Kit containing tumor neoantigen nanoformulation and dendritic cell vaccine and use thereof

Abstract

The present disclosure relates to a kit, which includes a tumor neoantigen nanoformulation and a dendritic cell vaccine. The kit disclosed in the present disclosure can significantly improve the uptake and presentation of antigens by antigen-presenting cells, activate and enhance the body's anti-tumor immune response, and effectively inhibit tumor progression. The combined therapy of tumor neoantigen nanoformulation and dendritic cell vaccine provides a new idea for personalized and precise treatment of cancer, which has clinical practical significance.

Description

Technical Field

[0001] The present disclosure belongs to the field of biomedicine, and more specifically, to a kit comprising a tumor neoantigen nanoformulation and a dendritic cell vaccine and uses thereof. Background Art

[0002] Data show that malignant tumors are one of the major diseases that endanger human health and seriously affect the quality of life of patients. In clinical practice, although surgical treatment, radiotherapy and chemotherapy can alleviate the progression of malignant tumors in the short term, the individual prognosis of patients varies greatly, and some patients still experience recurrence or metastasis. Cancer immunotherapy is another important anti-tumor method after the above three treatment strategies. Cancer immunotherapy activates the patient's own anti-tumor immune response to effectively kill tumor cells, and has shown significant therapeutic effects in clinical practice. At the same time, immunotherapy can induce a lasting immune memory effect in the patient's body, which can effectively inhibit the recurrence and metastasis of tumors.

[0003] Tumor neoantigens are abnormal proteins with specific amino acid site mutations produced by "non-synonymous gene mutations" in tumor cells. They are not expressed in normal cells and are specific targets that can be recognized by the body's immune system. By performing high-throughput sequencing on the patient's tumor genome, the patient's specific mutation site is screened out, and then an mRNA or protein peptide neoantigen vaccine targeting the site is prepared to achieve personalized immunity for tumor patients. However, neoantigen peptide drugs have poor stability, fast metabolic clearance rate in the body, lack of targeting in the body distribution, and low uptake by antigen-presenting cells. These characteristics limit their application and anti-tumor efficacy.

[0004] Dendritic cell vaccine is a cellular immunotherapy method with wide application potential. Under the stimulation of tumor-associated antigens or tumor-specific antigens, mature dendritic cells can take up and cross-present tumor antigens, activate cytotoxic T cell (CTLs) immune response and kill tumor cells. The proportion of dendritic cells in peripheral blood and tumor tissue is very low, so in vitro engineering of dendritic cell vaccines and adoptive therapy of patients can enhance the immune response of CTLs and improve the effect of immunotherapy. Summary of the invention

[0005] An object of the present disclosure is to provide a kit comprising a tumor neoantigen nanoformulation and a dendritic cell vaccine.

[0006] Another object of the present disclosure is to provide use of the kit in preparing a drug for preventing or treating malignant tumors.

[0007] Another object of the present disclosure is to provide use of a combination of a tumor neoantigen nanoformulation and a dendritic cell vaccine in the preparation of a medicament for preventing or treating malignant tumors.

[0008] According to one aspect of the present disclosure, a kit is provided, which comprises a tumor neoantigen nanoformulation and a dendritic cell vaccine.

[0009] According to another aspect of the present disclosure, there is provided use of the kit in preparing a medicament for preventing or treating malignant tumors.

[0010] According to another aspect of the present disclosure, there is provided use of a combination of a tumor neoantigen nanoformulation and a dendritic cell vaccine in the preparation of a medicament for preventing or treating malignant tumors.

[0011] Beneficial Effects

[0012] The kit disclosed in the present invention can overcome the shortcomings of existing tumor neoantigen vaccines and dendritic cell vaccines, and can be effectively used for personalized treatment of malignant tumors. The combination therapy of tumor neoantigen nanoformulation and dendritic cell vaccine, which prepares tumor neoantigen into nanoformulation and then combines it with engineered dendritic cell vaccine, can effectively improve the body's anti-tumor immune response, and has significant effects in various types of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 These are transmission electron micrographs of tumor neoantigen liposomes and PLGA nanoparticles. A and B in the figure are transmission electron micrographs of liposomes and PLGA nanoparticles, respectively.

