the mass (g) - volume (ml) ratio is 1:(4-20), 1:(6-20), 1:(8-20), 1:(10-20), 1:(12-20), 1:(14-20), 1:(16-20), 1:(18-20), 1:(2-18), 1:(4-18), 1:(6-18), 1:(8-18), 1:(10-18), 1:(12-18), 1:(14-18), 1:(16-18), 1:(2-16), 1:(4-16), 1:(6-16), 1:(8-16), 1:(10-16), 1:(12-18). The precipitate is dissolved in 5-50 mM Tris-HCl solution at a ratio of 1:9, preferably 1:9. In some embodiments, the concentration of Tris-HCl is, for example, 5–10 mM, 5–15 mM, 5–20 mM, 5–25 mM, 5–30 mM, 5–35 mM, 5–40 mM, 5–45 mM, 5–50 mM, 10–15 mM, 10–20 mM, 10–25 mM, 10–30 mM, 10–35 mM, 10–40 mM, 10–45 mM, 10–50 mM, 15–20 mM, 15–25 mM, 15–30 mM, 15–35 mM, 15–40 mM. 15–45 mM, 15–50 mM, 20–25 mM, 20–30 mM, 20–35 mM, 20–40 mM, 20–45 mM, 20–50 mM, 25–30 mM, 25–35 mM, 25–40 mM, 25–45 mM, 25–50 mM, 30–35 mM, 30–40 mM, 30–45 mM, 30–50 mM, 35–40 mM, 35–45 mM, 35–50 mM, 40–45 mM, 40–50 mM, or 45–50 mM, preferably 20 mM. In a further preferred embodiment, the NaCl concentration in the Tris-HCl solution is, for example, 50–100 mM, 50–150 mM, 50–200 mM, 100–150 mM, 100–200 mM, or 150–200 mM, preferably 200 mM.

[0017] In some embodiments, the ConA-Sepharose gel column chromatography includes the following steps:

[0018] Step 2-1: Load the initial extract onto a ConA-Sepharose gel column;

[0019] Step 2-2: Elute with 5-50 mM Tris-HCl solution containing 50-200 mM NaCl until the UV absorption of the elution product is below 0.01 at a detection wavelength of 280 nm;

[0020] Steps 2-3: Continue elution with a 5-50 mM Tris-HCl solution containing 8% α-pyranoside and 50-200 mM NaCl, and collect the eluent (preferably, collect the flow-through of the 0-3 (e.g., 0.5-2) column volumes) to obtain the ConA-Sepharose isolate.

[0021] In some embodiments, in step 2-2, the concentration of Tris-HCl is, for example, 5–10 mM, 5–15 mM, 5–20 mM, 5–25 mM, 5–30 mM, 5–35 mM, 5–40 mM, 5–45 mM, 5–50 mM, 10–15 mM, 10–20 mM, 10–25 mM, 10–30 mM, 10–35 mM, 10–40 mM, 10–45 mM, 10–50 mM, 15–20 mM, 15–25 mM, 15–30 mM, 15–35 mM, 15–40 mM, 15–40 mM, 10–45 mM, 10–50 mM, 15–20 mM, 15–25 mM, 15–30 mM, 15–35 mM, 15–40 mM, 15–40 mM, 10–40 mM, 10–50 mM, 15–20 mM, 15–25 mM, 15–30 mM, 15–35 mM, 15–40 ... 0mM, 15-45mM, 15-50mM, 20-25mM, 20-30mM, 20-35mM, 20-40mM, 20-45mM, 20-50mM, 25-30mM, 25-35mM, 25-40mM, 25-45mM, 25-50mM, 30-35mM, 30-40mM, 30-45mM, 30-50mM, 35-40mM, 35-45mM, 35-50mM, 40-45mM, 40-50mM, or 45-50mM, preferably 20mM. In a further preferred embodiment, the NaCl concentration in the Tris-HCl solution is, for example, 50–100 mM, 50–150 mM, 50–200 mM, 100–150 mM, 100–200 mM, or 150–200 mM, preferably 200 mM.

[0022] In some embodiments, the HiTrap Q ion exchange chromatography includes the following steps:

[0023] Step 3-1: Load the ConA-Sepharose isolate onto a HiTrap Q ion exchange column;

[0024] Step 3-2: Clean the HiTrap Q ion exchange column with 5-50 mM Tris-HCl;

[0025] Step 3-3: Elute with 5-50 mM Tris-HCl containing NaCl (300 mM-1000 mM), and collect the eluent until the absorbance of the elution product is less than 100 mA at a detection wavelength of 280 nm; optionally, concentrate the collected eluent (e.g., by ultrafiltration) and dilute with 20-100 mM phosphate buffer at pH 6-8 to obtain the HiTrap Q isolate.

[0026] In some implementations, in step 3-2, the following concentrations are used: 5-10 mM, 5-15 mM, 5-20 mM, 5-25 mM, 5-30 mM, 5-35 mM, 5-40 mM, 5-45 mM, 5-50 mM, 10-15 mM, 10-20 mM, 10-25 mM, 10-30 mM, 10-35 mM, 10-40 mM, 10-45 mM, 10-50 mM, 15-20 mM, 15-25 mM, 15-30 mM, 15-35 mM, 15-40 mM, 15-45 mM, 15- The HiTrap Q ion exchange column can be cleaned with Tris-HCl at concentrations of 50 mM, 20–25 mM, 20–30 mM, 20–35 mM, 20–40 mM, 20–45 mM, 20–50 mM, 25–30 mM, 25–35 mM, 25–40 mM, 25–45 mM, 25–50 mM, 30–35 mM, 30–40 mM, 30–45 mM, 30–50 mM, 35–40 mM, 35–45 mM, 35–50 mM, 40–45 mM, 40–50 mM, or 45–50 mM, preferably 20 mM.

[0027] In some embodiments, in step 3-3, the concentration of Tris-HCl is, for example, 5–10 mM, 5–15 mM, 5–20 mM, 5–25 mM, 5–30 mM, 5–35 mM, 5–40 mM, 5–45 mM, 5–50 mM, 10–15 mM, 10–20 mM, 10–25 mM, 10–30 mM, 10–35 mM, 10–40 mM, 10–45 mM, 10–50 mM, 15–20 mM, 15–25 mM, 15–30 mM, 15–35 mM, 15–40 ...40 mM, 10–50 mM, 15–20 mM, 15–25 mM, 15–30 mM, 15–40 mM, 15–40 mM, 15–40 mM, 1 0mM, 15-45mM, 15-50mM, 20-25mM, 20-30mM, 20-35mM, 20-40mM, 20-45mM, 20-50mM, 25-30mM, 25-35mM, 25-40mM, 25-45mM, 25-50mM, 30-35mM, 30-40mM, 30-45mM, 30-50mM, 35-40mM, 35-45mM, 35-50mM, 40-45mM, 40-50mM, or 45-50mM, preferably 20mM. In a further preferred embodiment, the NaCl concentration in the Tris-HCl is, for example, 300–350 mM, 300–400 mM, 300–450 mM, 300–500 mM, 300–550 mM, 300–600 mM, 300–650 mM, 300–700 mM, 300–750 mM, 300–800 mM, 300–850 mM, 300–900 mM. M, 300~950mM, 300~1000mM, 350~400mM, 350~450mM, 350~500mM, 350~550mM, 350~600mM, 350 ~650mM, 350~700mM, 350~750mM, 350~800mM, 350~850mM, 350~900mM, 350~950mM, 350~1000m M, 400~450mM, 400~500mM, 400~550mM, 400~600mM, 400~650mM, 400~700mM, 400~750mM, 400~ 800mM, 400~850mM, 400~900mM, 400~950mM, 400~1000mM, 450~500mM, 450~550mM, 450~600mM , 450~650mM, 450~700mM, 450~750mM, 450~800mM, 450~850mM, 450~900mM, 450~950mM, 450~1 000mM, 500~550mM, 500~600mM, 500~650mM, 500~700mM, 500~750mM, 500~800mM, 500~850mM,500~900mM, 500~950mM, 500~1000mM, 550~600mM, 550~650mM, 550~700mM, 550~750mM, 550~800mM, 550~850mM, 550~900mM, 550~950mM, 550~1000mM, 600~650mM, 600~700mM, 600~750mM, 600~800mM, 600~850mM, 600~900mM, 6 00~950mM, 600~1000mM, 650~700mM, 650~750mM, 650~800mM, 650~850mM, 65 0~900mM, 650~950mM, 650~1000mM, 700~750mM, 700~800mM, 700~850mM, 70 0~900mM, 700~950mM, 700~1000mM, 750~800mM, 750~850mM, 750~900mM, 750 ~950mM, 750~1000mM, 800~850mM, 800~900mM, 800~950mM, 800~1000mM, 850~900mM, 850~950mM, 850~1000mM, 900~950mM, 900~1000mM, or 950~1000mM.

[0028] In some preferred embodiments, in steps 3-3, the pH of the phosphate buffer solution is, for example, 6.2–8, 6.4–8, 6.6–8, 6.8–8, 7.0–8, 7.2–8, 7.4–8, 7.6–8, 7.8–8, 6.2–7.8, 6.4–7.8, 6.6–7.8, 6.8–7.8, 7.0–7.8, 7.2–7.8, 7.4–7.8, 7.6–7.8, 6.2–7.6, 6.4–7.6, 6.6–7.6, 6.8–7.6, 7.0–7. pH 6, 7.2–7.6, 7.4–7.6, 6.2–7.4, 6.4–7.4, 6.6–7.4, 6.8–7.4, 7.0–7.4, 7.2–7.4, 6.2–7.2, 6.4–7.2, 6.6–7.2, 6.8–7.2, 7.0–7.2, 6.2–7.0, 6.4–7.0, 6.6–7.0, 6.8–7.0, 6.2–6.8, 6.4–6.8, 6.6–6.8, 6.2–6.6, 6.4–6.6, or 6.2–6.4, preferably pH 6.8.

[0029] In some preferred embodiments, in step 3-3, the concentration of the phosphate buffer is, for example, 20-30 mM, 20-40 mM, 20-50 mM, 20-60 mM, 20-70 mM, 20-80 mM, 20-90 mM, 30-40 mM, 30-50 mM, 30-60 mM, 30-70 mM, 30-80 mM, 30-90 mM, 30-100 mM, 40-50 mM, 40-60 mM, 4... 0–70 mM, 40–80 mM, 40–90 mM, 40–100 mM, 50–60 mM, 50–70 mM, 50–80 mM, 50–90 mM, 50–100 mM, 60–70 mM, 60–80 mM, 60–90 mM, 60–100 mM, 70–80 mM, 70–90 mM, 70–100 mM, 80–90 mM, 80–100 mM, or 80–100 mM, preferably 50 mM.

[0030] In some embodiments, the CHT ion exchange chromatography includes the following steps:

[0031] Step 4-1: Load the HiTrap isolate onto a CHT ion exchange column;

[0032] Step 4-2: Clean the CHT ion exchange column with 5-50 mM Tris-HCl;

[0033] Step 4-3: Elute with 20 mM Tris-HCl containing 300 mM to 1000 mM NaCl, and collect the eluent until the absorbance value is below 100 mA; optionally, concentrate the collected eluent (e.g., ultrafiltration concentration) and dilute with 50 mM phosphate buffer at pH 6.8 to obtain the complex.

[0034] In some embodiments, in step 4-2, the concentration of Tris-HCl is, for example, 5–10 mM, 5–15 mM, 5–20 mM, 5–25 mM, 5–30 mM, 5–35 mM, 5–40 mM, 5–45 mM, 5–50 mM, 10–15 mM, 10–20 mM, 10–25 mM, 10–30 mM, 10–35 mM, 10–40 mM, 10–45 mM, 10–50 mM, 15–20 mM, 15–25 mM, 15–30 mM, 15–35 mM, 15–40 mM, 15–40 mM, 10–50 mM, 15–20 mM, 15–25 mM, 15–30 mM, 15–35 mM, 15–40 mM, 15–40 mM, 10–40 mM, 10–50 mM, 15–20 mM, 15–25 mM, 15–30 mM, 15–35 mM, 15–40 ... 0mM, 15-45mM, 15-50mM, 20-25mM, 20-30mM, 20-35mM, 20-40mM, 20-45mM, 20-50mM, 25-30mM, 25-35mM, 25-40mM, 25-45mM, 25-50mM, 30-35mM, 30-40mM, 30-45mM, 30-50mM, 35-40mM, 35-45mM, 35-50mM, 40-45mM, 40-50mM, or 45-50mM, preferably 20mM.

