A dendritic cell therapeutic vaccine preparation and its preparation method and application

Abstract

The present invention provides a dendritic cell therapeutic vaccine preparation and a preparation method and application thereof. Specifically, the preparation of the present invention comprises a therapeutically effective dose of antigen-sensitized mature human dendritic cells and a DC preservation solution. The active ingredient antigen-sensitized mature human dendritic cells in the preparation of the present invention can induce the patient's body to produce an antigen-specific cellular immune response. In addition, the preparation of the present invention has good stability when stored under liquid nitrogen and 80°C, can be directly subcutaneously injected for treatment after resuscitation, is convenient for clinical application, and shows good safety and tolerance.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a dendritic cell therapeutic vaccine preparation and a preparation method and application thereof. Background Art

[0002] In recent years, with the aging of the population and the extension of life expectancy, the incidence of prostate cancer has increased year by year. It has now surpassed bladder malignancies to become the malignant tumor with the highest incidence in the urinary system. Almost all patients with advanced prostate cancer will progress to castration-resistant prostate cancer (CRPC) after receiving endocrine therapy. Metastasis is the main cause of death in prostate cancer patients. Compared with the 100% 5-year survival rate of patients confined to the primary lesion, the 5-year survival rate of metastatic prostate cancer is only 29.8% (Nat Rev Urol. 2018;15(7):403-421). In the metastatic CRPC (mCRPC) stage, prostate cancer tumor cells themselves begin to secrete androgens, which promote the growth and reproduction of tumors in a self-sufficient manner. As a result, although existing treatment methods (surgical castration, drug castration, anti-androgens, etc.) can effectively block androgens from the testicles and adrenal glands, the disease still progresses rapidly. At the same time, the patient's symptoms will gradually worsen. If effective treatment methods are not taken, the patient's natural course of the disease is short. Therefore, when the disease progresses to the mCRPC stage, effective treatment must be taken as soon as possible. It is worth noting that abiraterone and enzalutamide are both anti-androgen inhibitors, and patients usually develop tolerance after taking them for 2 to 3 years [J ClinOncol. 2018;36(25):2639-2646]; docetaxel is a cytotoxic drug with strong side effects, and elderly patients often cannot tolerate and accept it. Therefore, mCRPC patients who have failed standard treatment lack effective treatments and urgently need to develop new treatments.

[0003] In April 2010, DENDREON's Sipuleucel-T was approved by the FDA as the first new tumor immunotherapy drug for the treatment of asymptomatic and mildly symptomatic mCRPC. This is an autologous tumor vaccine, and the preparation process is as follows: (1) About 3 days before infusion, the patient's PBMCs are separated and enriched by standard leukocyte separation. The monocytes are separated by density gradient centrifugation; (2) the monocytes are incubated in vitro with recombinant human prostate acid phosphatase-granulocyte macrophage stimulating factor fusion protein (Recombinant Fusion protein of Human Prostate Acid Phosphate and Granulocyte-macrophage Colony-Stimulating Factor, PAP-GM-CSF (code PA2024 in Sipuleucel-T product) for 36-44 hours, washed and formulated into a preparation; (3) the final product is transported to the outpatient center and returned to the patient. In addition to APC, the final product of Sipuleucel-T also includes T cells, B cells, natural killer cells and other cells. A single dose of Sipuleucel-T contains at least 50 million PGM-sensitized CD54 + APC is suspended in 250 ml of lactated Ringer's injection. The drug was approved based on a phase III multicenter randomized double-blind clinical trial (D9902B). 512 patients with mild or asymptomatic metastatic CRPC were randomly assigned to receive Sipuleucel-T or placebo in a 2:1 ratio. The median survival of the vaccine group was 25.8 months, compared with 21.7 months in the control group. Sipuleucel-T treatment reduced the risk of death by 22% (HR=0.78; 95% CI 0.61-0.98; P=0.03) (N EnglJ Med, 2010. 363(5): p. 411-22).

[0004] Based on the current research status of tumor immunotherapy drugs, this field still needs more effective and safer tumor therapeutic drugs. Summary of the invention

[0005] The object of the present invention is to provide a dendritic cell therapeutic vaccine preparation that can be stably stored and is safe and effective, as well as a preparation method and application thereof.

[0006] The first aspect of the present invention provides a dendritic cell therapeutic vaccine preparation, the preparation comprising:

[0007] (a) antigen-sensitized mature human dendritic cells; and

[0008] (b) DC preservation solution, with a pH of 6.3-7.3 and an osmotic pressure molar concentration of 1350-1600 mOsmol / kg;

[0009] The DC preservation solution contains: 30%~32% (v / v) compound electrolyte injection, 5%~8% (w / v) human albumin, 1% (w / v) dextran 40, 2.0625% (w / v) glucose, 0.14% (w / v) sodium chloride and 7.5%~10% (v / v) DMSO, based on the total volume of the DC preservation solution.

[0010] In another preferred embodiment, the antigen loading amount in the antigen-sensitized mature human dendritic cells is not less than 60%.

[0011] In another preferred embodiment, the concentration of antigen-sensitized mature human dendritic cells in the preparation is 2×10 6 / ml~2×10 7 / ml.

[0012] In another preferred embodiment, the antigen is recombinant human prostatic acid phosphatase-granulocyte macrophage stimulating factor fusion protein, referred to as PGM antigen.

[0013] In another preferred embodiment, the antigen-sensitized mature human dendritic cells are PGM antigen-sensitized mature human dendritic cells, namely, PGM-DCs.

[0014] In another preferred example, the amino acid sequence of the PGM antigen is shown in SEQ ID NO: 1.

[0015] In another preferred embodiment, the final concentration of the PGM antigen sensitization is 50 μg / ml-100 μg / ml.

[0016] In another preferred embodiment, the PGM antigen loading in the PGM-DC is not less than 60%.

[0017] In another preferred embodiment, the concentration of PGM-DC in the preparation is 2×10 6 / ml~2×10 7 / ml.

[0018] In another preferred embodiment, the antigen-sensitized mature human dendritic cells are glioblastoma antigen-sensitized mature human dendritic cells.

[0019] In another preferred embodiment, the DC preservation solution contains 5% (w / v) human albumin, based on the total volume of the DC preservation solution.

[0020] In another preferred embodiment, the DC preservation solution contains 7.5% (v / v) DMSO, based on the total volume of the DC preservation solution.

[0021] In another preferred embodiment, the DC preservation solution contains 30%~32% (v / v) compound electrolyte injection, 5% (w / v) human albumin, 1% (w / v) dextran 40, 2.0625% (w / v) glucose, 0.14% (w / v) sodium chloride and 7.5% (v / v) DMSO, based on the total volume of the DC preservation solution.

[0022] In another preferred embodiment, the DC preservation solution is prepared using dextran 40 glucose injection or dextran 40 sodium chloride injection.

[0023] In another preferred embodiment, the DC preservation solution is prepared using 50% glucose injection or 10% glucose injection.

[0024] In another preferred embodiment, the DC preservation solution is prepared using solid sodium chloride of drug substance grade.

[0025] In another preferred embodiment, the preparation has the following characteristics:

[0026] (1) The preparation can be stable for 24 months under liquid nitrogen conditions;

[0027] (2) The preparation is stable at -80°C ± 2°C for 6 months; and / or

[0028] (3) After cryopreservation and resuscitation, the preparation can be stable for 3 hours at 2°C to 8°C, and for up to 5 hours.