[0014] Figure 2 This is a particle size characterization diagram of tumor neoantigen PLGA nanoformulation.

[0015] Figure 3 The particle size and monodispersity of tumor neoantigen PLGA nanoparticles change over time.

[0016] Figure 4 This is a flow cytometric graph of the uptake of tumor neoantigen PLGA nanoformulation by DC2.4 cells.

[0017] Figure 5 The results are the flow cytometry results of tumor neoantigen PLGA nanoparticles inducing the maturation of bone marrow-derived dendritic cells (BMDCs) and the expression of CD80 and CD86 molecules. BMDCs treated with PBS were used as a control.

[0018] Figure 6 The distribution of tumor neoantigen PLGA nanoparticles in the abdominal lymph nodes of mice 24 hours after subcutaneous injection; PLGA nanoparticles encapsulate the fluorescent probe DiR as an imaging fluorescent molecule.

[0019] Figure 7The figure shows the distribution of engineered dendritic cell vaccine and tumor neoantigen PLGA nanoparticles in the lymph nodes of the mouse model after subcutaneous injection. The dendritic cell vaccine was labeled with Cy5, and the mouse lymph nodes were frozen and sectioned using laser confocal microscopy. A and B in the figure show the in vitro distribution of PLGA nanoparticles in mouse lymph nodes and the distribution of dendritic cell vaccine in mouse lymph node sections, respectively.

[0020] Figure 8 These are the experimental results of tumor neoantigen PLGA nanoparticles combined with engineered dendritic cell vaccines, which induced acquired T cell immune responses in a mouse model.

[0021] Fig. 9 It is a dendritic cell vaccine prepared by combining tumor neoantigen PLGA nanoparticles with engineered materials, which induces the secretion of tumor necrosis factor α (TNF-α) and interferon γ (IFN-γ) in a mouse model.

[0022] Fig.10 This is a pharmacodynamic evaluation of a tumor neoantigen PLGA nanoparticle-engineered dendritic cell vaccine in a B16-OVA melanoma C57BL / 6 tumor-bearing mouse model.

[0023] Fig.11 This is a pharmacodynamic evaluation of a tumor neoantigen PLGA nanoparticle-engineered dendritic cell vaccine in a 4T1 breast cancer BALB / C tumor-bearing mouse model.

[0024] Fig.12 It is a schematic diagram of the combined use of the tumor neoantigen nanoformulation and dendritic cell vaccine disclosed in the present invention. DETAILED DESCRIPTION

[0025] In order to enable persons with ordinary knowledge in the art to understand the characteristics and effects of the present invention, the following is a general description and definition of the terms and expressions mentioned in the specification and the scope of the patent application. Unless otherwise specified, all technical and scientific words used in the text have the common meanings understood by those skilled in the art for the present invention. In case of conflict, the definitions in this specification shall prevail.

[0026] As used herein, the terms "include", "comprising", "having", "containing" or any other similar terms are open-ended transitional phrases, which are intended to cover non-exclusive inclusions. For example, a composition or article containing multiple elements is not limited to the elements listed herein, but may also include other elements that are not explicitly listed but are generally inherent to the composition or article. In addition, unless otherwise expressly stated, the term "or" refers to an inclusive "or" rather than an exclusive "or". For example, any of the following situations satisfies the condition "A or B": A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist). In addition, as used herein, the terms "include", "comprising", "having", and "containing" should be interpreted as having been specifically disclosed and simultaneously covering closed or semi-closed transitional phrases such as "consisting of" and "consisting essentially of".

[0027] In this article, all features or conditions defined in the form of numerical ranges or percentage ranges are only for brevity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be deemed to have covered and specifically disclosed all possible secondary ranges and individual values ​​within the range, especially integer values. For example, the range description of "1 to 8" should be deemed to have specifically disclosed all secondary ranges such as 1 to 7, 2 to 8, 2 to 6, 3 to 6, 4 to 8, 3 to 8, etc., especially secondary ranges defined by all integer values, and should be deemed to have specifically disclosed individual values ​​such as 1, 2, 3, 4, 5, 6, 7, 8, etc. within the range. Unless otherwise specified, the above interpretation method applies to all contents of the entire present invention, regardless of whether the range is broad or not.