[0035] In some implementations, in steps 4-3, the concentration is, for example, 300-350 mM, 300-400 mM, 300-450 mM, 300-500 mM, 300-550 mM, 300-600 mM, 300-650 mM, 300-700 mM, 300-750 mM, 300-800 mM, 300-850 mM, 300-900 mM, 300-950 mM, 300-1000 mM, 350-400 mM, 350-450 mM, 350-500 mM, 350-550 mM, 350-600 mM, 350-650 mM, 350-700 mM, 350-750 mM, 350- 800mM, 350~850mM, 350~900mM, 350~950mM, 350~1000mM, 400~450mM, 400~5 00mM, 400~550mM, 400~600mM, 400~650mM, 400~700mM, 400~750mM, 400~800 mM, 400~850mM, 400~900mM, 400~950mM, 400~1000mM, 450~500mM, 450~550m M, 450~600mM, 450~650mM, 450~700mM, 450~750mM, 450~800mM, 450~850mM, 450~900mM, 450~950mM, 450~1000mM, 500~550mM, 500~600mM, 500~650mM, 5 00~700mM, 500~750mM, 500~800mM, 500~850mM, 500~900mM, 500~950mM, 500 ~1000mM, 550~600mM, 550~650mM, 550~700mM, 550~750mM, 550~800mM, 550~ 850mM, 550~900mM, 550~950mM, 550~1000mM, 600~650mM, 600~700mM, 600~7 50mM, 600~800mM, 600~850mM, 600~900mM, 600~950mM, 600~1000mM, 650~70 0mM, 650~750mM, 650~800mM, 650~850mM, 650~900mM, 650~950mM, 650~1000 mM, 700~750mM, 700~800mM, 700~850mM, 700~900mM, 700~950mM, 700~1000m M, 750~800mM, 750~850mM, 750~900mM, 750~950mM, 750~1000mM, 800~850mM,Elution with PB at 800–900 mM, 800–950 mM, 800–1000 mM, 850–900 mM, 850–950 mM, 850–1000 mM, 900–950 mM, 900–1000 mM, or 950–1000 mM, preferably 500 mM.

[0036] In some implementations, in step 4-3, the concentrations are 5-10 mM, 5-15 mM, 5-20 mM, 5-25 mM, 5-30 mM, 5-35 mM, 5-40 mM, 5-45 mM, 5-50 mM, 10-15 mM, 10-20 mM, 10-25 mM, 10-30 mM, 10-35 mM, 10-40 mM, 10-45 mM, 10-50 mM, 15-20 mM, 15-25 mM, 15-30 mM, 15-35 mM, 15-40 mM, 15-45 mM. Diluted with phosphate buffer at 15–50 mM, 20–25 mM, 20–30 mM, 20–35 mM, 20–40 mM, 20–45 mM, 20–50 mM, 25–30 mM, 25–35 mM, 25–40 mM, 25–45 mM, 25–50 mM, 30–35 mM, 30–40 mM, 30–45 mM, 30–50 mM, 35–40 mM, 35–45 mM, 35–50 mM, 40–45 mM, 40–50 mM, or 45–50 mM, preferably 20 mM. In a further preferred embodiment, the pH of the phosphate buffer solution is, for example, 6.2–8, 6.4–8, 6.6–8, 6.8–8, 7.0–8, 7.2–8, 7.4–8, 7.6–8, 7.8–8, 6.2–7.8, 6.4–7.8, 6.6–7.8, 6.8–7.8, 7.0–7.8, 7.2–7.8, 7.4–7.8, 7.6–7.8, 6.2–7.6, 6.4–7.6, 6.6–7.6, 6.8–7.6, 7.0–7.6, 7.2– pH values ​​are 7.6, 7.4–7.6, 6.2–7.4, 6.4–7.4, 6.6–7.4, 6.8–7.4, 7.0–7.4, 7.2–7.4, 6.2–7.2, 6.4–7.2, 6.6–7.2, 6.8–7.2, 7.0–7.2, 6.2–7.0, 6.4–7.0, 6.6–7.0, 6.8–7.0, 6.2–6.8, 6.4–6.8, 6.6–6.8, 6.2–6.6, 6.4–6.6, or 6.2–6.4, with pH 6.8 being preferred.

[0037] The heat shock protein gp96 is a highly expressed molecular chaperone protein in cells, playing important roles in various aspects such as the folding of newborn proteins and the degradation of damaged proteins, viral and tumor antigen presentation and T cell activation, damage-associated molecular patterns (DAMPs)-mediated innate immunity, initiation of anti-tumor and antiviral T cell immunity, and regulation of viral replication as a host factor, driving inflammation-cancer transformation and tumorigenesis. In some embodiments, gp96 has the amino acid sequence shown in SeqID No. 1.

[0038] In some embodiments, the antigen in the heat shock protein gp96-antigen complex of the present invention is selected from one or more polypeptides as shown in Seq ID No. 2 to 72 (e.g., more than 1, more than 5, more than 10, more than 15, more than 20, more than 25, more than 30, more than 35, more than 40, more than 45, more than 50, more than 55, more than 60, more than 65, or more than 70), and the polypeptide is a tumor-associated antigen or a protein highly expressed in tumor tissue.

[0039] In some embodiments, peptide microarray enrichment combined with mass spectrometry identification shows that gp96 in the complex binds to 71 tumor-associated antigens as indicated by SeqID Nos. 2–72. In some embodiments, the peptide-binding site of the heat shock protein gp96 is located at amino acids 624–630, which are highly conserved on gp96, allowing for non-covalent binding of the antigen peptide.

[0040] The complex obtained by the method of this invention has less impurity content, such as residual nucleic acids and other proteins, which greatly improves the purity of the heat shock protein gp96-antigen complex and enables better activation of immune cells. SDS-PAGE gel analysis shows that the purity of the complex described in this invention is above 80%, for example above 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0041] Activated dendritic cells

[0042] Gp96 is the only known natural adjuvant derived from mammals. It acts on APCs (antigen-presenting cells, mainly dendritic cells) to activate other immune cells, producing immune factors and thus enhancing the body's immune response. In the complex, gp96 can effectively present various bound tumor-associated antigens to dendritic cells, while simultaneously activating them as a natural adjuvant, promoting dendritic cell maturation and the secretion of related cytokines, thereby effectively stimulating tumor-specific CTLs and enhancing the body's non-specific immune response. Therefore, this invention also relates to dendritic cells activated by the aforementioned complex.

[0043] In another aspect, the present invention provides a population of DC cells,

[0044] (1) Provide monocytes;

[0045] (2) The monocytes are cultured in a DC cell culture medium containing 0.1-5 μM glucocorticoids for 3-10 days (e.g., 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days, preferably 6 days) to obtain mature DC cells.

[0046] (3) Administer at 20–100 μg / 1×10 7 The ratio of DC cells was adjusted, the complex described above was added, and the cells were cultured for 6–48 h to obtain the DC cell population.

[0047] "Monocytes" refer to CD14+ mononuclear leukocytes capable of differentiating into dendritic cells. Monocytes can be derived from any mammal, but are preferably human monocytes. Samples used to provide monocytes include, but are not limited to, blood, blood fractions (e.g., white blood cells (WBCs), erythrocyte sedimentation rate (ESR) amber layer, peripheral blood mononuclear cells (PBMCs), and monocyte leukocyte removal products. In some embodiments, the monocytes described in step (1) are derived from peripheral blood or umbilical cord blood, preferably peripheral blood. In some embodiments, monocytes are provided together with other peripheral blood mononuclear cells (PBMCs), for example, as a product of apheresis. Methods for isolating and enriching dendritic cell precursors such as monocytes and conventional dendritic cells from various sources, including blood and bone marrow, are known in the art. For example, monocytes and conventional dendritic cells can be isolated by collecting heparinized blood, by apheresis or leukocyte removal, by preparing ESR amber layer, rosette formation, centrifugation, density gradient centrifugation, differential cell lysis, filtration, panning, and fluorescence activation. Cell sorting or immunomagnetic separation is used for separation. In some preferred embodiments, monocytes are separated from monocyte leukacidosis. Methods of leukacidosis are known in the art. Leukacidosis is a procedure that removes leukocytes from a subject's blood and then returns the remainder to the subject. The product of leukacidosis is typically a blood fraction rich in PBMCs with low levels of contaminated red blood cells, granulocytes, and platelets. Methods and devices for performing leukacidosis are well known in the art. Monocyte-derived dendritic cell precursors and / or differentiated conventional dendritic cells can be separated from healthy subjects or from subjects requiring immune stimulation, such as cancer patients or other subjects for whom cellular immune stimulation may be beneficial or desired (e.g., subjects with bacterial or viral infections). Dendritic cell precursors and / or immature dendritic cells can also be obtained from HLA-matched healthy individuals for administration to HLA-matched subjects requiring immune stimulation.

[0048] In some implementations, the enrichment of monocytes can be performed on monocytes or PBMCs, including, for example, centrifugation, panning, tangential flow filtration, Ficoll density gradient, diluted Ficoll density gradient centrifugation, diluted Percoll density gradient centrifugation, antibody panning, magnetic cell sorting, positive or negative immunomagnetic selection, etc. Additionally, once isolated from the subject, monocytes (e.g., purified monocytes, enriched monocytes, PBMCs containing monocytes, etc.) can optionally be incubated, for example, at a temperature of 1°C to 34°C for a period of time, for example, approximately 1 to 96 hours after their isolation from the subject. Viable monocytes are highly purified, for example, with a purity exceeding 90%, 95%, or even 99%, as determined by flow cytometry using the monocyte marker CD14 and viability staining.

[0049] After isolating, purifying, and / or enriching monocytes, they are induced to differentiate into dendritic cells. Therefore, in a further embodiment, the method of the present invention includes a culture and / or differentiation step to obtain immature DCs.

[0050] In some implementations, in step (2), at 1×10 5 ~1×10 8 cells / ml (e.g., 1×10⁻⁶) 5 cells / ml, 1×10 6 cells / ml, 1×10 7 cells / ml, or 1×10 8 Cells / ml, preferably 2-4 × 10⁻⁶ 6 The mononuclear cells were seeded into the DC cell culture medium at a density of (cells / ml).

[0051] In some embodiments, one or more of PHA, IL-2, IL-4, GM-CSF, and TNF-α are added during the culture process described in step (2) (e.g., on days 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the culture). In some embodiments, monocytes are cultured in the presence of at least granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) (referred to as differentiation medium), and this lasts for about 1 to 4 days, for example, 1 to 3 days or 2 to 3 days, or even more particularly for 80, 75, 74, 73, 72, 71, 70 hours or less, more particularly for at least 48 hours and up to 72 hours. Limits of + / - 4 hours or + / - 2 hours are acceptable. In some embodiments, GM-CSF and IL-4 are added on day 0 of the culture, for example, at final concentrations of 50–100 ng / ml for GM-CSF and IL-4, respectively. In some implementations, monocytes are exposed to GM-CSF and IL-4 for approximately 1 to 4 days, such as 1 to 3 days or 2 to 3 days (based on a 24-hour day), during which time the DC precursors differentiate into immature dendritic cells. The concentrations of GM-CSF and IL-4 are 50–100 ng / ml, such as 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, or 100 ng / ml.

[0052] Precursor and / or immature dendritic cells are cultured using a combination of (at least) the above factors. This can be done by adding said factors to a culture medium. The culture medium can be any suitable type, i.e., supplemented with or without any other supplements, such as proteins, amino acids, or antibiotics. In some embodiments, the culture medium is RPMI 1640.

[0053] After monocytes differentiate into immature dendritic cells, the immature dendritic cells can mature into mature dendritic cells. Therefore, in some embodiments, the method for preparing the DC cell population described herein includes a maturation step, such as adding TNF-α to the (differentiated) immature DCs. The maturation time of DC cells in the presence of TNF-α is 1 to 3 days, preferably 1 to 2 days, more preferably about 1 day (24 hours). In some embodiments, TNF-α is added on day 6 of culture, preferably at a final concentration of 10–50 ng / ml, more preferably 20 ng / ml. The culture conditions are suitable for the immature DCs to form a mature DC population. In particular, TNF-α is added to the culture medium during the last 24 hours ± 4 hours (especially ± 2 hours) of cell culture before harvesting and / or stimulation. In some embodiments, the culture medium is RPMI 1640.