[0029] In another preferred embodiment, "stable presence" means that key quality parameters of the preparation, such as appearance, cell viability, cell density, cell phenotype, and biological activity, have no significant changes.

[0030] In another preferred embodiment, the preparation is a prostate cancer dendritic cell therapeutic vaccine preparation, which comprises the aforementioned PGM-DC and the aforementioned DC preservation solution.

[0031] In another preferred embodiment, the preparation is an injection.

[0032] The second aspect of the present invention provides a method for preparing the dendritic cell therapeutic vaccine preparation according to the first aspect of the present invention, the method comprising the following steps:

[0033] (S1) provide antigen-sensitized mature human dendritic cells;

[0034] (S2) resuspending the antigen-sensitized mature human dendritic cells in a DC preservation solution to obtain the dendritic cell therapeutic vaccine preparation.

[0035] In another preferred embodiment, the antigen-sensitized mature human dendritic cells are prepared by the following steps:

[0036] (S1a) providing antigens for priming dendritic cells;

[0037] (S1b) Providing mononuclear cells from the patient's autologous peripheral blood, which are cultured in vitro into immature dendritic cells;

[0038] (S1c) sensitizing the immature dendritic cells with the antigen and inducing them into mature dendritic cells in vitro;

[0039] (S1d) sensitizing the mature dendritic cells with the antigen, thereby obtaining the antigen-sensitized mature human dendritic cells.

[0040] In another preferred embodiment, in step (S1b), the immature dendritic cells are obtained by inducing monocytes from the patient's autologous peripheral blood in vitro with IL-4 and GM-CSF.

[0041] In another preferred embodiment, in step (S1c), the mature dendritic cells are obtained by adding 5-15 μg / ml PGE2, 500-1500 IU / ml TNFα and 1-10 μg / ml CD40L to the culture system of immature dendritic cells and continuing to culture for 16 to 60 hours.

[0042] In another preferred embodiment, the antigen is a PGM antigen, and its amino acid sequence is shown in SEQ ID NO: 1.

[0043] In another preferred embodiment, the concentration of the PGM antigen-sensitized immature dendritic cells is 50 μg / ml to 100 μg / ml, preferably 50 μg / ml.

[0044] In another preferred embodiment, before resuspending the antigen-sensitized mature human dendritic cells in DC preservation solution, the cells are washed with physiological saline to remove residual impurities.

[0045] In another preferred embodiment, the cells are washed three times with physiological saline to remove residual impurities in the antigen-sensitized mature human dendritic cells.

[0046] The third aspect of the present invention provides a use of the dendritic cell therapeutic vaccine preparation according to the first aspect of the present invention in the preparation of a medicament for treating a disease.

[0047] In another preferred embodiment, the disease is cancer or tumor.

[0048] In another preferred embodiment, the disease is prostate cancer or glioblastoma.

[0049] In a fourth aspect of the present invention, a cell preservation solution suitable for dendritic cells is provided, wherein the pH of the cell preservation solution is 6.3-7.3, and the osmotic pressure molar concentration is 1350-1600 mOsmol / kg; and the cell preservation solution contains: 30%-32% (v / v) compound electrolyte injection, 5%-8% (w / v) human albumin, 1% (w / v) dextran 40, 2.0625% (w / v) glucose, 0.14% (w / v) sodium chloride and 7.5%-10% (v / v) DMSO, based on the total volume of the cell preservation solution.

[0050] In another preferred embodiment, the cell preservation solution contains 5% (w / v) human albumin, based on the total volume of the cell preservation solution.

[0051] In another preferred embodiment, the cell preservation solution contains 7.5% to 10% (v / v) DMSO, based on the total volume of the cell preservation solution.

[0052] In another preferred embodiment, the cell preservation solution contains 7.5% (v / v) DMSO, based on the total volume of the cell preservation solution.

[0053] In another preferred embodiment, the cell preservation solution contains 30%~32% (v / v) compound electrolyte injection, 5% (w / v) human albumin, 1% (w / v) dextran 40, 2.0625% (w / v) glucose, 0.14% (w / v) sodium chloride and 7.5% (v / v) DMSO, based on the total volume of the cell preservation solution.

[0054] In another preferred embodiment, the cell preservation solution is prepared using dextran 40 glucose injection or dextran 40 sodium chloride injection.

[0055] In another preferred embodiment, the cell preservation solution is prepared using 50% glucose injection solution or 10% glucose injection solution.

[0056] In another preferred embodiment, the cell preservation solution is used to prepare a dendritic cell therapeutic vaccine preparation.

[0057] The fifth aspect of the present invention provides a use of the cell preservation solution as described in the fourth aspect of the present invention in the preparation of a dendritic cell therapeutic vaccine preparation.

[0058] The sixth aspect of the present invention provides a composition comprising: (i) the dendritic cell therapeutic vaccine preparation according to the first aspect of the present invention; and (ii) a pharmaceutically acceptable carrier.

[0059] In another preferred embodiment, the composition includes a pharmaceutical composition or a vaccine composition.

[0060] The seventh aspect of the present invention provides a method for treating a disease, comprising the step of administering the dendritic cell therapeutic vaccine preparation as described in the first aspect of the present invention to a subject in need thereof.

[0061] In another preferred embodiment, the disease is cancer or tumor.

[0062] In another preferred embodiment, the disease is prostate cancer or glioblastoma.

[0063] In another preferred embodiment, the disease is prostate cancer, and the method comprises the step of administering the prostate cancer dendritic cell therapeutic vaccine preparation as described in the first aspect of the present invention to a subject in need thereof.

[0064] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 The amino acid sequence of the PGM antigen is shown. The amino acids before the GS linker peptide are the PAP sequence, and the amino acids after the GS linker peptide are the GM-CSF sequence.

[0066] Figure 2 Shown is a comparison of PGM antigen loading after DC cells were sensitized with different PGM antigens.

[0067] Figure 3 The figure shows the comparison of cell survival rates after storage at -80°C ± 2°C for 3 months under different prostate cancer dendritic cell therapeutic vaccine formulations.

[0068] Figure 4 The figure shows the comparison of cell phenotype changes of the finished product of prostate cancer dendritic cell therapeutic vaccine preparation after 0 month and 24 months of storage in liquid nitrogen.

[0069] Figure 5 It was shown that PGM antigen-sensitized C57BL / 6J mouse dendritic cells could effectively inhibit the growth of mouse prostate cancer cells (RM-1).

[0070] Figure 6 It was shown that PGM antigen-sensitized C57BL / 6J mouse dendritic cells could effectively prolong the survival of RM-1 tumor-bearing mice. DETAILED DESCRIPTION

[0071] After extensive and in-depth research, the inventors unexpectedly developed a dendritic cell therapeutic vaccine preparation that can be stably present and preserved for the first time. The vaccine preparation of the present invention includes antigen-sensitized mature human DC, and a DC preservation solution with a pH range of 6.3-7.3 and an osmotic pressure molar concentration of 1350-1600mOsmol / kg. The DC preservation solution is composed of a compound electrolyte injection, human serum albumin, dextran 40, glucose, sodium chloride, and DMSO in a certain proportion. Experiments have shown that the vaccine preparation of the present invention can be stably stored for 24 months under liquid nitrogen conditions and for 6 months under -80°C ± 2°C temperature conditions, and its appearance, visible foreign matter, cell viability, cell density, cell phenotype, biological activity and other quality attributes have no significant changes; after the vaccine preparation of the present invention is revived, it is placed at room temperature for 5 hours, and the cell survival rate does not decrease significantly. Therefore, the dendritic cell therapeutic vaccine preparation of the present invention can be used as a long-term preservation and convenient clinical therapeutic drug. On this basis, the present invention is completed.