[0028] If the quantity or other numerical value or parameter is expressed as a range, a preferred range or a series of upper and lower limits, it should be understood that all ranges consisting of any upper limit or preferred value of the range and the lower limit or preferred value of the range have been specifically disclosed herein, regardless of whether these ranges are disclosed separately. In addition, if a numerical range is mentioned herein, unless otherwise specified, the range should include its endpoints and all integers and fractions within the range.

[0029] In this document, under the premise of achieving the purpose of the invention, numerical values ​​should be understood to have the accuracy of the number of significant digits of the numerical value. For example, the number 40.0 should be understood to cover the range from 39.50 to 40.49.

[0030] In this article, for the use of Markush groups or optional terms to describe the features or examples of the present invention, those skilled in the art should understand that all subgroups of elements in the Markush group or option list or any individual element can also be used to describe the present invention. For example, if X is described as "selected from the group consisting of X1, X2 and X3", it also means that the claim that X is X1 and the claim that X is X1 and / or X2 have been fully described. Furthermore, for the use of Markush groups or optional terms to describe the features or examples of the present invention, those skilled in the art should understand that any combination of all subgroups of elements in the Markush group or option list or individual elements can also be used to describe the present invention. Accordingly, for example, if X is described as "selected from the group consisting of X1, X2 and X3", and Y is described as "selected from the group consisting of Y1, Y2 and Y3", it means that the claim that X is X1 or X2 or X3 and Y is Y1 or Y2 or Y3 has been fully described.

[0031] The following detailed description is merely illustrative in nature and is not intended to limit the present invention and its use. In addition, this document is not limited by any theory described in the above prior art or invention summary or the following detailed description or examples.

[0032] According to one embodiment of the present disclosure, a kit is provided, which comprises a tumor neoantigen nanoformulation and a dendritic cell vaccine as active ingredients.

[0033] According to one embodiment of the present disclosure, the tumor neoantigen nanoformulation refers to a nanoformulation prepared by mixing tumor neoantigens and excipients, which may include liposomes encapsulating tumor neoantigen messenger ribonucleic acid (mRNA) or tumor neoantigen protein polypeptides, polylactic acid-glycolic acid copolymer (PLGA) nanoparticles, polylactic acid copolymer (PLA) nanoparticles and polycaprolactone copolymer (PCL) nanoparticles.

[0034] According to one embodiment of the present disclosure, the tumor neoantigen nanoformulation is selected from tumor neoantigen liposomes and tumor neoantigen PLGA / PLA / PCL nanoparticles.

[0035] In the present invention, the tumor neoantigen (Neoantigen) refers to a neoantigen encoded by a mutated gene of a tumor cell, which is mainly an abnormal gene, polypeptide or protein that is different from the gene, polypeptide or protein expressed by a normal cell, produced by a gene point mutation, a deletion mutation, a gene fusion, etc. For example, the hydrophilic antigen peptide OVA (SIINFEKL), the hydrophobic antigen peptide B-M27 (LCPGNKYGM), B-M30 (DWENVSPELNSTDQ), 4-M27 (FAICFSCLLAHALNLIKLVRGRKPLSW), 4-M32 (SHRSCSHQTSAPSPKALAHNGTPRNAI) used in the examples.

[0036] According to one embodiment of the present disclosure, the tumor neoantigen liposome is prepared by the following method: the tumor neoantigen and the excipients (for example, using nanotechnology and high shear conditions) are made into a nanoformulation. The distribution range of its hydrated average particle size can be 50 to 700 nm. In one embodiment, the tumor neoantigen nanoformulation is a liposome made by using hydrogenated soybean phospholipids (HSPC), cholesterol (Chol), and polyethylene glycol distearoylphosphatidylethanolamine (DSPE-PEG) as excipients. In another embodiment, the tumor neoantigen nanoformulation is a nanoparticle made by using polylactic acid-glycolic acid copolymer (PLGA) and polyvinyl alcohol (PVA) as excipients. In another embodiment, the tumor neoantigen nanoformulation is a nanoparticle made by using polylactic acid copolymer (PLA) and polyvinyl alcohol (PVA) as excipients. In another embodiment, the tumor neoantigen nanoformulation is a nanoparticle made by using polylactic acid copolymer (PLA) and polyvinyl alcohol (PVA) as excipients. In yet another embodiment, the tumor neoantigen nanoformulation is a nanoparticle made by using polycaprolactone copolymer (PCL) and polyvinyl alcohol (PVA) as excipients.