[0054] In some particularly preferred embodiments, the addition of a glucocorticoid compound during DC induction can enhance the stimulatory effect on PBMCs. Therefore, a glucocorticoid compound is added during the DC induction process. In some embodiments, the glucocorticoid compound is dexamethasone. The concentration of glucocorticoid compounds is 0.1–5 μM, for example 0.1–0.5 μM, 0.1–1 μM, 0.1–1.5 μM, 0.1–2 μM, 0.1–2.5 μM, 0.1–3 μM, 0.1–3.5 μM, 0.1–4 μM, 0.1–4.5 μM, 0.1–5 μM, 0.5–1 μM, 0.5–1.5 μM, 0.5–2 μM, 0.5–2.5 μM, 0.5–3 μM, 0.5–3.5 μM, 0.5–4 μM, 0.5–4.5 μM, 0.5–5 μM, 1–1.5 μM, 1–2 μM, 1–2.5 μM, 1–3 μM, 1–3.5 μM, 1–4 μM, 1–4 .5μM, 1~5μM, 1.5~2μM, 1.5~2.5μM, 1.5~3μM, 1.5~3.5μM, 1.5~4μM, 1.5~ 4.5μM, 1.5~5μM, 2~2.5μM, 2~3μM, 2~3.5μM, 2~4μM, 2~4.5μM, 2~5μM, 2.5~ 3μM, 2.5~3.5μM, 2.5~4μM, 2.5~4.5μM, 2.5~5μM, 3~3.5μM, 3~4μM, 3~4.5 μM, 3~5 μM, 3.5~4 μM, 3.5~4.5 μM, 3.5~5 μM, 4~4.5 μM, 4~5 μM, or 4.5 μM~5 μM.

[0055] In some implementations, the specific conditions for cultivation in steps (2) and (3) are 37°C and 5% CO2.

[0056] The proportion of CD83+ DC cells in the DC cell population prepared by the above method is greater than 70%, for example, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%.

[0057] In some embodiments, the proportion of DC cells with an IFN-γ+ELISPOT spot number greater than 500 CD83+ in PBMCs stimulated with the DC cell population is greater than 70%.

[0058] Pharmaceutical compositions, vaccine adjuvants, immunomodulators, vaccine formulations

[0059] In another aspect, the present invention provides a pharmaceutical composition comprising the aforementioned complex or DC cell population, and one or more pharmaceutically acceptable carriers.

[0060] In another aspect, the present invention provides a vaccine adjuvant, immunomodulator, or immunomodulator containing the aforementioned complex and / or DC cell population, and one or more pharmaceutically acceptable carriers.

[0061] In some implementations, the vaccine adjuvant, immunomodulator, or immunomodulator is an injectable (e.g., a solution or lyophilized powder for injection).

[0062] In some implementation schemes, the content of DC cells per unit dose is not less than 1×10⁻⁶. 4 (e.g., not less than 1×10) 4 No fewer than 3 × 10 4 No fewer than 5 × 10 4 No fewer than 7 × 10 4 No fewer than 1×10 5 No fewer than 3 × 10 5 No fewer than 5 × 10 5 No fewer than 7 × 10 5 No fewer than 1×10 6 No fewer than 3 × 10 6 No fewer than 5 × 10 6 No fewer than 7 × 10 6 No fewer than 1×10 7 No fewer than 3 × 10 7 No fewer than 5 × 10 7 No fewer than 7 × 10 7 No fewer than 1×10 8 No fewer than 3 × 10 8 No fewer than 5 × 10 8 No fewer than 7 × 10 8 No fewer than 1×10 9 No fewer than 3 × 10 9 No fewer than 5 × 10 9 No fewer than 7 × 10 9 No fewer than 1×10 10 No fewer than 3 × 10 10 No fewer than 5 × 10 10 One or no less than 7×10 10 For example, 1×10 5 ~1×10 8 indivual).

[0063] Methods for preparing dendritic cell vaccines are known to those skilled in the art. Suitable formulations for administration may include aqueous isotonic sterile injectable solutions containing antioxidants, buffers, antibacterial agents, and solutes that make the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions, wherein the suspensions may include suspending agents, solubilizers, thickeners, stabilizers, preservatives, immunostimulants, cytokines, and adjuvants.

[0064] Dendritic cell drug compositions / vaccines can be administered by various methods, such as, but not limited to, injection (e.g., subcutaneous, intradermal, intravenous, intralymphatic, intra-articular, intramuscular, intraperitoneal), continuous infusion, sustained release from implants, etc. DC vaccines can be administered at specific intervals. In one embodiment, DCs are administered at intervals of 2 to 4 weeks, particularly at intervals of two weeks. Dendritic cell vaccines can be administered with physiologically acceptable carriers, buffers, diluents, adjuvants, immunomodulators, etc. Preferably, the dendritic cell vaccine is autologous to the patient to whom it is administered, or is maximally HLA-matched.

[0065] The effective dose of cells administered to a subject is the amount that effectively achieves the desired beneficial therapeutic response in the subject, or inhibits the growth of cancer cells, while maintaining a good tolerability profile (minimal toxicity). The amount sufficient to achieve this effect is defined as the “therapeutic effective dose.” This dose will be determined by the biological and / or clinical activity of the resulting dendritic cells and, optionally, the patient’s condition. The size of this dose will also be determined by the presence, nature, and extent of any adverse side effects associated with the administration of specific cells to a particular patient. In determining the effective dose of cells for the treatment or prevention of diseases such as cancer (e.g., glioma, cholangiocarcinoma, etc.), the physician (or investigator) needs to evaluate the immune response against the target contained in the vaccine (i.e., immune surveillance) and assess the clinical progression of the tumor using measurable parameters (radiological tumor burden, tumor markers, circulating tumor cells, plasma circulating tumor DNA, or other alternative markers of disease burden or disease activity according to routine or immune-related RECIST criteria).

[0066] Typically, the dosage administered depends on the yield of DCs obtained from one round of leukocyte removal and the number of subsequent vaccinations required. In some implementations, the dosage is, for example, 5-100 × 10⁶ per round of vaccination. 6DC vaccination is administered 2 to 8 times, particularly 2 to 6 times, and even more particularly 2 to 4 times. The toxicity of DC vaccination is generally low and is related to the route of administration (intravenous administration has a greater risk of acute side effects compared to intradermal administration). Injections can be repeated 2, 3, 4, 5, or 6 times at intervals of 1, 2, or 3 weeks, and should be given intravenously or by intradermal or subcutaneous injection near a lymph node, or directly into the lymph node. Booster injections may be given after a pause of, for example, 1 to several months.

[0067] Optionally, a biological response modifier may be added to allow treatment via the DCs of the present invention. For example, cells may optionally be administered with an adjuvant or a cytokine such as GM-CSF, IL-12, IFN-α, or IL-2.

[0068] Uses, Immunotherapy

[0069] In another aspect, the present invention provides the use of the complex or DC cell population described herein in the preparation of a medicament, wherein the medicament has at least one of the following functions:

[0070] (1) Enhance the subject's immune response (e.g., non-specific immune response);

[0071] (2) Inducing the generation of tumor-specific CTL cells;

[0072] (3) Anti-tumor (e.g., slowing down or stopping the growth of established tumor lesions).

[0073] In another aspect, the present invention provides a method for enhancing a subject’s immune response (e.g., a nonspecific immune response), comprising the step of administering an effective amount of the complex or DC cell population described herein to the subject.

[0074] In another aspect, the present invention provides a method for inducing the generation of tumor-specific CTL cells, comprising the step of administering an effective amount of the complex described herein or a population of DC cells to a subject.

[0075] In another aspect, the present invention provides a method for anti-tumor activity (e.g., slowing or stopping the growth of an established tumor lesion), comprising the step of administering an effective amount of the complex or DC cell population described herein to a subject.

[0076] In some implementations, the tumors described herein are selected from gliomas and cholangiocarcinomas.

[0077] Unless otherwise stated, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art, and the immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA technologies referred to herein are conventional techniques in their respective fields. See, for example, Sambrook and Green, Molecular Cloning: A Laboratory Mannual, 4th edition (2012); Current Protocols in Molecular Biology (edited by F.M.A. Susubel et al.); Methods in Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (edited by M.J. MacPherson, B.D. Hames, and G.G. Taylor (1995)); Antibodies, A Laboratory Mannual (edited by Harlow and Lane (1988); and Culture of Animal Cells: A Mannual of Basic Technique and Specialized Applications, 6th edition (edited by R.R. Freshney (2010)).

[0078] Unless otherwise specified, the singular forms “a / an” and “the” used in this document include the referents of the plural forms.

[0079] "About" or "approximately" means within an acceptable margin of error for a particular value as determined by a person skilled in the art, which is partly dependent on the limitations of the measurement system. For example, in some embodiments, "about" means within one or more standard deviations. Alternatively, particularly with respect to biological systems or processes, "about" means within an order of magnitude of the referred value, preferably within five times the referred value, and more preferably within two times the referred value.

[0080] "Cell" generally refers to a biological cell, which can be the basic structural, functional, and / or biological unit of a living organism. It can originate from any organism having one or more cells. Some non-limiting examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaea cells, cells of unicellular eukaryotes, protozoan cells, cells from plants, algal cells, fungal cells, animal cells (e.g., invertebrate cells, vertebrate cells, mammalian cells), and so on. In some embodiments, the cell may not be derived from a natural organism; for example, it may be an artificial cell.

[0081] Dendritic cells, or DCs, are the most powerful professional antigen-presenting cells (APCs) in the body. They can efficiently take up, process, and present antigens. Immature DCs have strong migration capabilities, while mature DCs can effectively activate naïve T cells and are at the center of initiating, regulating, and maintaining the immune response.

[0082] "Antigen" refers to any molecule or fragment thereof that can be bound by a selective binder (e.g., a receptor or antibody). In some embodiments, antigen specifically refers to any substance that can elicit an immune response and can be used in animals to produce molecules or fragments thereof capable of binding to said antigen.

[0083] "Tumor-associated antigens" refer to antigens that are abnormally highly expressed in tumor cells but expressed at low levels in normal tissue cells. Through peptide microarray enrichment combined with mass spectrometry, the complexes extracted from ex vivo animal tissues described in this paper contain multiple tumor antigens, including 71 highly expressed proteins from tumor tissues as shown in Seq ID Nos. 2–72. These are tumor-associated antigens, and some of these antigens are specific surface antigens of glioma and cholangiocarcinoma, involved in specific immune responses.

[0084] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably in this document and refer to a polymer of at least two amino acid residues linked by one or more peptide bonds. This term does not imply a specific length of the polymer, nor is it intended to suggest or distinguish whether a polypeptide is produced using recombinant technology, chemical or enzymatic synthesis, or is naturally occurring.

[0085] "Subject" refers to a healthy individual or an individual who has or is suspected of having a certain disease. This can be a human or a non-human mammal, preferably a human. It typically includes healthy volunteers, patients, those being tested, and those being treated.

[0086] "Treatment / treating" generally refers to achieving the desired pharmacological and / or physiological effects, and covers the treatment of any disease in mammals, especially humans, including:

[0087] (1) To prevent the occurrence of disease or symptoms in subjects who may be susceptible to the disease or symptoms but have not yet been diagnosed with the disease or symptoms;

[0088] (2) Suppress disease symptoms, that is, prevent their development; or

[0089] (3) To alleviate disease symptoms, that is, to cause the disease or symptoms to subside.

[0090] The effect can be preventative in terms of complete or partial prevention of the disease or its symptoms, and / or therapeutic in terms of partial or complete stabilization or cure of the disease and / or side effects attributable to the disease. Furthermore, in addition to primary or initial therapy, vaccines can be used as "adjunctive therapy" to maximize their effectiveness in a therapeutic setting, or as a "maintenance" or "consolidative" approach after initial therapy to maximize disease control and delay disease recurrence.

[0091] "Cancer" refers to any kind of disease caused by malignant tumors.