[0072] the term

[0073] In order to make the present invention more easily understood, some technical and scientific terms are specifically defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0074] As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0075] As used herein, the term "treatment" refers to administering an internal or external therapeutic agent, including a vaccine for tumors of the present invention and a composition thereof, to a patient who has one or more symptoms of a disease, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, the amount of the therapeutic agent that is effective in alleviating one or more symptoms of the disease (therapeutically effective amount) is administered to the patient.

[0076] Prostatic acid phosphatase (PAP)

[0077] Prostatic acid phosphatase (PAP) is the most abundant enzyme in human prostate tissue, with a molecular weight of 100KD. It is composed of two catalytically active subunits bound by non-covalent bonds to form an active enzyme dimer. The activity of human prostatic acid phosphatase (hPAP) mainly depends on the secondary structure of its protein. PAP exists in two forms in the body: secretory PAP (SPAP) and transmembrane PAP (TM-PAP). S-PAP is synthesized and secreted by prostate epithelial cells, stored in the seminal vesicles, and participates in semen liquefaction and reproduction. Its production and secretion are regulated by androgens and are tissue-specific. Under normal conditions, S-PAP is extremely low in the blood. It only enters the blood in large quantities in disease states (such as prostate cancer) and can produce a tumor suppressor effect. Therefore, for nearly fifty years, PAP has been used as a tumor marker for prostate cancer and a target for specific immunotherapy for clinical diagnosis and treatment.

[0078] Recombinant human prostatic acid phosphatase-granulocyte macrophage stimulating factor fusion protein (PGM antigen)

[0079] The recombinant human prostatic acid phosphatase-granulocyte macrophage stimulating factor fusion protein (PGM antigen) has a total of 483 amino acids. The N-terminus (1-354) is a PAP protein molecule with three pairs of disulfide bonds (C129-C340, C183-C281, C315-C319) and three N-glycosylation sites (Asn at positions 62, 188 and 301). The C-terminal 127 amino acids are GM-CSF protein molecules, which are connected to the PAP protein molecule through the intermediate linker Gly-Ser. There are two pairs of disulfide bonds (C410-C452, C444-C477), four O-glycosylation sites (Ser at positions 361, 363 and 365; Thr at position 366), and two N-glycosylation sites (Asn at positions 383 and 393). Its theoretical molecular weight is: 55607.40kDa (see Figure 1 ). Key active sequence: PAP is the antigen region of DC sensitized during the preparation of prostate cancer dendritic cell therapeutic vaccine preparations. GM-CSF is an adjuvant-like cytokine that can promote DC cell proliferation and differentiation, and enhance DC cell uptake and presentation of PAP antigen by binding to corresponding receptors on the DC surface. The amino acid sequence of PGM antigen is shown in SEQ ID NO 1.

[0080] KELKFVTLVFRHGDRSPIDTFPTDPIKESSWPQGFGQLTQLGMEQHYELGEYIRKRYRKFLNESYKHEQVYIRSTDVDRTLMSAMTNLAALFPPEGVSIWNPILLWQPIPVHTVPLSEDQLLYLPFRNCPRFQELESETLKSEEFQKRLHPYKDFIATLGKLSGLHGQDLFGIWSKVYDPLYCESVHNFTLPSWATEDTMTKLRELSELSLLSLYGIHKQKEKSRLQGGVLVNEILNHMKRATQIPSYKKLIMYSAHDTTVSGLQMALDVYNGLLPPYASCHLTELYFEKGEYFVEMYYRNETQHEPYPLMLPGCSPSCPLERFAELVGPVIPQDWSTECMTTNSHQGTEDSTDGSAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE(SEQ ID NO: 1)

[0081] Dendritic cells (DC)

[0082] Dendritic cells (DC) were first discovered by Ralph M. Steinman in the United States in 1973. They are named for the many dendritic or pseudopodia-like protrusions they extend when mature. DC is the antigen-presenting cell with the strongest antigen-presenting function known so far. Its most important feature is that it can stimulate the activation and proliferation of naive T cells, while Mφ and B cells can only stimulate activated T cells or memory T cells. Therefore, DC is the initiator of specific immune response and occupies a unique position in the immune system. DC is widely distributed in organs throughout the body except the brain. The number is small, accounting for less than 1% of peripheral blood mononuclear cells. According to the maturation state of DC, DC can be divided into DC precursors, immature DC, migratory DC and mature DC. DC precursor cells enter the epithelial sites of various solid organs and non-lymphoid tissues through the blood circulation or lymphatic circulation, and differentiate and develop into immature DC (iDC) under the action of certain cytokines. Under normal circumstances, most DCs in the body are in an immature state. They only express low levels of MHC class II molecules, co-stimulatory molecules and adhesion molecules, and have a weak ability to activate T cells in vitro; but they express more FcR and pathogen receptors, and have a strong ability to absorb and process antigens. After iDCs absorb antigens or are stimulated by certain factors (mainly inflammatory factors such as LPS, IL1β and TNFα), immature DCs begin to differentiate and mature. Mature DCs (mDCs) express a large number of MHC class II molecules and co-stimulatory molecules (CD80, CD40, CD86, etc.), and can effectively present processed antigens to naive T cells in the form of antigen peptide-MHC molecule complexes and activate them.

[0083] Dendritic cell therapeutic vaccine preparations

[0084] The present invention provides a dendritic cell therapeutic vaccine preparation, which comprises a therapeutically effective dose of antigen-sensitized mature human DC, and a DC preservation solution with a pH range of 6.3-7.3 and an osmotic pressure molar concentration of 1350-1600 mOsmol / kg, wherein the DC preservation solution is prepared by a compound electrolyte injection, human serum albumin, dextran 40, glucose, sodium chloride, and DMSO in a certain proportion.

[0085] In a preferred example of the present invention, the concentration of the compound electrolyte injection in the preparation is 30-32% (v / v); the concentration of human albumin is 5%-8% (w / v), preferably 5%; the concentration of dextran 40 is 1% (w / v); the concentration of glucose is 2.0625% (w / v); the concentration of sodium chloride is 0.14% (w / v); and the concentration of DMSO is 7.5%-10% (v / v), preferably 7.5%.

[0086] In one embodiment of the present invention, a prostate cancer dendritic cell therapeutic vaccine preparation is provided, which comprises a therapeutically effective dose of the aforementioned PGM antigen-sensitized mature human DC (i.e., PGM-DC), and the aforementioned DC preservation solution; wherein the PGM antigen loading amount in the PGM-DC is not less than 60%, and the concentration of the PGM-DC in the preparation is 2×10 6 / ml~2×10 7 / ml; the concentration of the compound electrolyte injection in the DC preservation solution is 30-32% (v / v); the concentration of human albumin is 5%-8% (w / v), preferably 5%; the concentration of dextran 40 is 1% (w / v); the concentration of glucose is 2.0625% (w / v); the concentration of sodium chloride is 0.14% (w / v); the concentration of DMSO is 7.5%-10% (v / v), preferably 7.5%.