[0037] According to one embodiment of the present disclosure, the tumor neoantigen liposome is prepared by the following method:

[0038] (1) Accurately weigh HSPC, Chol and DSPE-PEG in a mass ratio of HSPC:Chol:DSPE-PEG=(5-6):(0.5-3):(1-3) and dissolve them in an organic phase; accurately weigh a hydrophobic tumor neoantigen polypeptide in a mass ratio of 1:(25-5) to HSPC and dissolve them in an organic phase; fully mix the organic phase of the tumor neoantigen polypeptide and the organic phase of the lipid;

[0039] (2) removing the organic solvent by rotary evaporation under reduced pressure at 35-45°C for 2-3 hours to form a film; dissolving the hydrophilic tumor neoantigen polypeptide or mRNA in the aqueous phase, and the mass ratio of the hydrophilic tumor neoantigen to HSPC is 1:(25-5); adding the aqueous phase containing the hydrophilic tumor neoantigen to the film-forming lipids, and hydrating at 30-90°C for 1 hour; then treating the obtained liposomes under high pressure or high shear conditions to obtain the final tumor neoantigen liposome nanoformulation.

[0040] According to one embodiment of the present disclosure, the tumor neoantigen PLGA / PLA / PCL nanoparticles are prepared by the following method:

[0041] (1) dissolving a water-soluble tumor neoantigen in sterile water at a final concentration of 0.1-10 mg / mL, and slowly adding the solution dropwise to the organic phase of PLGA / PLA / PCL under high pressure or high shear conditions, wherein the final concentration of PLGA / PLA / PCL is 0.5-5 mg / mL, and the mixing ratio of the aqueous phase to the organic phase is 1:(100-10);

[0042] (2) dissolving the hydrophobic tumor neoantigen in the organic phase of PLGA / PLA / PCL obtained in step (1) at a final concentration of 0.1-10 mg / mL, stirring thoroughly and preparing it uniformly under high pressure or high shear conditions;

[0043] (3) slowly adding the solution obtained in step (2) dropwise to a 10-fold volume of a sterile aqueous solution of PVA under high pressure or high shear conditions, with a final concentration of PVA of 0.2-2 mg / mL;

[0044] (4) The PLGA / PLA / PCL nanoparticles loaded with tumor neoantigens obtained in step (3) are dialyzed with flowing sterile deionized water for 12 hours, and then homogenized with high pressure or high shear force to obtain tumor neoantigen nanoformulations PLGA / PLA / PCL nanoparticles.

[0045] According to one embodiment of the present disclosure, the organic phase is one or more of ethanol, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, ethyl acetate, dichloromethane, chloroform, and acetone.

[0046] According to one embodiment of the present disclosure, the high pressure and high shear force conditions are: homogenization is performed under instrument conditions such as a high pressure homogenizer, an ultrasonic disruptor, a high shear mixer, or a high shear agitator.

[0047] According to one embodiment of the present disclosure, the dendritic cell vaccine is prepared by the following method: taking bone marrow-derived, spleen-derived or peripheral blood-derived monocytes, adding granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) to promote their differentiation, and purifying (for example, using magnetic bead sorting or flow sorting) to obtain the dendritic cell vaccine.

[0048] According to one embodiment of the present disclosure, the dendritic cell vaccine is prepared by the following method: after the patient's autologous tumor cell lysate is co-cultured in a serum-containing cell culture medium for 6 to 24 hours, lipopolysaccharide LPS at a concentration of 500 to 5000 ng / mL is added and cultured for 6 to 10 hours, and then the suspended dendritic cells are aspirated, centrifuged at 300g for 0.5 hours at 4 to 10°C, and redispersed in physiological saline to a cell suspension of 1 million to 10 million / mL, thereby obtaining an engineered dendritic cell vaccine. The patient's autologous tumor cell lysate is derived from the patient's own body, and after digestion and dispersion into a single cell suspension, it is treated with a hypochlorous acid solution for 0.5 to 4 hours, the hypochlorous acid concentration is 50 to 500 μM, and the co-culture number ratio with the dendritic cells is 1:10 to 1:1.