[0092] Gliomas originate from the neuroectoderm of the brain and account for approximately 51% of all intracranial tumors. They are among the most aggressive malignant brain tumors, characterized by high growth rates, high recurrence rates, and high invasiveness. In China, brain tumors rank 14th in incidence and 10th in mortality among all cancers. Due to the unique location of the lesions, brain tumors are difficult to treat and highly complex, remaining one of the most challenging and difficult research areas in cancer treatment. Currently, surgery, radiotherapy, and chemotherapy are the main treatments for gliomas. However, each method has its limitations. Surgical treatment can reduce the disability and mortality rates, but it is highly invasive and increases the risk of postoperative infection. While radiotherapy has improved the cure rate for glioma patients, it also causes some damage to normal brain tissue. As for chemotherapy, although it has achieved some success in treating gliomas, chemotherapy drugs still suffer from poor water solubility, insufficient stability, and significant adverse reactions. Moreover, due to its lack of selectivity and low targeting efficiency, it can also be toxic to healthy brain tissue or brain cells.

[0093] Cholangiocarcinoma is a malignant tumor originating in the bile ducts, accounting for 3% of all gastrointestinal tumors. Globally, malignant tumors of the liver and bile ducts account for 13% of cancer-related deaths, with 10-20% of these being cholangiocarcinoma. Over the past 30 years, the global incidence of cholangiocarcinoma has been gradually increasing. Due to its unique anatomical location and late-onset clinical symptoms, cholangiocarcinoma faces challenges in diagnosis and treatment, resulting in poor prognosis. Radical surgical treatment for cholangiocarcinoma is extremely difficult due to its anatomical structure, with a 5-year survival rate of less than 30%. Immunotherapy, particularly cellular immunotherapy, aims to treat cholangiocarcinoma and reduce recurrence and metastasis by regulating the body's immune function and inducing specific tumor immunity. It has become an important component of comprehensive cholangiocarcinoma treatment. Attached Figure Description

[0094] Figure 1 This shows the proportion of CD83+ DC cells as detected by flow cytometry.

[0095] Figure 2 The image shows the number of spots in DC-stimulated PBMCs after dexamethasone activation, as detected by IFN-γ+ELIPOT.

[0096] Figure 3 This study shows the effects of 25 types of glucocorticoids, small molecule compounds, and antibody drugs on DC-activated PBMCs.

[0097] Figure 4 This study demonstrates the effect of subcutaneous abdominal injection of gp96-antigen complex-activated dendritic cells (DCs) on tumor volume changes in HS 683 glioma mice.

[0098] Figure 5 This study demonstrates the effect of subcutaneous abdominal injection of gp96-antigen complex-activated dendritic cells (DCs) in RBE human cholangiocarcinoma mice on tumor volume changes. Detailed Implementation

[0099] The embodiments of the present invention will be further described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Reagents or instruments used in the examples, unless otherwise specified, are all commercially available conventional products. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al.'s Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or according to the conditions recommended in the manufacturer's instructions.

[0100] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0101] Female nude mice were derived from products of Beijing Vital River Laboratory Animal Co., Ltd.; in this example, they are referred to as mice.

[0102] HS 683 cells (human glioma cells) and RBE cells (human cholangiocarcinoma cells) were purchased from the Cell Center of Basic Medical Sciences, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. The cell line resource numbers were 3111C0001CCC000345 and 3131C0001000700179, respectively.

[0103] The gp96 monoclonal antibody is a product of Santa Cruz Biotechnology, catalog number sc-56399.

[0104] The horseradish peroxidase-labeled goat anti-rat monoclonal antibody is a product of Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., with product catalog number ZB-2307.

[0105] The HRP-labeled IgG antibody is a product of SEROTEC, catalog number STAR117P.

[0106] The HiTrap-Q Sepharose ion exchange chromatography column is a product of GE, with product catalog number 17-5053-01.

[0107] The Superdex 200 10 / 300GL molecular sieve chromatography column is a product of GE, with product catalog number 17517501.

[0108] The ConA agarose gel column is a GE product, catalog number 17-0440-01. The column measures 1.6 × 2.5 cm and is filled with Con A-Sepharose 4B medium.

[0109] The Hitrap Q anion exchange column is a GE product, catalog number 17-1153-01, with dimensions of 0.7 × 2.5 cm.

[0110] The 50kD and 3kD ultrafiltration tubes are products of Merck Millipore, with product catalog numbers UFC905096 and UFC500324, respectively.

[0111] The grinding buffer was 30 mM NaHCO3, with distilled water as the solvent and a pH of 7.4.

[0112] Solution A: 20 mM Tris-HCl, solvent is distilled water, pH value is 7.4.

[0113] Solution B: 20mM Tris-HCl, 1000mM NaCl, solvent is distilled water, pH value is 7.4.

[0114] Solution C: 20mM Tris-HCl, 200mM NaCl, solvent is distilled water, pH value is 7.4.

[0115] 1× Wash buffer is a pH 7.4, 0.01 mol / L PBS buffer containing 0.1% (v / v) Triton-X100.

[0116] BSA, PMSF, NaHCO3, MnCl2, CaCl2, NaCl2, Tris, methyl α-D-mannopyranoside, and dexamethasone are all products of Sigma-Aldrich, with product catalog numbers V900933, P7626, 792519, V900197, 793639, 746398, T1378, M6882, and D1756, respectively.

[0117] Sequence Information Summary

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124] Amino acid sequence of gp96 protein

[0125] MRALWVLGLCCVLLTFGSVRADDEVDVDGTVEEDLGKSREGSRTDD

[0126] EVVQREEEAIQLDGLNASQIRELREKSEKFAFQAEVNRMMKLIINSLY

[0127] KNKEIFLRELISNASDALDKIRLISLTDENALSGNEELTVKIKCDKEKNL

[0128] LHVTDTGVGMTREELVKNLGTIAKSGTSEFLNKMTEAQEDGQSTSEL

[0129] IGQFGVGFYSAFLVADKVIVTSKHNNDTQHIWESDSNEFSVIADPRGNT

[0130] LGRGTTITLVLKEEEASDYLELDTIKNLVKKYSQFINFPIYVWSSKTETV

[0131] EEPMEEEEAAKEEKEESDDEAAVEEEEEEKKPKTKKVEKTVWDWEL

[0132] MNDIKPIWQRPSKEVEEDEYKAFYKSFSKSEDDPMAYYIHFTAEGEVTF

[0133] KSILFVPTSAPRGLFDEYGSKKSDYIKLYVRRVFITDDFHDMPKYLN

[0134] FVKGVVDSDDLPLNVSRETLQQHKLLKVIRKKLVRKTLDMIKKIADD

[0135] KYNDTFWKEFGTNIKLGVIEDHSNRTRLAKLLRFQSSHHPTDITSLDQ

[0136] YVERMKEKQDKIYFMAGSSRKEAESSPFVERLLKKGYEVIYLTEPVDE

[0137] YCIQALPEFDGKRFQNVAKEGVKFDESEKTKESREAVKEFEPLLNW

[0138] MKDKALKDKIEKAVVSQRLTESPCALVASQYGWSGNMERIMKAQAY

[0139] QTGKDISTNYYASQKKTFEINPRHPLIRDMLRRIKEDEDDKTVLDLAV

[0140] VLFETATLRSGYLLPDTKAYGDRIERMLRLSLNIDPDAKVEEEPEEEPE

[0141] ETAEDTTEDTEQDEDEEMDVGTDEEEETAKESTAEKDEL

[0142] Example 1: Extraction of gp96 protein from placental tissue

[0143] The extraction steps for heat shock protein gp96 (hereinafter referred to as gp96) from tissues are as follows:

[0144] (1) Collect human placental tissue after delivery, cut it into small pieces, add grinding buffer at a mass-volume ratio of 1g:4mL, and then prepare tissue homogenate using a tissue homogenizer.

[0145] (2) After completing step (1), centrifuge at 7000 rpm for 1 hour to obtain supernatant A.

[0146] (3) After completing step (2), take the supernatant A and centrifuge at 7000 rpm for 50 min to obtain supernatant B.

[0147] (4) After completing step (3), take the supernatant B, remove impurities with ammonium sulfate, collect the precipitate, and add it to solution C at a mass-to-volume ratio (g / ml) of 1:9. Mix well to obtain the sample loading solution. The specific operation is as follows:

[0148] a. Removal of supernatant impurities and proteins

[0149] Measure the total volume of the homogenate supernatant, and slowly add ammonium sulfate powder at 4℃ at a ratio of 29.1g ammonium sulfate / 100ml (i.e., 50% saturated ammonium sulfate ratio). Mix the liquid with a magnetic stirrer to ensure that the ammonium sulfate dissolves quickly. Then let the solution stand overnight at 4℃, centrifuge at 7000rpm for 30min at 4℃, and discard the precipitate.

[0150] b. Collection of target protein from supernatant

[0151] Measure the volume of the supernatant, and then add ammonium sulfate powder at a ratio of 12.5g ammonium sulfate / 100ml (i.e., 70% saturated ammonium sulfate ratio). Add slowly, using a magnetic stirrer to ensure that the ammonium sulfate dissolves quickly after addition. Let stand at 4°C for at least 3 hours, centrifuge at 7000rpm for 30 minutes, collect the precipitate, and dissolve the precipitate by adding solution C at a ratio of precipitate:solution C = 1:9 (g / ml).

[0152] (5) After completing step (4), load the sample solution onto the ConA agarose gel column.

[0153] (6) After completing step (5), rinse the ConA agarose gel column with solution C. Monitor the UV absorption value in real time during the elution process. The detection wavelength is 280 nm until the UV absorption value of the elution product is lower than 0.01.

[0154] (7) After completing step (6), the ConA agarose gel column is eluted with solution C containing α-pyranose. The first 0.5 column volume of flow-through solution is discarded, and then 2 column volumes of the solution after passing through the column are collected. This is the ConA elution solution.

[0155] (8) After completing step (7), load the ConA elution buffer onto the Hitrap Q anion exchange column.

[0156] (9) After completing step (8), perform linear gradient elution with solutions A and B at a flow rate of 1 mL / min. Gradient elution program: linear gradient elution for 20 column volumes. Monitor the UV absorbance value in real time during elution at a detection wavelength of 280 nm, collect the elution peak, and obtain eluent A.

[0157] (10) After completing step (9), take eluent A, concentrate it by ultrafiltration using an ultrafiltration tube, and then replace it with 50 mMMPB buffer at pH 6.8 to obtain replacement buffer B. Load replacement buffer B onto the CHT ion chromatography column at a rate of 1 ml / min to 2 ml / min. Perform gradient elution using solutions A and B: rinse and elute for 5-10 column volumes, and collect eluent C after the desired gradient elution.

[0158] (11) After completing step (10), take the eluent C and perform ultrafiltration concentration and replace it in the protective buffer to obtain a high-purity heat shock protein gp96-antigen complex solution, wherein the gp96 concentration is 5 mg / mL.

[0159] The gp96-antigen complex solution was analyzed by SDS-PAGE electrophoresis and Western blot (using gp96 monoclonal antibody as primary antibody and HRP-labeled IgG antibody as secondary antibody). The results showed that the gp96-antigen complex solution displayed a single molecular weight band, corresponding to a molecular weight of 96 kDa. SDS-PAGE gel analysis showed a protein purity greater than 95%.

[0160] The gp96-antigen complex was treated with 0.3% trifluoroacetic acid in an ice bath for 30 minutes to promote the dissociation of gp96 from the placental-derived antigen peptide. The eluted peptide antigen was then concentrated and collected using a 5 kDa ultrafiltration tube, and added to the surface of a peptide enrichment chip for mass spectrometry identification of the peptide sequence.

[0161] Using peptide microarray enrichment combined with mass spectrometry, 71 tumor antigens were identified in the gp96-antigen complex extracted from the placenta. The amino acid sequences are shown in Seq ID No. 2–72. The amino acid sequence of the heat shock protein gp96 is shown in Seq ID No. 1.

[0162] Example 2: DC cells activated by gp96-antigen complex

[0163] Peripheral blood mononuclear cells (PBMCs) were obtained from anticoagulated fresh whole blood of healthy volunteers using human lymphocyte separation medium (Cellgro, 25-072-CI). The cell concentration was adjusted to 2.0 x 10⁻⁶ cells / mL using RPMI-1640 complete medium (Gibco, 12633012) containing 10% fetal bovine serum (Gibco, 10099-141-FBS). 7 / ml;

[0164] Preparation method of placental gp96 activated DC:

[0165] a. Collect 150-200ml of peripheral blood from healthy volunteers, transfer the collected blood sample into a 50ml centrifuge tube, centrifuge at 2000rpm for 5min, aspirate the plasma into 50ml centrifuge tubes, and inactivate the plasma in a 56℃ water bath for 30min for use in culturing dendritic cells.