[0087] Method for preparing dendritic cell therapeutic vaccine preparation

[0088] The present invention also provides a method for preparing the above-mentioned dendritic cell therapeutic vaccine preparation, which comprises the steps of providing antigen-sensitized mature human dendritic cells, and resuspending the antigen-sensitized mature human dendritic cells in a DC preservation solution.

[0089] In one embodiment of the present invention, a method for preparing the aforementioned prostate cancer dendritic cell therapeutic vaccine preparation is provided, the method comprising the steps of: (1) providing purified PGM antigen;

[0090] (2) Providing mononuclear cells from the peripheral blood of prostate cancer patients and culturing them into immature DCs in vitro;

[0091] (3) sensitizing immature DCs with the PGM antigen and inducing them into mature DCs in vitro;

[0092] (4) sensitizing mature DCs with the PGM antigen and washing with saline to remove residual impurities;

[0093] (5) resuspending the harvested PGM antigen-sensitized mature DCs in a DC preservation solution to obtain the prostate cancer dendritic cell vaccine preparation;

[0094] The concentration of the PGM antigen-sensitized immature DC is 50 μg / ml~100 μg / ml, preferably 50 μg / ml; after adding 5-15 μg / ml PGE2, 500-1500 IU / ml TNFα and 1-10 μg / ml CD40L to the immature DC culture system, mature DC is obtained after continuing to culture for 16-60 hours; and the preparation is washed 3 times with physiological saline to remove residual impurities in the preparation, including IL-4, free PGM antigen, etc.

[0095] Compositions and methods of administration

[0096] The present invention also provides a composition, comprising (i) the dendritic cell therapeutic vaccine preparation according to the first aspect of the present invention; and (ii) a pharmaceutically acceptable carrier.

[0097] The compositions of the present invention include pharmaceutical compositions or vaccine compositions.

[0098] The composition is prepared as a liquid preparation, including (but not limited to) injection, suspension, or spray.

[0099] The pharmaceutical composition of the present invention comprises (or contains) a therapeutically effective amount of antigen-sensitized mature human dendritic cells.

[0100] As used herein, the term "therapeutically effective amount" refers to an amount of a therapeutic agent that treats, alleviates or prevents a target disease or condition, or an amount that exhibits a detectable therapeutic or preventive effect. The effect can be detected, for example, by antigen levels. The therapeutic effect also includes a reduction in physiological symptoms. The exact effective amount for a subject depends on the subject's size and health, the nature and extent of the condition, and the therapeutic agent and / or combination of therapeutic agents selected for administration. Therefore, it is useless to specify an exact effective amount in advance. However, for a given condition, the effective amount can be determined by routine experimentation.

[0101] For the purposes of the present invention, an effective dosage is about 0.001 mg / kg to 10 mg / kg, preferably about 0.003 mg / kg to 0.1 mg / kg body weight administered to a subject.

[0102] The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier used for administering a therapeutic agent (e.g., a dendritic cell therapeutic vaccine formulation of the present invention). The term refers to such pharmaceutical carriers that do not induce the production of antibodies harmful to the individual receiving the composition and do not have excessive toxicity after administration. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, etc. These carriers are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable carriers or excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0103] The pharmaceutically acceptable carrier in the composition may include liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as wetting agents or emulsifiers, pH buffer substances, etc. may also be present in these carriers. Generally, the composition can be made into an injectable, such as a liquid solution or suspension; it can also be made into a solid form suitable for adding a solution or suspension, a liquid excipient before injection. Liposomes are also included in the definition of pharmaceutically acceptable carriers.

[0104] The vaccine composition of the present invention comprises an immunogenic antigen (e.g., a mature human dendritic cell sensitized by the antigen of the present invention), and is usually combined with a "pharmaceutically acceptable carrier", which includes any carrier that does not itself induce the production of antibodies that are harmful to the individual receiving the composition. Suitable carriers are usually large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, amino acid polymers, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), etc. These carriers are well known to those of ordinary skill in the art. In addition, these carriers can act as immunostimulants ("adjuvants").

[0105] Preferred adjuvants for enhancing the effect of the immune composition include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) water-in-oil emulsion formulations, for example: (a) Montanide ISA 51VG, (b) Montanide ISA720VG; (c) MF59; (3) saponin adjuvants; (4) Freund's complete adjuvant (CFA) and Freund's incomplete adjuvant (IFA); (5) cytokines, such as interleukins (such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (such as gamma interferon), macrophage colony stimulating factor (GM-CSF), etc.; (6) detoxified variants of bacterial ADP-ribosylating toxins (such as Escherichia coli heat-labile toxin LT); and (7) other substances that act as immunostimulants to enhance the effect of the composition.

[0106] Vaccine compositions including immunogenic compositions (for example, may include antigens, pharmaceutically acceptable carriers and adjuvants) generally contain diluents such as water, saline, glycerol, ethanol, etc. In addition, auxiliary substances such as wetting agents or emulsifiers, pH buffer substances, etc. may be present in such carriers. More specifically, vaccines including immunogenic compositions contain an immunogenic composition in an immunologically effective amount, as well as other required components as described above. "Immunologically effective amount" refers to an amount that is effective for treatment when administered to an individual as a single dose or as part of a continuous dose. The dosage can be based on the health and physiological condition of the individual being treated, the type of individual being treated (such as humans), the ability of the individual's immune system to synthesize antibodies, the desired degree of protection, the formulation of the vaccine, the treating physician's assessment of the medical condition, and other relevant factors. It is expected that the dosage will be within a relatively wide range and can be determined by routine experiments.

[0107] Typically, the vaccine composition or immunogenic composition can be prepared as an injectable, such as a liquid solution or suspension; it can also be prepared as a solid form suitable for being mixed with a solution or suspension or a liquid excipient before injection. The preparation can also be emulsified or encapsulated in a liposome to enhance the adjuvant effect.

[0108] Once formulated, the composition of the invention can be administered directly to a subject. The subject to be treated can be a mammal, especially a human.

[0109] When used as a vaccine, the vaccine composition of the present invention can be directly administered to an individual by known methods, usually using the same administration routes as conventional vaccines. Routes for administering the pharmaceutical composition or vaccine composition of the present invention include (but are not limited to): subcutaneous, intradermal, intramuscular or other parenteral administration routes. If necessary, the administration routes can be combined or adjusted according to the disease condition. The vaccine composition can be administered in a single dose or multiple doses, and can include the administration of booster doses to induce and / or maintain immunity. The vaccine composition should be administered in an "effective amount", that is, the amount of the vaccine composition is sufficient to induce an immune response in the selected administration route and can effectively improve the symptoms of the disease.

[0110] Representative diseases include (but are not limited to) tumors (or) cancers selected from the following groups, especially solid tumors: lung cancer, non-small cell lung cancer, colorectal cancer, breast cancer, liver cancer, gastric cancer, esophageal cancer, pancreatic cancer, melanoma, kidney cancer, prostate cancer, cervical cancer, ovarian cancer, nasopharyngeal cancer, oral cancer, osteosarcoma, brain glioma, bladder cancer, etc.