[0049] According to one embodiment of the present disclosure, the dendritic cell vaccine is prepared by the following method:

[0050] (1) After the tumor tissue was prepared into a single cell suspension by a tissue grinder, the cells were counted, centrifuged at 300-500 g for 10-30 minutes at 4°C, the supernatant was removed, and the suspension was redispersed into 1×10 cells using a 50-500 μM hypochlorous acid solution. 7 cells / mL and incubate for 0.5-2 hours;

[0051] (2) The obtained cell suspension was centrifuged at 500-1500 g for 0.5-1 hour at 4°C, the supernatant was removed, the cell suspension was washed and redispersed with serum-free cell culture medium, and the suspension was repeatedly frozen and thawed 3-5 times at 20 to -80°C for later use;

[0052] (3) The patient's own blood was collected and mononuclear cells were isolated using lymphocyte separation fluid and gradient density centrifugation. The mononuclear cells were dispersed into 1×10 6 -1×10 7 / mL and added to the cell culture plate for 8-24 hours; then the suspended cells and supernatant culture medium in each well were aspirated and discarded, and complete cell culture medium with a final concentration of GM-CSF and IL-4 of 10-100ng / mL was added and cultured for 2-3 days in a 37°C cell culture incubator with a carbon dioxide concentration of 5%; then half the volume of the culture medium in the well was replaced and cultured for another 2 days; on the 5th day, the suspended cells in the well were counted and the ratio of the number of tumor cells to the number of dendritic cells was calculated. For example, autologous tumor cell lysate is added at a concentration of 1:10-1:1 for co-culture; on the 6th day, lipopolysaccharide LPS at a concentration of 500-5000 ng / mL and IFN-γ at a concentration of 20-200 ng / mL are added to the wells and culture is continued for 4-16 hours; then the suspended dendritic cells are aspirated, centrifuged at 100-500g for 3-30 minutes at 4°C-10°C, and re-dispersed in physiological saline to a cell suspension of 1 million-10 million / mL to obtain a dendritic cell vaccine.

[0053] According to another embodiment of the present disclosure, there is provided use of the kit in preparing a drug for preventing or treating malignant tumors.

[0054] According to another embodiment of the present disclosure, the malignant tumors include: lung cancer, sarcoma, malignant melanoma, brain cancer, pancreatic cancer, liver cancer, breast cancer, bladder cancer, prostate cancer, gastric cancer, ovarian cancer, colorectal cancer, kidney cancer, esophageal cancer, neuroblastoma, cervical cancer, nasopharyngeal carcinoma, leukemia, multiple myeloma and lymphoma, and metastatic tumors caused by the above tumors.

[0055] Example

[0056] The present disclosure will be further described in detail below by way of examples. The sources of the raw materials used in the examples of the present disclosure are as follows:

[0057] Hydrogenated soybean phosphatidylcholine (HSPC), cholesterol (Chol), and PEGylated distearoylphosphatidylethanolamine (DSPE-PEG) were purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.; dimethylformamide solution and dichloromethane were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai); tumor neoantigen peptides OVA (GTA32313-0918), B-M27 (GT30732-1-0906), B-M30 (GT30732-2-0906), 4-M27 (GT30732-3-0906), and 4-M32 (GT30732-4-0906) were purchased from Hefei Guopeptide Technology Co., Ltd.; polyvinyl alcohol (PVA) was purchased from Aladdin Technology (China) Co., Ltd.; 1640 culture medium and fetal bovine blood FBS was purchased from Gibco, Canada; granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), lipopolysaccharide (LPS) and interferon-γ (IFN-γ) were purchased from Peprotech, USA; polylactic-co-glycolic acid (PLGA) and phosphate buffered saline (PBS) were purchased from Dalian Meilun Biotechnology Co., Ltd.; TNF-α, Anti-CD11C-FITC, Anti-CD86-PerCp-Cy5.5, Anti-CD80-PE, Anti-CD3-PerCp-Cy5.5, Anti-CD8-PE, Anti-CD4-FITC, and IFN-γ were purchased from eBioscience, UK.