[0166] b. Add Ficoll-Hypaque (density 1.077) lymphocyte separation medium to a 50ml sterile centrifuge tube, 20ml / tube (for isolating mononuclear cells);

[0167] c. Reduce the plasma sample to a certain volume with physiological saline, mix well, and slowly add it to Ficoll-Hypaque at a volume ratio of 1 to 1.5:1. Centrifuge at 2000 rpm at room temperature for 20 min.

[0168] d. Gently insert a flat-mouthed Pasteur pipette into the mononuclear cell layer, carefully aspirate the cells along the tube wall, and transfer them to another 50ml centrifuge tube. Add physiological saline to each tube to a final volume of 50ml, gently mix the cells, and centrifuge twice (first time, 1500 rpm for 7 min at room temperature; second time, 1200 rpm for 10 min at room temperature) to remove platelets and separation medium.

[0169] e. Culture the obtained peripheral blood mononuclear cells;

[0170] f. Take one sample of peripheral blood mononuclear cells, at a concentration of 2–4 x 10⁻⁴. 6 Cells per ml are seeded into 40 ml culture flasks and placed in a 37°C, 5% CO2 incubator. The cell concentration and number of flasks can be adjusted according to the cell count.

[0171] g. After incubation for 2 hours, gently shake the culture flask and discard the suspended cells (or collect the suspended cells for T cell culture). Wash the flask 1-2 times with phosphate buffer, add 40 ml of dendritic cell culture medium (containing 1 μM dexamethasone), and continue to incubate at 37°C in a 5% CO2 incubator.

[0172] h. On day 3, add 40 ml / bottle of dendritic culture medium (containing 1 μM dexamethasone);

[0173] i. On day 6, add TNF-α to a final concentration of 20 ng / ml and take samples for bacterial / fungal testing;

[0174] j. Collect 1×10 on day 6 7 Dendritic cells were added with 50 μg of gp96-antigen complex or culture medium as a control and co-stimulated in a 37°C, 5% CO2 incubator for 24 h. Flow cytometry analysis showed that the proportion of CD83+ DC cells was greater than 70% (see...). Figure 1 PBMCs were stimulated with dexamethasone-activated DCs, and the number of spots greater than 500 / 10 was detected by IFN-γ+ELISPOT. 5 PBMCs (see Figure 2 The effects of different glucocorticoids, small molecule compounds, and antibody drugs on DC-activated PBMCs are shown in [reference needed]. Figure 3 .

[0175] Flow cytometry detection:

[0176] (1) Take an appropriate amount of peripheral blood mononuclear cells that need to be stained (not exceeding 10).6 (100 cells) The cells were placed into a 1.5 mL EP tube and blocked with PBS containing 10% BSA for at least 10 minutes.

[0177] (2) Add 1 mL of PBS to the cells, centrifuge at 1500 rpm for five minutes, and discard the supernatant. Add another 1 mL of PBS to resuspend the cells, centrifuge at 1500 rpm for five minutes, and discard the supernatant.

[0178] (3) Resuspend the cells in 100 μL of PBS containing 10% BSA, add 5 μL of CD83 surface staining antibody, and incubate at 4°C for at least 30 minutes.

[0179] (4) Add 1 mL of PBS to the cells, centrifuge at 1500 rpm for five minutes, and discard the supernatant. Add another 1 mL of PBS to resuspend the cells, centrifuge at 1500 rpm for five minutes, and discard the supernatant.

[0180] (5) The cells were resuspended in 500 μL of PBS and directly analyzed by flow cytometer.

[0181] Enzyme-linked immunospot assay (ELISPOT)

[0182] (1) Pre-coating: Dilute the coating antibody to 20 mg / mL (1:200 dilution) with sterile PBS (pH 7.4), add 100 μL of the diluted coating antibody to each well of an ELISPOT plate, and incubate overnight at 4°C. All the above operations should be performed in a clean bench.

[0183] (2) Blocking: Discard the coating solution in the ELISPOT plate, wash 3 times with sterile PBS, 200 μL per well. After the last wash, pat dry, add 200 μL of RPMI 1640 medium containing 10% serum to each well, and incubate at room temperature for 30 minutes.

[0184] (3) Take an appropriate amount of PBMCs stimulated with DC and dilute them to a concentration of 3.5 million / mL with RPMI 1640 medium containing 10% serum. At the same time, add IL-2 and anti-mouse CD28 antibody to make their final concentrations 20 U / mL and 1 μg / mL, respectively.

[0185] (4) Add 200 μL of cells to each well, so that each well contains 500,000 cells. Use PMA and ionomycin as positive controls.

[0186] (5) Place in a cell culture incubator at 37℃ and 5% CO2 for 24 hours.

[0187] (6) After the culture is completed, remove the ELISPOT plate, discard the cells and culture medium, and pat dry.

[0188] (7) Add 200 μL of pre-cooled deionized water to each well, let stand for 3 minutes, discard, repeat once more, and pat dry.

[0189] (8) Add 200 μL of PBST to each well, let stand for 3 minutes, and then discard. Wash a total of 5 times, and pat dry on absorbent paper for the last wash.

[0190] (9) Prepare biotin-labeled anti-IFN-γ monoclonal antibody with PBS and dilute it 1:200. Add 100 μL to each well and incubate at room temperature for 2 hours.

[0191] (10) Discard the antibody, add 200 μL of PBST to each well, incubate for 3 minutes, and then discard. Wash a total of 5 times, and pat dry on absorbent paper for the last wash.

[0192] (11) Prepare avidin-labeled HRP with PBS and dilute it 1:100. Add 100 μL to each well and incubate at room temperature for 1 hour.

[0193] (12) Discard the liquid, add 200 μL of PBST to each well, let stand for 3 minutes, and then discard. Wash a total of 5 times, then wash twice with PBS, and finally pat dry on absorbent paper.

[0194] (13) Prepare AEC substrate and add it to ELISPOT plates, 100 μL per well.

[0195] (14) Let it stand at room temperature for 10 minutes to develop color.

[0196] (15) After the spots have developed to a suitable size, wash the plate three times with deionized water to stop the color development reaction. Then place the plate upside down on absorbent paper, pat off the small water droplets, and place it face up in a ventilated place to air dry naturally at room temperature.

[0197] (16) Use an automatic image analyzer to count the spots on the ELISPOT plate, record various parameters of the spots, and perform statistical analysis.

[0198] Example 3: The therapeutic effect of gp96-antigen complex-activated DCs on glioma.

[0199] Take 20 female nude mice aged 6-8 weeks, and reinfuse 10 mg / L of the drug into each mouse via the tail vein. 7 One healthy human PBMC cell (0.5 ml) was used to reconstruct the human immune system in mice; three days after infusion, all mice were subcutaneously inoculated with 5 × 10⁸ human PBMC cells. 6 HS 683 glioma cells; On the second day after tumor inoculation, mice were divided into two groups of 10 each, and treated as follows:

[0200] Group 1: Subcutaneous injection of gp96-antigen complex-activated dendritic cells (DCs) into the abdomen, three times (0.5 ml each time), with a single immunization dose of 10. 6 / Only;

[0201] Group 2: Human mononuclear cell-derived dendritic cells (DCs) unstimulated by gp96-antigen complex were reinfused via tail vein, and immunized three times (0.5 ml each time). The single immunization dose was 10. 6 / Only;

[0202] In both groups above: the first immunization was administered on day 2 after tumor cell inoculation; the second immunization was administered on day 9; and the third immunization was administered on day 16. Starting from the first day of immunization, tumor growth was observed daily, and tumor size was recorded. Tumor volume was calculated using the following formula: V = ab 2 / 2(V—volume, a—major diameter of tumor, b—minor diameter of tumor). Changes in tumor volume are shown in [reference needed]. Figure 4 .

[0203] Example 4: The therapeutic effect of gp96-antigen complex-activated DCs on cholangiocarcinoma

[0204] Take 20 female nude mice aged 6-8 weeks, and reinfuse 10 mg / L of the drug into each mouse via the tail vein. 7 Three days after infusion, each mouse was subcutaneously inoculated with 1×10⁶ PBMC cells from a healthy human. 6 RBE human cholangiocarcinoma cells; on the second day after tumor inoculation, mice were divided into two groups of 10 mice each, and were treated as follows:

[0205] Group 1: Subcutaneous injection of gp96-antigen complex-activated human mononuclear cell (DC) cells into the abdomen, immunized three times (0.5 ml each time), with a single immunization dose of 10. 6 / Only;

[0206] Group 2: Subcutaneous injection of human mononuclear cell-derived dendritic cells (DCs) into the abdomen, immunized three times (0.5 ml each time), with a single immunization dose of 10. 6 / Only;

[0207] In both groups above: the first immunization was administered on day 2 after tumor cell inoculation; the second immunization was administered on day 9; and the third immunization was administered on day 16. Starting from the first day of immunization, tumor growth was observed daily, and tumor size was recorded. Tumor volume was calculated using the following formula: V = ab 2 / 2(V—volume, a—major diameter of tumor, b—minor diameter of tumor). Changes in tumor volume are shown in [reference needed]. Figure 5 .