[0111] The amount of the pharmaceutical composition selected in each vaccine dose is determined by the amount that can induce an immunoprotective response without significant side effects. Standard research methods including observation of antibody titers and other responses in subjects can be used to determine the optimal dosage of a specific vaccine. The need for a booster dose can be determined by monitoring the level of immunity provided by the vaccine. After evaluating the antibody titer in the serum, it may be necessary to select a booster dose for immunization. Administration of the pharmaceutical composition or vaccine composition can increase the immune response to the present invention.

[0112] In addition, the vaccines of the present invention may be administered in combination with other immunomodulators, or with other therapeutic agents.

[0113] application

[0114] The present invention also provides the use of the aforementioned dendritic cell therapeutic vaccine preparation in the preparation of a drug for treating a disease. Specifically, the present invention provides the use of the aforementioned prostate cancer dendritic cell therapeutic vaccine preparation in the preparation of a drug for treating prostate cancer.

[0115] Compared with conventional formulations, the main innovation of the present invention is to develop and provide a stable formulation containing antigen-sensitized human DC active ingredients, and its main advantages include:

[0116] (1) The preparation of the present invention contains antigen-sensitized human DC (eg, PGM antigen-sensitized human DC) as its active ingredient, and can induce the body to produce an antigen-specific immune response.

[0117] (2) The PGM antigen used in the production process of the prostate cancer dendritic cell therapeutic vaccine preparation of the present invention is expressed in a CHO cell system, has a purity greater than 95%, is less likely to be contaminated by viruses, has better safety, and is of higher purity.

[0118] (3) The preparation of the present invention can produce multiple doses of cell therapy products by separating blood cells from the patient once, thus avoiding the risk and burden of repeated blood cell separation for the patient.

[0119] (4) The present invention has explored the formulation of the dendritic cell therapeutic vaccine preparation through experiments. The antigen-sensitized human DC in the preparation can be stored for a long time in liquid nitrogen or ultra-low temperature refrigerator (-80℃±2℃), and has good stability after resuscitation. After resuscitation, it can be directly injected subcutaneously, which is convenient for transportation and clinical use.

[0120] (5) The preparation of the present invention contains only 7.5% DMSO, and has been clinically proven to be safe and well tolerated.

[0121] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0122] Example 1 Preparation of Prostate Cancer Dendritic Cell Therapeutic Vaccine Stock Solution

[0123] Patients who were clinically diagnosed with prostate adenocarcinoma and voluntarily accepted the transfusion of autologous DC sensitized by antigens and met the requirements for blood cell separation had about 3-7L of whole blood separated by a blood cell separator. The white blood cells obtained by the blood cell separator were mainly peripheral blood mononuclear cells (PBMC), including monocytes and lymphocytes, with a small amount of red blood cells, granulocytes and platelets.

[0124] (1) Slowly add the collected white blood cells into a centrifuge tube pre-added with lymphocyte separation solution (the volume of cell suspension and separation solution are equal). After the centrifuge tube is strictly balanced, centrifuge at 750×g for 30 minutes.

[0125] (2) After centrifugation, use a flat-mouth Pasteur pipette to absorb the misty interface cells. The interface layer cells are mainly PBMCs. Collect the interface layer cells, resuspend them in physiological saline, centrifuge at 420×g for 15 minutes, and discard the supernatant.

[0126] (3) After centrifugation, PBMCs were resuspended in physiological saline and centrifuged at 150 × g for 8 minutes. The supernatant was discarded and this washing step was repeated twice.

[0127] (4) After centrifugation, collect the cells and resuspend them in RPMI 1640 medium. Count the cells on a hemocytometer and measure the cell survival rate using trypan blue staining. Resuspend the cells in RPMI 1640 medium to 4×10 6 ~6×10 6 / ml, inoculated into a T175 sterile cell culture flask, and placed in an incubator at 37°C, 5% carbon dioxide and saturated humidity for 2 to 20 hours.

[0128] (5) Gently shake the sterile culture flask of T175 to suspend the non-adherent cells, aspirate the suspended cells into a centrifuge tube, and then gently wash with RPMI 1640 medium to remove the non-adherent cells. The adherent cells are mainly monocytes (the suspended cells are mainly lymphocytes, which will be collected and frozen for subsequent biological activity determination).

[0129] (6) Add RPMI 1640 medium containing 100-250 ng / ml recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF), 100-500 ng / ml recombinant human interleukin-4 (rhIL-4), and 5%-10% (v / v) inactivated autologous plasma to the culture flask retaining adherent cells and culture in an incubator at 37°C, 5% carbon dioxide, and saturated humidity until the 5th day.

[0130] (7) On the fifth day of culture, the cell morphology was observed. Most cells had burr-like protrusions on their surface and aggregated into clusters. These cells were immature dendritic cells. The DC cells were collected by centrifugation at 420 × g for 5 minutes.

[0131] (8) The collected immature DCs were resuspended to 4×10 cells in RPMI 1640 medium containing 100-500 ng / ml rhIL-4 and 5% (v / v) inactivated autologous plasma. 5 ~6×10 5 / ml, add PGM antigen with a final concentration of 50-100μg / ml for sensitization, and continue to culture in an incubator at 37°C, 5% carbon dioxide and saturated humidity.

[0132] (9) On the 6th day of culture, add stimulatory factors to the cell culture flask: prepare according to the applicant's authorized patent (application number: 202111614627.2), add 5-15μg / ml PGE2, 500-1500IU / ml TNFα and 1-10μg / ml CD40L to the culture system, and continue to culture for 16-60 hours to obtain mature dendritic cells (if the proportion of CD83-positive cells is not less than 50%, it indicates that DCs are mature and can be harvested).

[0133] (10) Collect mature human DCs sensitized with PGM antigen, centrifuge at 420 × g for 5 min, and discard the supernatant.

[0134] Wash three times with sodium chloride injection, centrifuge at 420×g for 5 minutes, discard the supernatant, and after three washes, the harvested DCs are the original solution of prostate cancer dendritic cell therapeutic vaccine.

[0135] Comparative Example 1 Optimization of PGM antigen sensitization concentration

[0136] After the immature DCs were obtained according to step (7) of Example 1, 0 μg / ml, 5 μg / ml, 10 μg / ml, 25 μg / ml, 50 μg / ml, and 100 μg / ml of FITC-PGM (FITC fluorescently labeled PGM antigen) or FITC-BSA (FITC fluorescently labeled bovine serum albumin, as an irrelevant control protein) were added to the immature DC culture system and sensitized for 4 hours. The proportions of FITC-PGM positive cells detected by FACS were 0%, 23.2%, 59.7%, 76.6%, 94.1%, and 98.5%, respectively. The results showed that DC binding to PGM showed a certain dose-dependency. When 50 μg / ml of FITC-PGM antigen was added to the culture system, the DC loading reached more than 90%, which was basically saturated. After FITC-BSA was incubated with DC at the same concentration, the corresponding FITC positive cell proportion was significantly lower than that of the FITC-PGM group (see Tables 1 and Figure 2 ). The results suggest that PGM antigen can specifically bind to immature dendritic cells, and the sensitization concentration of PGM antigen is preferably 50-100 μg / ml, more preferably 50 μg / ml.