[0058] BF16-OVA tumor cells and 4T1 tumor cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0059] BALB / C mice: 4-6 weeks old, 18-22 g, were purchased from Shanghai Slack Laboratory Animal Co., Ltd. The feeding conditions and experimental procedures were strictly in accordance with the relevant requirements and standards of the Ethics Committee for the Management and Use of Laboratory Animals of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

[0060] Rotary evaporator (Heidlph, Germany); high pressure homogenizer (Microfluidizer, USA); transmission electron microscope (JEOL JEM-1230, Japan); laser particle size analyzer (ZEN3690, Malven, USA); desktop centrifuge (Eppendorf); carbon dioxide incubator (Thermo, USA); flow cytometer (BD, Calibur, USA);

[0061] Cell clean bench (BJ-1CD, Shanghai Boxun Industrial Co., Ltd.).

[0062] Example 1: Preparation and characterization of tumor neoantigen liposomes

[0063] Hydrogenated soybean phosphatidylcholine (HSPC), cholesterol (Chol) and polyethylene glycol distearoylphosphatidylethanolamine (DSPE-PEG) were accurately weighed, and the mass ratio was HSPC:Chol:DSPE-PEG=5:3:2 and dissolved in dichloromethane; hydrophobic tumor neoantigen polypeptides B-M27 and B-M30 (the mass ratio of B-M27 and B-M30 was 1:1) were accurately weighed, and the mass ratio of them to HSPC was 1:25, and dissolved in dichloromethane; the organic phase of the tumor neoantigen polypeptide and the organic phase of the lipid were fully mixed and uniformly mixed. The organic solvent was removed by rotary evaporation under reduced pressure at 40°C for 3 hours to form a film; the hydrophilic tumor neoantigen polypeptide OVA or mRNA was dissolved in the aqueous phase, and the mass ratio of them to HSPC was 1:25; the aqueous phase dissolved with hydrophilic tumor neoantigen was added to the film-forming lipids, and hydrated at 50°C for 1 hour; the obtained liposomes were then treated with a high-pressure homogenizer to obtain the final tumor neoantigen liposome nanoformulation.

[0064] In addition to using 4-M27 and 4-M32 (whose mass ratio is also 1:1) instead of the hydrophobic tumor neoantigen polypeptides B-M27 and B-M30, another tumor neoantigen liposome nanoformulation was prepared using the same method as above.

[0065] Figure 1 A is a transmission electron microscopic photograph of the tumor neoantigen liposomes containing B-M27 and B-M30 obtained by observation using a transmission electron microscope.

[0066] Example 2: Preparation and characterization of tumor neoantigen PLGA nanoparticles

[0067] The water-soluble tumor neoantigen (OVA) was dissolved in sterile water at a final concentration of 10 mg / mL, and 1 mL was slowly added to 10 mL of poly(lactic-co-glycolic acid) (PLGA) N,N-dimethylformamide solution under ultrasonic conditions, and the final concentration of PLGA was 5 mg / mL. The hydrophobic tumor neoantigens B-M27 and B-M30 (the mass ratio of B-M27 and B-M30 was 1:1) were further dissolved in the above-prepared PLGA N,N-dimethylformamide solution at a final concentration of 1 mg / mL, and stirred and mixed evenly. Take 1 mL of the PLGA solution containing tumor neoantigens and slowly add it to 10 mL of a sterile aqueous solution of polyvinyl alcohol PVA under ultrasonic conditions, and the final concentration of PVA was 2 mg / mL. The obtained PLGA nanoparticles loaded with tumor neoantigens were dialyzed with flowing sterile deionized water for 12 hours, and then homogenized with a high-pressure homogenizer to obtain tumor neoantigen nanoformulation PLGA nanoparticles (NP-Ag(B-M27 / B-M30)).

[0068] In addition to using 4-M27 and 4-M32 (whose mass ratio is also 1:1) instead of the hydrophobic tumor neoantigen polypeptides B-M27 and B-M30, the same method was used to prepare another tumor neoantigen nanoformulation PLGA nanoparticles, namely, NP-Ag (4-M27 / 4-M32).