[0208] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention. The full scope of the present invention is given by the appended claims and any equivalents. SEQUENCE LISTING <110> Foshan Rexiu Biotechnology Co., Ltd. <120> Activated dendritic cells and their application in cancer treatment <130> IDC200471 <160> 72 <170> PatentIn version 3.5 <210> 1 <211> 803 <212> PRT <213> artificial <220> <223> Amino acid sequence of heat shock protein gp96 <400> 1 Met Arg Ala Leu Trp Val Leu Gly Leu Cys Cys Val Leu Leu Thr Phe 1 5 10 15 Gly Ser Val Arg Ala Asp Asp Glu Val Asp Val Asp Gly Thr Val Glu 20 25 30 Glu Asp Leu Gly Lys Ser Arg Glu Gly Ser Arg Thr Asp Asp Glu Val 35 40 45 Val Gln Arg Glu Glu Glu Ala Ile Gln Leu Asp Gly Leu Asn Ala Ser 50 55 60 Gln Ile Arg Glu Leu Arg Glu Lys Ser Glu Lys Phe Ala Phe Gln Ala 65 70 75 80 Glu Val Asn Arg Met Met Lys Leu Ile Ile Asn Ser Leu Tyr Lys Asn 85 90 95 Lys Glu Ile Phe Leu Arg Glu Leu Ile Ser Asn Ala Ser Asp Ala Leu 100 105 110 Asp Lys Ile Arg Leu Ile Ser Leu Thr Asp Glu Asn Ala Leu Ser Gly 115 120 125 Asn Glu Glu Leu Thr Val Lys Ile Lys Cys Asp Lys Glu Lys Asn Leu 130 135 140 Leu His Val Thr Asp Thr Gly Val Gly Met Thr Arg Glu Glu Leu Val 145 150 155 160 Lys Asn Leu Gly Thr Ile Ala Lys Ser Gly Thr Ser Glu Phe Leu Asn 165 170 175 Lys Met Thr Glu Ala Gln Glu Asp Gly Gln Ser Thr Ser Glu Leu Ile 180 185 190 Gly Gln Phe Gly Val Gly Phe Tyr Ser Ala Phe Leu Val Ala Asp Lys 195 200 205 Val Ile Val Thr Ser Lys His Asn Asn Asp Thr Gln His Ile Trp Glu 210 215 220 Ser Asp Ser Asn Glu Phe Ser Val Ile Ala Asp Pro Arg Gly Asn Thr 225 230 235 240 Leu Gly Arg Gly Thr Thr Ile Thr Leu Val Leu Lys Glu Glu Ala Ser 245 250 255 Asp Tyr Leu Glu Leu Asp Thr Ile Lys Asn Leu Val Lys Lys Tyr Ser 260 265 270 Gln Phe Ile Asn Phe Pro Ile Tyr Val Trp Ser Ser Lys Thr Glu Thr 275 280 285 Val Glu Glu Pro Met Glu Glu Glu Glu Ala Ala Lys Glu Glu Lys Glu 290 295 300 Glu Ser Asp Asp Glu Ala Ala Val Glu Glu Glu Glu Glu Glu Lys Lys 305 310 315 320 Pro Lys Thr Lys Lys Val Glu Lys Thr Val Trp Asp Trp Glu Leu Met 325 330 335 Asn Asp Ile Lys Pro Ile Trp Gln Arg Pro Ser Lys Glu Val Glu Glu 340 345 350 Asp Glu Tyr Lys Ala Phe Tyr Lys Ser Phe Ser Lys Glu Ser Asp Asp 355 360 365 Pro Met Ala Tyr Ile His Phe Thr Ala Glu Gly Glu Val Thr Phe Lys 370 375 380 Ser Ile Leu Phe Val Pro Thr Ser Ala Pro Arg Gly Leu Phe Asp Glu 385 390 395 400 Tyr Gly Ser Lys Lys Ser Asp Tyr Ile Lys Leu Tyr Val Arg Arg Val 405 410 415 Phe Ile Thr Asp Asp Phe His Asp Met Met Pro Lys Tyr Leu Asn Phe 420 425 430 Val Lys Gly Val Val Asp Ser Asp Asp Leu Pro Leu Asn Val Ser Arg 435 440 445 Glu Thr Leu Gln Gln His Lys Leu Leu Lys Val Ile Arg Lys Lys Leu 450 455 460 Val Arg Lys Thr Leu Asp Met Ile Lys Lys Ile Ala Asp Asp Lys Tyr 465 470 475 480 Asn Asp Thr Phe Trp Lys Glu Phe Gly Thr Asn Ile Lys Leu Gly Val 485 490 495 Ile Glu Asp His Ser Asn Arg Thr Arg Leu Ala Lys Leu Leu Arg Phe 500 505 510 Gln Ser Ser His His Pro Thr Asp Ile Thr Ser Leu Asp Gln Tyr Val 515 520 525 Glu Arg Met Lys Glu Lys Gln Asp Lys Ile Tyr Phe Met Ala Gly Ser 530 535 540 Ser Arg Lys Glu Ala Glu Ser Ser Pro Phe Val Glu Arg Leu Leu Lys 545 550 555 560 Lys Gly Tyr Glu Val Ile Tyr Leu Thr Glu Pro Val Asp Glu Tyr Cys 565 570 575 Ile Gln Ala Leu Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala 580 585 590 Lys Glu Gly Val Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg 595 600 605 Glu Ala Val Glu Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp 610 615 620 Lys Ala Leu Lys Asp Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu 625 630 635 640 Thr Glu Ser Pro Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly 645 650 655 Asn Met Glu Arg Ile Met Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp 660 665 670 Ile Ser Thr Asn Tyr Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Asn 675 680 685 Pro Arg His Pro Leu Ile Arg Asp Met Leu Arg Arg Ile Lys Glu Asp 690 695 700 Glu Asp Asp Lys Thr Val Leu Asp Leu Ala Val Val Leu Phe Glu Thr 705 710 715 720 Ala Thr Leu Arg Ser Gly Tyr Leu Leu Pro Asp Thr Lys Ala Tyr Gly 725 730 735 Asp Arg Ile Glu Arg Met Leu Arg Leu Ser Leu Asn Ile Asp Pro Asp 740 745 750 Ala Lys Val Glu Glu Glu Pro Glu Glu Glu Pro Glu Glu Thr Ala Glu 755 760 765 Asp Thr Thr Glu Asp Thr Glu Gln Asp Glu Asp Glu Glu Met Asp Val 770 775 780 Gly Thr Asp Glu Glu Glu Glu Thr Ala Lys Glu Ser Thr Ala Glu Lys 785 790 795 800 Asp Glu Leu <210> 2 <211> 20 <212> PRT <213> artificial <220> <223> Antigen 1 <400> 2 Ile Glu Gln Asn Thr Lys Ser Pro Leu Phe Met Gly Lys Val Val Asn 1 5 10 15 Pro Thr Gln Lys 20 <210> 3 <211> 19 <212> PRT <213> artificial <220> <223> Antigen 2 <400> 3 Val Lys Lys Pro Pro Arg Gly Arg Lys Pro Ala Glu Lys Pro Leu Pro 1 5 10 15 Lys Pro Arg <210> 4 <211> 27 <212> PRT <213> artificial <220> <223> Antigen 3 <400> 4 Gly Phe Gly Phe Val Thr Phe Ser Ser Met Ala Glu Val Asp Ala Ala 1 5 10 15 Met Ala Ala Arg Pro His Ser Ile Asp Gly Arg 20 25 <210> 5 <211> 27 <212> PRT <213> artificial <220> <223> Antigen 4 <400> 5 Leu Gln Ala Gln Gly Val Glu Val Pro Ser Lys Asp Ser Leu Pro Lys 1 5 10 15 Lys Arg Pro Ile Tyr Glu Asp Lys Lys Arg Lys 20 25 <210> 6 <211> 37 <212> PRT <213> artificial <220> <223> Antigen 5 <400> 6 Ala Val Arg Asp Met Arg Gln Thr Val Ala Val Gly Val Ile Lys Ala 1 5 10 15 Val Asp Lys Lys Ala Ala Gly Ala Gly Lys Val Thr Lys Ser Ala Gln 20 25 30 Lys Ala Gln Lys Ala 35 <210> 7 <211> 8 <212> PRT <213> artificial <220> <223> Antigen 6 <400> 7 Gln Leu Arg Lys Leu Gln Leu Gln 1 5 <210> 8 <211> 19 <212> PRT <213> artificial <220> <223> Antigen 7 <400> 8 Thr Gln Asn Thr Tyr Gln His Phe Tyr Asp Gly Ser Glu Ile Val Val 1 5 10 15 Ala Gly Arg <210> 9 <211> 24 <212> PRT <213> artificial <220> <223> Antigen 8 <400> 9 Ala Thr Pro Ala Pro Ser Ala Pro Ala Ala Ala Ser Ala Thr Ser Pro 1 5 10 15 Ser Pro Ala Pro Ser Ser Gly Asn 20 <210> 10 <211> 39 <212> PRT <213> artificial <220> <223> Antigen 9 <400> 10 Lys Glu Ala Gly Glu Gly Gly Glu Ala Glu Ala Pro Ala Ala Glu Gly 1 5 10 15 Gly Lys Asp Glu Ala Ala Gly Gly Ala Ala Ala Ala Ala Ala Glu Ala 20 25 30 Gly Ala Ala Ser Gly Glu Gln 35 <210> 11 <211> 25 <212> PRT <213> artificial <220> <223> Antigen 10 <400> 11 Val Ala Phe Gln Asp Val Ala Gln Asn Pro Ala Asn Met Ser Lys Tyr 1 5 10 15 Gln Ser Asn Pro Lys Val Met Asn Leu 20 25 <210> 12 <211> 17 <212> PRT <213> artificial <220> <223> Antigen 11 <400> 12 Thr Ile Lys Pro Asp Val Gln Lys Ser Lys Glu Tyr Phe Ser Lys Gln 1 5 10 15 Lys <210> 13 <211> 15 <212> PRT <213> artificial <220> <223> Antigen 12 <400> 13 Phe Val Thr Ala Ala Gln Thr Ser Gly Leu Pro Ser Ser Val Arg 1 5 10 15 <210> 14 <211> 22 <212> PRT <213> artificial <220> [[ID=३३]]<223> Antigen 13 <400> 14 Ala Ser Asp Pro Leu Asp Thr Leu Gly Ser Glu Gly Ala Leu Ser Pro 1 5 10 15 Gly Gly Val Ala Ser Leu 20 <2१०> 15 <211> 27 <212> PRT <213> artificial <220> <223> Antigen 14 <400> 15 Phe Gln Ala Pro Asp Leu Arg Gly Ile Ser Glu Gln Ser Leu Val Val 1 5 10 15 Ser Gly Val Gln His Gln Ser Thr Leu Glu Leu 20 25 <210> 16 <211> 27 <212> PRT<00००764> It should be noted that there seems to be an error in the original text where "२१०" and "००००764" are present. They are likely incorrect notations and might need to be corrected in the original source for a more accurate translation.<213> artificial <220> <223> Antigen 15 <400> 16 Leu Arg Glu Pro Val Arg Ala Pro Ala Val Ala Val Ala Pro Thr Pro 1 5 10 15 Val Gln Pro Pro Ile Ile Val Ala Pro Val Ala 20 25 <210> 17 <211> 22 <212> PRT <213> artificial <220> <223> Antigen 16 <400> 17 Ile Arg Glu Phe Asp Asn Met Ala Lys Val Gln Asp Glu Lys Arg Lys 1 5 10 15 Ser Lys Gln Lys Leu Gly 20 <210> 18 <211> 24 <212> PRT <213> artificial <220> <223> Antigen 17 <400> 18 Ala Ser Thr Ala Ser Gln Leu His Ser Asn Val Val Asn Tyr Val Gln 1 5 10 15 Gln Ile Val Ala Pro Lys Gly Ser 20 <210> 19 <211> 20 <212> PRT <213> artificial <220> <223> Antigen 18 <400> 19 Lys Val Ser Met Glu Ala Val Gln Lys Asn Gln Gly Arg Lys Lys Gln 1 5 10 15 Val Val Gln Phe 20 <210> 20 <211> 15 <212> PRT <213> artificial <220> <223> Antigen 19 <400> 20 Tyr Trp Val Asn Gly Gln Val Pro Asp Gly Val Ser Lys Val Leu 1 5 10 15 <210> 21 <211> 15 <212> PRT <213> artificial <220> <223> Antigen 20 <400> 21 Ile Ser Lys Lys Glu Met Gln Pro Thr His Pro Ile Arg Leu Gly 1 5 10 15 <210> 22 <211> 13 <212> PRT <213> artificial <220> <223> Antigen 21 <400> 22 Val Arg Gln Ile Asn Arg Lys Thr Pro Val Pro Gly Lys 1 5 10 <210> 23 <211> 17 <212> PRT <213> artificial <220> <223> Antigen 22 <400> 23 Leu Glu Gly Lys Pro Leu Glu Ala Ser Arg Ala Leu Pro Ala Lys Pro 1 5 10 15 Arg <210> 24 <211> 14 <212> PRT <213> artificial <220> <223> Antigen 23 <400> 24 Val Leu Gln Ala Thr Val Val Ala Val Gly Ser Gly Ser Lys 1 5 10 <210> 25 <211> 29 <212> PRT <213> artificial <220> <223> Antigen 24 <400> 25 Glu Pro Ser Pro Gly Thr Leu Pro Arg Lys Ala Gly Val Phe Ser Asp 1 5 10 15 Leu Ser Asn Gln Glu Leu Lys Ala Val His Ser Phe Leu 20 25 <210> 26 <211> 29 <212> PRT <213> artificial <220> <223> Antigen 25 <400> 26 Ser Arg Pro Gly Gly Arg Ala Ser Val Asp Thr Lys Glu Ala Glu Gly 1 5 10 15 Ala Pro Gln Val Glu Ala Gly Lys Arg Leu Glu Glu Leu 20 25 <210> 27 <211> 20 <212> PRT <213> artificial <220> <223> Antigen 26 <400> 27 Ser Val Gln Arg Gln Phe Phe Pro Thr Asp Glu Asp Glu Ile Gly Ala 1 5 10 15 Ala Lys Ala Leu 20 <210> 28 <211> 15 <212> PRT <213> artificial <220> <223> Antigen 27 <400> 28 Asp Leu Gly Asn Ile Arg Ala Glu Pro Leu Asn Ser Val Ala His 1 5 10 15 <210> 29 <211> 15 <212> PRT <213> artificial <220> <223> Antigen 28 <400> 29 Lys Ile Gly Ile Val Gly Leu Pro Asn Val Gly Lys Ser Thr Phe 1 5 10 15 <210> 30 <211> 11 <212> PRT <213> artificial <220> <223> Antigen 29 <400> 30 Asp Leu Pro Ala Pro Val Thr Pro Gln Pro Arg 1 5 10 <210> 31 <211> 17 <212> PRT <213> artificial <220> <223> Antigen 30 <400> 31 Val Ala Val Val Thr Gly Ser Thr Ser Gly Ile Gly Phe Ala Ile Ala 1 5 10 15 Arg <210> 32 <211> 20 <212> PRT <213> artificial <220> <223> Antigen 31 <400> 32 Ala Ala Pro Ala Val Gln Thr Lys Thr Lys Lys Thr Leu Ala Lys Pro 1 5 10 15 Asn Ile Arg Asn 20 <210> 33 <211> 33 <212> PRT <213> artificial <220> <223> Antigen 32 <400> 33 His Gly Glu Gly Ser Ser Ser Gly Lys Ala Thr Gly Asp Glu Thr Gly 1 5 10 15 Ala Lys Val Glu Arg Ala Asp Gly Tyr Glu Pro Pro Val Gln Glu Ser 20 25 30 Val <210> 34 <211> 19 <212> PRT <213> artificial <220> <223> Antigen 33 <400> 34 Ser Ala Ser Leu Gly Thr Arg Gln Ala Glu Pro Glu Leu Asp Leu Arg 1 5 10 15 Ser Ile Lys <210> 35 <211> 30 <212> PRT <213> artificial <220> <223> Antigen 34 <400> 35 Pro Ala Ala Asp Asn Ile Glu Met Leu Pro His Tyr Glu Pro Ile Pro 1 5 10 15 Phe Ser Ser Ser Met Asn Glu Ser Ala Pro Thr Gly Ile Thr 20 25 30 <210> 36 <211> 27 <212> PRT <213> artificial <220> <223> Antigen 35 <400> 36 Asn Tyr Asp Ile Pro Asn Leu Ala Lys Lys Leu Glu Glu Ile Lys Lys 1 5 10 15 Asp Leu Asp Ala Lys Lys Lys Pro Pro Ser Ala 20 25 <210> 37 <211> 23 <212> PRT <213> artificial <220> <223> Antigen 36 <400> 37 Ala Val Pro Met Gln His Asn Asn Arg Pro Thr Gln Pro Leu Lys Gly 1 5 10 15 Arg Thr Val Arg Ala Ser Phe 20 <210> 38 <211> 18 <212> PRT <213> artificial <220> <223> Antigen 37 <400> 38 Ala Ala Ile Pro Lys Asp Lys Ala Ile Leu Asp Ile Glu Arg Pro Asp 1 5 10 15 Leu Met <210> 39 <211> 15 <212> PRT <213> artificial <220> <223> Antigen 38 <400> 39 Ile Arg Glu Val Ala Asn Lys Val Lys Val Pro Leu Gln Asp Leu 1 5 10 15 <210> 40 <211> 18 <212> PRT <213> artificial <220> <223> Antigen 39 <400> 40 Thr Lys Leu Ala Asn Pro His Tyr Gln Pro Glu Leu Gln Ala Gln Ala 1 5 10 15 Thr Leu <210> 41 <211> 56 <212> PRT <213> artificial <220> <223> antigen 40 <400> 41 Thr Pro Ala Val Pro Val Glu Ser Lys Pro Asp Lys Pro Ser Gly Lys 1 5 10 15 Ser Gly Met Asp Ala Ala Leu Asp Asp Leu Ile Asp Thr Leu Gly Gly 20 25 30 Pro Glu Glu Thr Glu Glu Glu Asn Thr Thr Tyr Thr Gly Pro Glu Val 35 40 45 Ser Asp Pro Met Ser Ser Thr Tyr 50 55 <210> 42 <211> 38 <212> PRT <213> artificial <220> <223> antigen 41 <400> 42 Gly Ile Leu Gly Tyr Thr Glu His Gln Val Val Ser Ser Asp Phe Asn 1 5 10 15 Ser Asp Thr His Ser Ser Thr Phe Asp Ala Gly Ala Gly Ile Ala Leu 20 25 30 Asn Asp His Phe Val Lys 35 <210> 43 <211> 38 <212> PRT <213> artificial <220> <223> antigen42 <400> 43 Glu Pro Val Arg Thr Ser Arg Glu His Pro Val Pro Leu Leu Pro Ile 1 5 10 15 Arg Gln Thr Leu Pro Glu Asp Asn Glu Glu Pro Pro Ala Leu Pro Pro 20 25 30 Arg Thr Leu Glu Gly Leu 35 <210> 44 <211> 18 <212> PRT <213> artificial <220> <223> antigen43 <400> 44 Trp Thr Ala Asn Val Gly Lys Gly Gln Pro Ser Val Leu Gln Val Val 1 5 10 15 Leo <210> 45 <211> 20 <212> PRT <213> artificial <220> <223> antigen44 <400> 45 Pro Glu Lys Arg Pro Phe Glu Arg Leu Pro Ala Asp Val Ser Pro Ile 1 5 10 15 Asn Tyr Ser Leo 20 <210> 46 <211> 32 <212> PRT <213> artificial <220> <223> Antigen 45 <400> 46 Lys Pro Ala Ala Ala Ala Ala Pro Gly Thr Ala Glu Lys Leu Ser Pro 1 5 10 15 Lys Ala Ala Thr Leu Ala Glu Arg Ser Ala Gly Leu Ala Phe Ser Leu 20 25 30 <210> 47 <211> 22 <212> PRT <213> artificial <220> <223> Antigen 46 <400> 47 Thr Ser Trp Glu Arg Val Ser Thr Glu Val Arg Asp Tyr Val Tyr Arg 1 5 10 15 Gln Glu Ala Arg Leu Glu 20 <210> 48 <211> 32 <212> PRT <213> artificial <220> <223> Antigen 47 <400> 48 Ser Asp Gly Val Pro Ser Asp Ser Val Glu Ala Ala Lys Asn Ala Ser 1 5 10 15 Asn Thr Glu Lys Leu Thr Asp Gln Val Met Gln Asn Pro Arg Val Leu 20 25 30 <210> 49 <211> 21 <212> PRT <213> artificial <220> <223> Antigen 48 <400> 49 Met Ile Glu Gln Asn Thr Lys Ser Pro Leu Phe Met Gly Lys Val Val 1 5 10 15 Asn Pro Thr Gln Lys 20 <210> 50 <211> 25 <212> PRT <213> artificial <220> <223> Antigen 49 <400> 50 Lys Thr Asp Thr Ser His His Asp Gln Asp His Pro Thr Phe Asn Lys 1 5 10 15 Ile Thr Pro Asn Leu Ala Glu Phe Ala 20 25 <210> 51 <211> 30 <212> PRT <213> artificial <220> <223> Antigen 50 <400> 51 Glu Ala Asp Glu Arg Glu Pro Thr Glu Ser Thr Gln Gln Leu Asn Lys 1 5 10 15 Pro Glu Val Leu Glu Val Thr Leu Asn Arg Pro Phe Leu Phe 20 25 30 <210> 52 <211> 25 <212> PRT <213> artificial <220> <223> antigen51 <400> 52 Ala Leu Ala Gly Asn Gln Asp Lys Arg Lys Glu Val Leu Lys Ser Leu 1 5 10 15 Asn Glu Glu Ala Val Lys Lys Asp Asn 20 25 <210> 53 <211> 25 <212> PRT <213> artificial <220> <223> antigen52 <400> 53 Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp 1 5 10 15 Leu Arg Gln Gly Leu Leu Pro Val Leu 20 25 <210> 54 <211> 38 <212> PRT <213> artificial <220> <223> antigen53 <400> 54 Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys Ala Thr Glu His Leu 1 5 10 15 Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg Gln 20 25 30 Gly Leu Leu Pro Val Leu 35 <210> 55 <211> 27 <212> PRT <213> artificial <220> <223> Antigen 54 <400> 55 Gln Arg Gln Trp Ala Gly Leu Val Glu Lys Val Gln Ala Ala Val Gly 1 5 10 15 Thr Ser Ala Ala Pro Val Pro Ser Asp Asn His 20 25 <210> 56 <211> 21 <212> PRT <213> artificial <220> <223> Antigen 55 <400> 56 Ile Thr Val Lys His Arg Lys Gln Gln Val Leu Glu Thr Val Ala Gly 1 5 10 15 Lys Arg Ser Tyr Arg 20 <210> 57 <211> 35 <212> PRT <213> artificial <220> <223> Antigen 56 <400> 57 Glu Pro Ser Pro Gly Thr Leu Pro Arg Lys Ala Gly Val Phe Ser Asp 1 5 10 15 Leu Ser Asn Gln Glu Leu Lys Ala Val His Ser Phe Leu Trp Ser Lys 20 25 30 Lys Glu Leu 35 <210> 58 <211> 24 <212> PRT <213> artificial <220> <223> Antigen 57 <400> 58 Leu Glu Tyr Arg Glu Val Val Asp Gly Leu Glu Lys Ala Ile Tyr Lys 1 5 10 15 Gly Pro Gly Ser Glu Ala Gly Pro 20 <210> 59 <211> 24 <212> PRT <213> artificial <220> <223> Antigen 58 <400> 59 Lys Leu Ser Asn Asn Ala Leu Ser Gly Leu Pro Gln Gly Val Phe Gly 1 5 10 15 Lys Leu Gly Ser Leu Gln Glu Leu 20 <210> 60 <211> 20 <212> PRT <213> artificial <220> <223> Antigen 59 <400> 60 Pro Ala Leu Gln Gly Ala Gln Thr Lys Met Ser Ala Ser Asp Pro Asn 1 5 10 15 Ser Ser Ile Phe 20 <210> 61 <211> 28 <212> PRT <213> artificial <220> <223> antigen 60 <400> 61 Thr Glu Glu Asp Lys Ala Thr Ile Thr Ser Leu Trp Gly Lys Val Asn 1 5 10 15 Val Glu Asp Ala Gly Gly Glu Thr Leu Gly Arg Leu 20 25 <210> 62 <211> 27 <212> PRT <213> artificial <220> <223> antigen 61 <400> 62 Val Leu Ser Pro Ala Asp Lys Thr Asn Val Lys Ala Ala Trp Gly Lys 1 5 10 15 Val Gly Ala His Ala Gly Glu Tyr Gly Ala Glu 20 25 <210> 63 <211> 29 <212> PRT <213> artificial <220> <223> antigen 62 <400> 63 Val Gly Asn Lys Ser Asp Leu Arg His Leu Arg Ala Val Pro Thr Asp 1 5 10 15 Glu Ala Arg Ala Phe Ala Glu Lys Asn Asn Leu Ser Phe 20 25 <210> 64 <211> 19 <212> PRT <213> artificial <220> <223> antigen 63 <400> 64 Ile Lys Gln Leu Ala Lys Ser Val Arg Asp Arg Tyr Ala Arg Ser Pro 1 5 10 15 Lys Glu Lys <210> 65 <211> 21 <212> PRT <213> artificial <220> <223> antigen 64 <400> 65 Ala Ala Pro Ser Glu Pro Ser Glu Pro Ser Arg Pro Ser Pro Gln Pro 1 5 10 15 Lys Pro Arg Thr Pro 20 <210> 66 <211> 42 <212> PRT <213> artificial <220> <223> antigen 65 <400> 66 Arg Ser Trp Ser Pro Thr Gly Glu Arg Leu Gly Glu Asp Pro Tyr Tyr 1 5 10 15 Thr Glu Asn Gly Gly Gly Gln Gly Tyr Ser Ser Gly Pro Gly Thr Ser 20 25 30 Pro Glu Ala Gln Gly Lys Ala Ser Val Asn 35 40 <210> 67 <211> 35 <212> PRT <213> artificial <220> <223> antigen 66 <400> 67 Ser Asn Glu Asn His Gly Ile Ala Gln Arg Ile Tyr Gly Asn Gln Asp 1 5 10 15 Thr Ser Ser Gln Leu Lys Lys Phe Tyr Asn Gln Val Ser Thr Pro Leu 20 25 30 Leu Arg Asn 35 <210> 68 <211> 27 <212> PRT <213> artificial <220> <223> antigen 67 <400> 68 Leu Ser Ser Trp Leu Gln Ser Asp Asp Glu Pro Glu Lys Glu Arg Leu 1 5 10 15 Arg Gln Arg Ala Gln Ala Leu Ala Val Ser Tyr 20 25 <210> 69 <211> 23 <212> PRT <213> artificial <220> <223> antigen 68 <400> 69 Ile Ala Asn Met Pro Glu Ser Gly Pro Ser Tyr Glu Phe His Leu Thr 1 5 10 15 Arg Gln Glu Ile Val Ser Leu 20 <210> 70 <211> 34 <212> PRT <213> artificial <220> <223> antigen 69 <400> 70 Phe Ser Glu Thr Gly Ala Gly Lys His Val Pro Arg Ala Val Phe Val 1 5 10 15 Asp Leu Glu Pro Thr Val Ile Asp Glu Val Arg Thr Gly Thr Tyr Arg 20 25 30 Gln Leu <210> 71 <211> 47 <212> PRT <213> artificial <220> <223> antigen 70 <400> 71 Thr Val Thr Asp Tyr Gly Lys Asp Leu Met Glu Lys Val Lys Ser Pro 1 5 10 15 Glu Leu Gln Ala Glu Ala Lys Ser Tyr Phe Glu Lys Ser Lys Glu Gln 20 25 30 Leu Thr Pro Leu Ile Lys Lys Ala Gly Thr Glu Leu Val Asn Phe 35 40 45 <210> 72 <211> 32 <212> PRT <213> artificial <220> <223> antigen 71 <400> 72 Val Lys Ser Pro Glu Leu Gln Ala Glu Ala Lys Ser Tyr Phe Glu Lys 1 5 10 15 Ser Lys Glu Gln Leu Thr Pro Leu Ile Lys Lys Ala Gly Thr Glu Leu 20 25 30