[0137] Table 1. Optimization results of the concentration of immature dendritic cells sensitized with PGM antigen

[0138]

[0139] Comparative Example 2 Washing process optimization

[0140] According to the steps described in Example 1, three different prostate cancer patient-derived PGM antigen-sensitized mature human DCs were harvested and washed 5 times with normal saline. During the first wash, the volume of normal saline was basically the same as the harvested cell culture supernatant; during the second wash, the volume of normal saline used for washing was reduced to 1 / 2~1 / 3; during the third, fourth, and fifth washes, all cells were collected in a 50ml centrifuge tube and washed with 35~45ml normal saline. The results showed that when the washing was washed for the third time, the residual amount of IL-4 and PGM antigens in the washing supernatant had basically reached the minimum value, and the residual amount did not decrease significantly after continued washing. Considering that increasing the number of washes will have a certain effect on cell viability and cell yield, and considering that there are too many operating steps, the risk of cell contamination increases. Therefore, preferably, the DC harvested by washing 3 times with normal saline is the prostate cancer dendritic cell therapeutic vaccine stock solution.

[0141] Example 2 Screening of formulations for prostate cancer dendritic cell therapeutic vaccine preparations

[0142] According to the grouping in Table 2, different cell preservation solutions were prepared with compound electrolyte injection (Shanghai Baxter Medical Products Co., Ltd.), human albumin [Grifols, specification 10g / bottle (20%, 50ml)], 10% dextran 40 0.9% sodium chloride injection (Sichuan Kelun Pharmaceutical Co., Ltd.), 50% glucose injection (China Otsuka Pharmaceutical Co., Ltd.), sodium chloride (Tianjin Haiguang Pharmaceutical Co., Ltd.), and DMSO (OriGen) in a certain proportion. After preparation, the mixture was sterilized by filtration with a 0.22μm filter membrane. The compound electrolyte injection mainly maintains the pH buffer system of the cell preservation solution and provides electrolytes; human albumin and dextran 40 are mainly used as non-permeable cryoprotectants to maintain a certain osmotic pressure and minimize the solution damage that may occur during cell cryopreservation; glucose provides a certain amount of energy for cryopreserved cells to maintain cell survival; sodium chloride is used as an osmotic pressure regulator; DMSO is the most commonly used permeable cryoprotectant for cell cryopreservation, which reduces the temperature of water crystallization and prevents ice crystal damage during cell cryopreservation.

[0143] Table 2. Cell preservation solution formulation research group (total volume of 1L)

[0144]

[0145] The mature DCs sensitized with PGM antigen in Example 1 were cultured at a density of 1×10 7 / ml were cryopreserved with the prepared cell preservation solution at -80℃±2℃. The corresponding cells were revived after 3 months and the cell survival rate was calculated.

[0146] The experimental results are as follows Figure 3 As shown in Table 3, the cell survival rates of Group 1 and Group 5 were 94% and 95%, respectively (P>0.05). When DMSO was reduced to 5% (v / v) in Group 4, the cell survival rate was 81%, which was significantly decreased compared with Group 1 and Group 5 (P<0.05). The results suggest that the reduction of DMSO concentration will affect the freezing effect of the cell preservation solution and reduce the cell survival rate. Therefore, the DMSO concentration in the cell preservation solution is preferably 7.5%~10% (v / v). Considering that DMSO has a certain irritation to the skin when the vaccine is subcutaneously injected, the DMSO concentration is more preferably 7.5%. Group 2 and Group 3 were respectively down-regulated and up-regulated in human serum albumin concentration. The average cell survival rates of Group 2 and Group 3 were 85% (P<0.05) and 91% (P>0.05), respectively. The results suggest that the reduction of human serum protein concentration will affect the freezing effect of the cell preservation solution and reduce the cell survival rate. Therefore, the concentration of human serum albumin in the cell preservation solution is preferably 5% to 8% (w / v). Considering the production cost, more preferably, the concentration of human serum albumin is 5%.

[0147] Table 3. Cell viability of the cell preservation solution formula research group (total volume is 1L)

[0148]

[0149] In summary, Group 1 is preferred as the formulation for prostate cancer dendritic cell therapeutic vaccine preparations. The cell preservation fluid prepared according to the formulation of Group 1 is named DC preservation fluid, which has the following quality attributes. The test results of three consecutive batches of DC preservation fluid all meet the quality standards, as shown in Table 4 for details.

[0150] Table 4. Statistical table of the test results of three batches of DC preservation solution

[0151]

[0152] In addition, DC preservation solution can be stably stored for 24 months at 2~8℃, see Table 5 for details.

[0153] Table 5. DC storage solution stability study results at 2~8℃

[0154]

[0155] It is worth noting that the raw materials in the DC preservation solution can be adjusted, such as 10% dextran 40 0.9% sodium chloride injection can be replaced with dextran 40 glucose injection, human albumin can be replaced with a concentration of 25%, and 50% glucose injection can be replaced with 10% glucose injection. The final concentration of each component after configuration only needs to be maintained as shown below, and it will not affect the quality attributes of the cell preservation solution: 30%~32% (v / v) compound electrolyte injection, 5% (w / v) human albumin, 1% (w / v) dextran 40, 2.0625% (w / v) glucose, 0.14% (w / v) sodium chloride, and 7.5% (v / v) DMSO.

[0156] Comparative Example 3 Comparison of cryopreservation effects of cell preservation solutions

[0157] The PGM antigen-sensitized mature human dendritic cells (PGM-DC) prepared in Example 1 were resuspended in the DC preservation solution in Example 2, as well as the commonly available commercial cell preservation solution CryoStor® CS10 (Cat. No.: 07930, manufacturer: Stemcell), Cellbanker2 (ZENOAQ, Japan) and GMP-grade vitrified cell freezing solution (Cat. No.: NC1011, manufacturer: Youkang Biotechnology) to a density of about 1×10 7 / ml, and then cryopreserved at -80℃±2℃. After 3 months, the corresponding cells were revived and the cell survival rate was detected.

[0158] Table 6. Comparison of long-term stability of DC storage medium and commercial cell storage medium at -80℃±2℃

[0159]

[0160] The results showed that the PGM-DC cells frozen in DC preservation solution, CryoStor® CS10 and Cellbanker2 preservation solution were revived in an ultra-low temperature refrigerator (-80℃±2℃) for 3 months, and the average cell survival rate was ≥85%, which was significantly higher than that of the GMP-grade vitrified cell cryopreservation solution group (see Table 6). The GMP-grade vitrified cell cryopreservation solution is not suitable for the cryopreservation of human dendritic cells. However, it was unexpectedly found that after the cells in each group were revived, they were placed at 2~8℃ for resuscitation stability research. After 5 hours of cell resuscitation in the DC cell preservation solution group, there was no significant decrease in cell survival rate, while after 1 hour of cell resuscitation in the CryoStor® CS10 and Cellbanker2 groups, the cell viability dropped to <80% (see Tables 7 and 8).