[0069] Except for not adding hydrophobic tumor neoantigens B-M27 and B-M30, another tumor neoantigen nanoformulation PLGA nanoparticles, namely, NP-Ag(OVA), was prepared by the same method as above.

[0070] Figure 1 B is a transmission electron microscope photo of NP-Ag (B-M27 / B-M30) obtained by observing the obtained PLGA nanoparticles using a transmission electron microscope. PLGA is a spherical nanoparticle. The particle size of NP-Ag (B-M27 / B-M30) was measured using a laser particle size analyzer, and the particle size was about 240nm. The results are shown in Figure 2 .

[0071] Example 3: Preparation of a kit containing tumor neoantigen nanoformulation (NP-Ag) and dendritic cell vaccine (DCV)

[0072] The kit, namely DCV-NP-Ag, is obtained by mixing equal volumes of the prepared NP-Ag (OVA) or NP-Ag (B-M27 / B-M30) and DCV.

[0073] The same method as above was used to prepare additional kits, except that 4-M27 and 4-M32 were used instead of the hydrophobic tumor neoantigen polypeptides B-M27 and B-M30.

[0074] Example 4: Study on the uptake of tumor neoantigen PLGA nanoformulation by DC2.4 cells

[0075] FITC-labeled OVA (SIINFEKL) was used as a model peptide. FITC ) were encapsulated in PLGA nanoparticles, and NP-Ag(OVA FITC ) and PBS were co-incubated with DC2.4 cells for 24 h, and the uptake of PLGA nanoparticles by DC2.4 cells was analyzed by flow cytometry (FCM). Figure 4 shown.

[0076] Example 5: Engineering preparation of dendritic cell vaccines

[0077] Mononuclear cells were isolated from the tibia and humerus of mice; the mononuclear cells were dispersed into 2 × 10 cells / mL using complete medium containing 10% serum. 6 / mL and added to a 12-well cell culture plate at 1mL / well for 12 hours; then the suspended cells and supernatant culture medium in each well were aspirated and discarded, and complete cell culture medium with a final concentration of 10-100ng / mL of granulocyte-macrophage colony stimulating factor GM-CSF and IL-4 was added thereto and cultured for 2-3 days, and the culture conditions were a 37°C cell culture incubator with a carbon dioxide concentration of 5%; then half the volume of the culture medium in the well was replaced and cultured for another 2 days; on the 5th day, the suspended cells in the well were counted, and autologous tumor cell lysate was added for co-culture at a ratio of 1:1 between the number of tumor cells and the number of dendritic cells; on the 6th day, lipopolysaccharide LPS at a concentration of 1000ng / mL and IFN-γ at a concentration of 100ng / mL were added to the well and cultured for another 8 hours. The suspended dendritic cells were then aspirated, centrifuged at 300 g for 0.5 hours at 4°C, and redispersed in physiological saline to form a cell suspension of 1 million to 10 million / mL, thereby obtaining an engineered dendritic cell vaccine.

[0078] Example 6: Quantitative study on the maturation of bone marrow-derived dendritic cells (BMDCs) induced by tumor neoantigen PLGA nanoparticles

[0079] Mononuclear cells were isolated from the tibia and humerus of mice; the mononuclear cells were dispersed into 2 × 10 cells / mL using complete medium containing 10% serum. 6 And add 1mL / well into 12-well cell culture plate to adhere to the wall for 12 hours; then discard the suspended cells and supernatant culture medium in each well, add GM-CSF and IL-4 with a final concentration of 10-20ng / mL complete cell culture medium for 2-3 days, the culture condition is 37℃ cell culture incubator, 5% carbon dioxide concentration; then replace half volume of culture medium in the well and continue to culture for 2 days; on the 5th day, count the suspended cells in the well and re-plate at 1 million per well, add phosphate buffered saline (PBS), tumor neoantigen nanoformulation (NP-Ag (OVA)) and BMDC cells for 24 hours, and use flow cytometry to detect the degree of BMDC maturation. The results are as follows Figure 5 As shown, NP-Ag(OVA) enhanced the maturation of BMDCs by 15%-20% compared with PBS.