Claims

1. A population of dendritic cells (DCs) prepared by the following method: (1) Provide monocytes; (2) The monocytes were cultured in a DC cell culture medium containing 0.1-5 μM glucocorticoids for 3-10 days to obtain mature DC cells; (3) 20~100μg / 1×10 7 The proportion of DC cells was adjusted, and heat shock protein gp96-antigen complex was added. The cells were cultured for 6-48 hours, and the DC cell population was collected. in, The heat shock protein gp96-antigen complex was prepared by the following method: human placental tissue homogenate was sequentially separated by ConA-Sepharose gel column chromatography, HiTrap Q ion exchange chromatography and CHT ion exchange chromatography to obtain the heat shock protein gp96-antigen complex; wherein the amino acid sequence of the heat shock protein gp96 in the heat shock protein gp96-antigen complex is as shown in SEQ ID NO.1, and the antigen is as shown in SEQ ID NO.2~72.

2. The DC cell population of claim 1, wherein in step (2), the monocytes are cultured in the DC cell culture medium for 3, 4, 5, 6, 7, 8, 9 or 10 days.

3. The DC cell population of claim 1, wherein in step (2), the mononuclear cells are cultured in the DC cell culture medium for 6 days.

4. The DC cell population according to claim 1, characterized in that... One or more of the following: 1) The mononuclear cells mentioned in step (1) are derived from peripheral blood or umbilical cord blood; 2) The glucocorticoid compound mentioned in step (2) is dexamethasone; 3) In step (2), press 1×10 5 ~1×10 8 The mononuclear cells were seeded into the DC cell culture medium at a density of cells / ml; 4) Add one or more of PHA, IL-2, IL-4, GM-CSF and TNF-α during the culture process described in step (2); 5) The DC cell culture medium mentioned in step (2) is RPMI 1640; 6) The specific cultivation conditions described in steps (2) and (3) are both 37°C. o C. 5% CO2.

5. The DC cell population of claim 1, wherein in step (1), the monocytes are derived from peripheral blood.

6. The DC cell population of claim 1, wherein in step (2), the cells are grown at a ratio of 1 × 10⁻⁶. 5 cells / ml, 1×10 6 cells / ml, 1×10 7 pcs / ml or 1×10 8 The mononuclear cells were seeded into the DC cell culture medium at a density of cells / ml.

7. The DC cell population of claim 1, wherein in step (2), the concentration is 2~4×10⁻⁶. 6 The mononuclear cells were seeded into the DC cell culture medium at a density of cells / ml.

8. The DC cell population of claim 1, wherein one or more of PHA, IL-2, IL-4, GM-CSF and TNF-α are added on day 0, day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9 or day 10 of the culture in step (2).

9. The DC cell population of claim 1, wherein GM-CSF and IL-4 are added on day 0 of the culture in step (2).

10. The DC cell population of claim 9, wherein the final concentration of GM-CSF is 50~100 ng / ml.

11. The DC cell population of claim 9 or 10, wherein the final concentration of IL-4 is 50-100 ng / ml.

12. The DC cell population of claim 1, wherein TNF-α is added on day 6 of the culture in step (2).

13. The DC cell population of claim 12, wherein the final concentration of TNF-α is 10-50 ng / ml.

14. The DC cell population according to any one of claims 1-10 and 12-13, wherein the proportion of CD83+ DC cells is greater than 70%.

15. The DC cell population of claim 14, wherein the proportion of CD83+ DC cells is greater than 80%.

16. The DC cell population of claim 14, wherein the proportion of CD83+ DC cells is greater than 90%.

17. The DC cell population of claim 14, wherein the proportion of CD83+ DC cells is greater than 95%.

18. The DC cell population of claim 14, wherein the proportion of CD83+ DC cells is greater than 99%.

19. The DC cell population of claim 14, wherein PBMCs stimulated with said DC cell population have an IFN-γ+ELISPOT spot count greater than 500 / 10. 5 Each PBMC has a CD83+ DC cell ratio greater than 95%.

20. The DC cell population of claim 1, wherein the method for preparing the heat shock protein gp96-antigen complex further comprises one or more of the following: (1) The homogenate of the human placental tissue was prepared by the following method: human placental tissue was added to a 10-50 mM NaHCO3 solution with a pH of 6-8 at a mass (g)-volume (ml) ratio of 1:4 to 1:8 and ground to obtain the tissue homogenate; (2) Before separation, the procedure also includes a preliminary purification of the tissue homogenate by the following steps: Step 1-1: Homogenize the tissue solution with 2-6 ml of water. o Centrifuge at C and collect the supernatant; Step 1-2: Using ammonium sulfate fractionation precipitation, the supernatant obtained in Step 1-1 is precipitated at 2~6 °C. o C. Add ammonium sulfate and collect the precipitate obtained when the concentration of ammonium sulfate is 50%~70% to obtain the initial extract; (3) The ConA-Sepharose gel column chromatography includes the following steps: Step 2-1: Load the initial extract onto a ConA-Sepharose gel column; Step 2-2: Elute with 5-50 mM Tris-HCl solution containing 50-200 mM NaCl until the UV absorption of the elution product is below 0.01 at a detection wavelength of 280 nm; Steps 2-3: Elute further with 5-50 mM Tris-HCl solution containing 8% α-pyranoside and 50-200 mM NaCl, collect the eluent to obtain ConA-Sepharose isolate; (4) The HiTrap Q ion exchange chromatography includes the following steps: Step 3-1: Load the ConA-Sepharose isolate onto a HiTrap Q ion exchange column; Step 3-2: Clean the HiTrap Q ion exchange column with 5~50 mM Tris-HCl; Step 3-3: Elute with 5-50 mM Tris-HCl containing 300 mM-1000 mM NaCl, collect the eluent until the absorbance of the elution product is less than 100 mA when the detection wavelength is 280 nm, and obtain the HiTrap Q isolate; (5) The CHT ion exchange chromatography includes the following steps: Step 4-1: Load the HiTrap Q isolate onto a CHT ion exchange column; Step 4-2: Clean the CHT ion exchange column with 5~50 mM PB; Step 4-3: Elute with 300mM~1000mM MPB, collect the eluent until the absorbance value is below 100mA, and obtain the heat shock protein gp96-antigen complex.

21. The DC cell population of claim 20, wherein step 1-1 comprises: The tissue homogenate was heated at 2-6°C. o C. Centrifuge at 1000rpm~10000rpm for 0.5h~1h and collect the supernatant.

22. The DC cell population of claim 21, wherein the collected supernatant is subjected to 2-6 [units of temperature / temperature]. o C. Centrifuge again at 1000rpm~10000rpm for 0.5h~1h, and collect the supernatant.

23. The DC cell population of claim 20, wherein in steps 1-2, the precipitate is dissolved in a 5-50 mM Tris-HCl solution containing 50-200 mM NaCl at a mass (g) to volume (ml) ratio of 1:(2-20).

24. The DC cell population of claim 20, wherein in steps 2-3, the eluent collected is the flow-through fluid collected from the 0th to 3rd column volumes.

25. The DC cell population of claim 24, wherein in steps 2-3, the eluent collected is the flow-through fluid collected from the first 0.5 to 2 column volumes.

26. The DC cell population of claim 20, in step 3-3, the collected eluent is concentrated and diluted with 20-100 mM phosphate buffer at pH 6-8 to obtain HiTrap Q isolate.

27. The DC cell population of claim 20, in step 4-3, the collected eluent is concentrated and diluted with 5-50 mM phosphate buffer at pH 6-8 to obtain the heat shock protein gp96-antigen complex.

28. The DC cell population of claim 26 or 27, wherein the concentration is ultrafiltration concentration.

29. The DC cell population of claim 1, wherein the heat shock protein gp96-antigen complex has a purity of 80% or higher.

30. The DC cell population of claim 1, wherein the heat shock protein gp96-antigen complex has a purity of 90% or higher.

31. The DC cell population of claim 1, wherein the heat shock protein gp96-antigen complex has a purity of 95% or higher.

32. The DC cell population of claim 1, wherein the heat shock protein gp96-antigen complex has a purity of 99% or higher.

33. A pharmaceutical composition comprising a population of DC cells as described in any one of claims 1-32, and one or more pharmaceutically acceptable carriers.

34. The pharmaceutical composition of claim 33, wherein it is an injectable preparation.

35. The pharmaceutical composition of claim 34, wherein it is an injection solution or a lyophilized powder for injection.

36. The pharmaceutical composition of claim 34 or 35, wherein the content of DC cells per unit dose is not less than 1 × 10⁻⁶. 4 indivual.

37. The pharmaceutical composition of claim 36, wherein the DC cell content per unit dose is not less than 1 × 10⁻⁶. 5 indivual.

38. The pharmaceutical composition of claim 36, wherein the DC cell content per unit dose is not less than 1 × 10⁻⁶. 6 indivual.

39. The pharmaceutical composition of claim 36, wherein the DC cell content per unit dose is not less than 1 × 10⁻⁶. 7 indivual.

40. The pharmaceutical composition of claim 36, wherein the DC cell content per unit dose is not less than 1 × 10⁻⁶. 8 indivual.

41. The pharmaceutical composition of claim 36, wherein the DC cell content per unit dose is not less than 1 × 10⁻⁶. 9 indivual.

42. The pharmaceutical composition of claim 36, wherein the DC cell content per unit dose is not less than 1 × 10⁻⁶. 10 indivual.

43. The pharmaceutical composition of claim 36, wherein the DC cell content per unit dose is 1 × 10⁻⁶. 5 ~1×10 8 indivual.

44. A vaccine adjuvant, immunomodulator, or vaccine formulation comprising a population of DC cells as described in any one of claims 1-32, and one or more pharmaceutically acceptable carriers.

45. The vaccine adjuvant, immunomodulator, or vaccine preparation of claim 44 is an injectable formulation.

46. ​​The vaccine adjuvant, immunomodulator, or vaccine preparation according to claim 45 is an injectable solution or a lyophilized powder for injection.

47. The vaccine adjuvant, immunomodulator, or vaccine formulation according to claim 45 or 46, wherein the DC cell content per unit dose is not less than 1 × 10⁻⁶. 4 indivual.

48. The vaccine adjuvant, immunomodulator, or vaccine formulation according to claim 47, wherein the DC cell content per unit dose is not less than 1 × 10⁻⁶. 5 indivual.

49. The vaccine adjuvant, immunomodulator, or vaccine formulation according to claim 47, wherein the DC cell content per unit dose is not less than 1 × 10⁻⁶. 6 indivual.

50. The vaccine adjuvant, immunomodulator, or vaccine formulation according to claim 47, wherein the DC cell content per unit dose is not less than 1 × 10⁻⁶. 7 indivual.

51. The vaccine adjuvant, immunomodulator, or vaccine formulation according to claim 47, wherein the DC cell content per unit dose is not less than 1 × 10⁻⁶. 8 indivual.

52. The vaccine adjuvant, immunomodulator, or vaccine formulation according to claim 47, wherein the DC cell content per unit dose is not less than 1 × 10⁻⁶. 9 indivual.

53. The vaccine adjuvant, immunomodulator, or vaccine formulation according to claim 47, wherein the DC cell content per unit dose is not less than 1 × 10⁻⁶. 10 indivual.

54. The vaccine adjuvant, immunomodulator, or vaccine formulation according to claim 47, wherein the DC cell content per unit dose is 1 × 10⁻⁶. 5 ~1×10 8 indivual.

55. Use of the DC cell population according to any one of claims 1-32 in the preparation of a medicament, wherein the medicament is used to treat glioma and cholangiocarcinoma.

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