[0161] Table 7. Comparison of DC preservation solution and commercial cell preservation solution recovery stability (1 hour)

[0162]

[0163] Table 8. DC preservation solution recovery stability study

[0164]

[0165] Example 3 Preparation and stability study of prostate cancer dendritic cell therapeutic vaccine preparation

[0166] (1) Prepare DC preservation solution according to the formula in Example 2, sterilize and filter with a pore size of 0.22 μm, and dispense into sterile vials at a rate of 10 to 40 ml / bottle, and store at 2 to 8°C. After passing the quality inspection, use it for the preparation of prostate cancer dendritic cell therapeutic vaccine preparations.

[0167] (2) The preparation of therapeutic vaccine for prostate cancer dendritic cells is single-person single-batch, and the production scale of each batch is determined by the number of mature DCs sensitized by PGM antigens finally harvested from each prostate cancer patient. The stock solution of therapeutic vaccine for prostate cancer dendritic cells was prepared according to Example 1. According to the number of DCs and the configuration density, the corresponding volume of DC preservation solution was added to resuspend the DC cells, and then filled into AT sterile cryopreservation bottles (Aseptic Technologies, Belgium), and sealed with M1 laser sealer (Aseptic Technologies, Belgium).

[0168] Transfer the sterile cryopreserved AT vials to a programmed cooling box and place them in a -80°C ultra-low temperature freezer for programmed cooling. If stored for a short period of time (≤ 6 months), they can continue to be stored in a -80°C ultra-low temperature freezer. For long-term storage, transfer them to liquid nitrogen for storage.

[0169] 3.2 Finished Product Testing

[0170] After the frozen prostate cancer dendritic cell therapeutic vaccine preparation was thawed, the following tests were performed:

[0171] (1) Appearance and visible foreign matter

[0172] The inspector adjusts the position so that the test sample is at the clear visual distance of the eye, and the distance between the test sample and the human eye is 25 cm. Remove the container label and wipe the outer wall of the container. Hold the container neck, gently rotate and flip the container to suspend the visible foreign matter in the liquid medicine, and be careful not to generate bubbles in the liquid medicine. Visually inspect under black and white backgrounds respectively, and repeat 3 times, with a total time limit of 20 seconds.

[0173] There should be no foreign matter other than cells in the test sample, and the appearance should be a white or light yellow suspension.

[0174] (2) Cell survival rate and cell density

[0175] Cell density and cell viability were measured using a fluorescent staining-based cell counter, the NucleoCounter® NC-250™ cell analyzer, and the AO / DAPI dye was Solution 18 that came with the instrument.

[0176] The cell viability of the test article should be no less than 80%, and the cell density should be 80%~120% of the labeled amount.

[0177] (3) Phenotype

[0178] The revived cells were washed with saline and resuspended in saline and placed in 6 flow cytometry labeled tubes (about 1×10 5 / 50μl / tube). Label the 6 labeled tubes separately: Alex Flour488 anti-human HLA-DR and PE anti-human CD86 (double labeling), FITC anti-human CD14, APC anti-human CD83 flow cytometry antibodies and corresponding isotype controls, and label them for 15 minutes at room temperature in the dark. After incubation, add 0.5ml of normal saline, centrifuge at 420×g for 5 minutes to wash, and discard the supernatant; wash twice in total. Discard the supernatant, resuspend the cell pellet with 200μl of normal saline, and detect it on a flow cytometer.

[0179] Detection of HLA-DR in test cell + CD86 + The ratio should not be less than 80%, CD14+ The ratio should not exceed 5%, CD83 + The proportion should not be less than 60%.

[0180] (4) Biological activity

[0181] CellTrace TM After CFSE (Cat. No.: C34554, ThermoFisher) labeling, mix with revived PGM-DC at a ratio of 10:1, and co-culture in a 37°C, 5% CO2 incubator for 72-96 hours in RMPI 1640 complete medium containing 5% FBS and 100 IU / ml IL-2. After co-culture, collect cells and label with isotype antibody control and APC-labeled anti-human CD8 antibody, and incubate at room temperature for 15 minutes. Wash cells once with saline, resuspend with 200 μl saline, and detect CD8 by FACS. + The proportion of FITC-negative cells in T lymphocytes (no PGM-DC stimulation, only CFSE-labeled T cells) is the CD8 + Proliferation ratio in T lymphocytes.

[0182] The test article induces CD8 + The proliferation ratio of T lymphocytes minus the negative control CD8 + The T lymphocyte proliferation rate should not be less than 15%.

[0183] (5) Bacterial endotoxin test

[0184] According to the 2020 edition of the Chinese Pharmacopoeia, Part IV, General Rules 1143, it should be less than 5EU / ml.

[0185] (6) Sterility test

[0186] Refer to the General Rules 1101 of Part IV of the 2020 Edition of the Chinese Pharmacopoeia, and the test sample should be sterile.

[0187] (7) Mycoplasma test

[0188] Refer to "General Chapter 3301 Mycoplasma Test Method" of the 2020 edition of the "Chinese Pharmacopoeia" and conduct a mycoplasma test, which should be negative.

[0189] 3.3 Stability study of finished product

[0190] The finished product of the preparation was selected. After the initial inspection (0 month) was completed as described in 3.2, the finished product stored in liquid nitrogen was extracted at 3 months, 6 months, 12 months, 18 months and 24 months, and all items were tested as described in 3.2. The test results are shown in Table 9: The appearance, visible foreign matter, cell survival rate, cell density, phenotype ( Figure 4 ), biological activity and other key quality parameters are within the controllable range of quality. At the same time, a stability study of the finished product of the preparation in an ultra-low temperature refrigerator (-80℃±2℃) was carried out. The finished product of the preparation was selected. After the initial inspection (0 month) was completed by the method described in 3.2, the finished product stored in the ultra-low temperature refrigerator (-80℃±2℃) was sampled in March and June, respectively. The key quality parameters were tested as described in 3.2. The test results are shown in Table 10: After the finished product of prostate cancer dendritic cell therapeutic vaccine was stored in an ultra-low temperature refrigerator (-80℃±2℃) for 6 months, the key quality parameters such as appearance, visible foreign matter, cell viability, cell density, phenotype, and biological activity were all within the controllable range of quality.

[0191] Table 9. Long-term stability study of finished prostate cancer dendritic cell therapeutic vaccine in liquid nitrogen storage

[0192]

[0193] Table 10. Storage stability study of finished prostate cancer dendritic cell therapeutic vaccine at -80℃±2℃

[0194]

[0195] Example 4 In vivo pharmacodynamic study of the effectiveness of prostate cancer dendritic cell therapeutic vaccine preparations in mice

[0196] C57BL / 6J mouse dendritic cells sensitized with 0 and 20 μg / ml PGM antigen (abbreviated as DC0 and DC20) were prepared. Three days after C57BL / 6J mice were subcutaneously inoculated with mouse prostate cancer cells (RM-1 cells) in the abdomen, C57BL / 6J mice bearing RM-1 tumor cells were immunized with sodium chloride injection (NS group), DC0, and DC20, respectively. 2×10 6 / mouse / time, once a week, subcutaneous injection, 100ml each time, three times in total. The NS group was subcutaneously injected with the same volume of normal saline. The tumor growth and survival of C57BL / 6J mice were observed.

[0197] The results showed that the growth of RM-1 tumor in the DC20 group was significantly slower than that in the NS and DC0 groups (P<0.001, see Figure 5 At the same time, the comparison of survival time showed that the OS of the DC20 group was significantly prolonged (P<0.01, see Figure 6), which conceptually verified the effectiveness of a prostate cancer dendritic cell therapeutic vaccine.

[0198] Example 5 Clinical study to evaluate the effectiveness and safety of a prostate cancer dendritic cell therapeutic vaccine formulation

[0199] Twelve patients with metastatic castration-resistant prostate cancer (mCRPC) who failed standard treatment were randomly divided into three groups, with three patients in each group. All patients voluntarily received the prostate cancer dendritic cell therapeutic vaccine transfusion treatment and met the relevant requirements for blood cell separation. The patient's leukocytes were obtained by apheresis using a blood cell separator, and the finished product of the prostate cancer dendritic cell therapeutic vaccine was prepared according to Examples 1 and 2.

[0200] All patients were divided into 3 groups according to Table 11 and given corresponding doses of prostate cancer dendritic cell therapeutic vaccine by subcutaneous injection once every 2 weeks for a total of 3 times. Safety evaluation was performed 2 weeks after the third injection and the ELISPLOT IFN-γ method was used to detect the in vitro immune response of peripheral blood PBMCs to PGM antigens before and after vaccine administration.

[0201] Table 11. Long-term stability study of finished prostate cancer dendritic cell therapeutic vaccine in liquid nitrogen storage

[0202]

[0203] Clinical results showed that after administration of the vaccine to 12 subjects, no SAE (serious adverse events) and DLT (dose-limiting toxicity) occurred during the reporting period, and no vaccine-related AEs of grade 3 or above occurred. In cohort 1, cohort 2, and cohort 3, 1, 1, and 6 subjects, respectively, showed positive immune responses to PGM antigens after administration of the vaccine, and the immune response showed a certain positive correlation with the vaccine dose.

[0204] Example 6 Application of DC preservation solution in other DC cell therapy products

[0205] The DC preservation solution configured according to the formula in Example 2 was used to freeze cells other than PGM-DC, and had the same good freezing effect. In this embodiment, the DC preservation solution was used to prepare a glioblastoma dendritic cell vaccine. For the preparation method of a glioblastoma dendritic cell vaccine, please refer to the patent authorized by the applicant (application number: 202311725263.4). The results of the long-term freezing stability study in liquid nitrogen showed that mature human dendritic cells induced and differentiated in vitro from peripheral blood mononuclear cells of glioblastoma patients frozen in DC preservation solution had good stability after 12 months of liquid nitrogen storage (Table 12).

[0206] Table 12. Long-term stability study of glioblastoma dendritic cell therapeutic vaccine preparations stored in liquid nitrogen

[0207]

[0208] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A dendritic cell therapeutic vaccine preparation, characterized in that: The preparation comprises: (a) antigen-sensitized mature human dendritic cells; and (b) DC preservation solution, with a pH of 6.3-7.3 and an osmotic pressure molar concentration of 1350-1600 mOsmol / kg; The DC preservation solution is composed of the following components: 30% to 32% v / v compound electrolyte injection, 5% to 8% w / v human albumin, 1% w / v dextran 40, 2.0625% w / v glucose, 0.14% w / v sodium chloride and 7.5% to 10% v / v DMSO, and the remainder is water for injection, calculated based on the total volume of the DC preservation solution; Wherein, the antigen-sensitized mature human dendritic cells are mature human dendritic cells sensitized with PGM antigen, the amino acid sequence of the PGM antigen is shown in SEQ ID NO: 1, and the final concentration of the PGM antigen sensitization is 50 μg / ml to 100 μg / ml; Furthermore, the antigen-sensitized mature human dendritic cells are prepared by the following steps: (S1a) providing PGM antigen for sensitizing dendritic cells; (S1b) Providing mononuclear cells from the patient's autologous peripheral blood, which are cultured in vitro into immature dendritic cells; (S1c) sensitizing the immature dendritic cells with the PGM antigen and inducing them into mature dendritic cells in vitro; (S1d) sensitizing the mature dendritic cells with the PGM antigen to obtain the antigen-sensitized mature human dendritic cells.

2. The preparation according to claim 1, characterized in that The concentration of antigen-sensitized mature human dendritic cells in the preparation is 2×10 6 / ml~2×10 7 / ml.

3. The preparation according to claim 1, characterized in that The DC preservation solution is composed of the following components: 31.25% v / v compound electrolyte injection, 5% w / v human albumin, 1% w / v dextran 40, 2.0625% w / v glucose, 0.14% w / v sodium chloride and 7.5% v / v DMSO, and the remainder is water for injection, calculated based on the total volume of the DC preservation solution.

4. The preparation according to claim 1, characterized in that The DC preservation solution is composed of the following components: 30%~32% v / v compound electrolyte injection, 5% w / v human albumin, 1% w / v dextran 40, 2.0625% w / v glucose, 0.14% w / v sodium chloride and 7.5% v / v DMSO, and the remainder is water for injection, calculated based on the total volume of the DC preservation solution.

5. A method for preparing the dendritic cell therapeutic vaccine preparation according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: (S1) provide antigen-sensitized mature human dendritic cells; (S2) resuspending the antigen-sensitized mature human dendritic cells in a DC preservation solution to obtain the dendritic cell therapeutic vaccine preparation; Wherein, the antigen-sensitized mature human dendritic cells are prepared by the following steps: (S1a) providing PGM antigen for sensitizing dendritic cells; (S1b) Providing mononuclear cells from the patient's autologous peripheral blood, which are cultured in vitro into immature dendritic cells; (S1c) sensitizing the immature dendritic cells with the PGM antigen and inducing them into mature dendritic cells in vitro; (S1d) sensitizing the mature dendritic cells with the PGM antigen to obtain the antigen-sensitized mature human dendritic cells.

6. The method according to claim 5, characterized in that In step (S1c), the mature dendritic cells are obtained by adding 5-15 μg / ml PGE2, 500-1500 IU / ml TNFα and 1-10 μg / ml CD40L to the culture system of immature dendritic cells and continuing to culture for 16 to 60 hours.

7. Use of the dendritic cell therapeutic vaccine preparation according to any one of claims 1 to 4 in the preparation of a medicament for treating prostate cancer.

8. Use of a cell preservation solution in preserving dendritic cells, wherein: The pH of the cell preservation solution is 6.3-7.3, and the osmotic pressure molar concentration is 1350-1600 mOsmol / kg; and the cell preservation solution is composed of the following components: 30%-32% v / v compound electrolyte injection, 5%-8% w / v human albumin, 1% w / v dextran 40, 2.0625% w / v glucose, 0.14% w / v sodium chloride and 7.5%-10% v / v DMSO, and water for injection, based on the total volume of the cell preservation solution.

9. The use according to claim 8, characterized in that The pH of the cell preservation solution is 6.3-7.3, and the osmotic pressure molar concentration is 1350-1600 mOsmol / kg; and the cell preservation solution is composed of the following components: 30%-32% v / v compound electrolyte injection, 5% w / v human albumin, 1% w / v dextran 40, 2.0625% w / v glucose, 0.14% w / v sodium chloride and 7.5% v / v DMSO, and water for injection, based on the total volume of the cell preservation solution.

10. A composition, characterized in that The composition comprises: (i) the dendritic cell therapeutic vaccine preparation according to any one of claims 1 to 4; and (ii) a pharmaceutically acceptable carrier.

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