[0080] Example 7: Evaluation of the distribution of tumor neoantigen PLGA nanoparticles and engineered dendritic cell vaccines in mouse lymph nodes

[0081] Using DiR as a fluorescent molecule, the fluorescent probe DiR was encapsulated in PLGA (OVA) nanoparticles. After subcutaneous injection for 24 hours, the mice were imaged and analyzed using the small animal in vivo optical imaging system (IVIS). Figure 6 shown.

[0082] Using cell metabolism and click chemistry techniques, dendritic cell vaccine (DCV) was labeled with fluorescent probe Cy5 (DCV-Cy5). After subcutaneous injection, the distribution of DCV in mice was monitored using the in vivo optical imaging system (IVIS), and the infiltration of DCV in isolated lymph nodes was detected using confocal fluorescence microscopy (CLSM). The results are as follows: Figure 7 shown.

[0083] Example 8: Study on the effect of tumor neoantigen PLGA nanoparticles and engineered dendritic cell vaccine on inducing acquired T cell immune response in mice

[0084] BALB / C mice were immunized with PBS and DCV-NP-Ag (OVA) (dendritic cell vaccine-tumor neoantigen nanoformulation) on the 1st and 5th days. Blood, lymph nodes or spleen of mice were collected on the 8th to 10th days, and then stained with Anti-CD3-PerCp-Cy5.5, Anti-CD8-PE, and Anti-CD4-FITC. The acquired T cell immune response in mice was detected by flow cytometry. (OVA) The results are as follows: Figure 8 , as shown in 9.

[0085] Example 9: Study on the combined effect of tumor neoantigen PLGA nanoparticles and engineered dendritic cell vaccine on the growth of melanoma and breast cancer

[0086] On days 1 and 5, C57BL / 6 mice were immunized with PBS, tumor neoantigens (Ag, B-M27 and B-M30), NP-Ag (B-M27 / B-M30), DCV, and DCV-NP-Ag (B-M27 / B-M30), respectively (5-10 mice per group). On day 10, B16-OVA tumor cells (1.0×10 6 -2.0×10 6 When the tumor volume is about 100-200mm 3 The mice were monitored for tumors every two days.

[0087] The results of the reagents involving B-M27 and B-M30 hydrophobic tumor neoantigen peptides are as follows Fig.10As shown. The experimental results show that DCV-NP-Ag (B-M27 / B-M30) can significantly prevent the formation of melanoma in mice. However, DCV and NP-Ag (B-M27 / B-M30) alone have no significant effect on preventing the formation of melanoma in mice. This shows that the combination of tumor neoantigen nanoformulation and dendritic cell vaccine achieves a synergistic effect.

[0088] BALB / C mice were inoculated with 4T1 tumor cells (1.0×10 6 -2.0×10 6 cells / mouse), and wait until the tumor volume grows to about 100-200mm 3 At the same time, mice were immunized with PBS, NP-Ag (4-M27 / 4-M32), DCV, and DCV-NP-Ag (4-M27 / 4-M32) (5-10 mice per group) and tumor growth was monitored every two days. The results of the reagents involving 4-M27 and 4-M32 hydrophobic tumor neoantigen peptides are shown in Figure 2. Fig.11 The study showed that DCV-NP-Ag (4-M27 / 4-M32) could significantly inhibit the growth of breast cancer in mice compared with other groups.

Claims

1. A kit comprising a tumor neoantigen nanoformulation and a dendritic cell vaccine, in, The tumor neoantigen nanoparticle preparation is tumor neoantigen PLGA nanoparticles. Wherein, the tumor neoantigen PLGA nanoparticles are prepared by the following method: (1) Dissolve the water-soluble tumor neoantigen in sterile water at a final concentration of 0.1-10 mg / mL, and slowly add it dropwise to the organic phase solution of PLGA under high pressure or high shear conditions. The final concentration of PLGA is 0.5-5 mg / mL, and the mixing ratio of the aqueous phase to the organic phase is 1:(100-10); (2) dissolving the hydrophobic tumor neoantigen in the organic phase of PLGA obtained in step (1) at a final concentration of 0.1-10 mg / mL, stirring thoroughly and preparing it uniformly under high pressure or high shear conditions; (3) slowly adding the solution obtained in step (2) dropwise into a 10-fold volume of a sterile aqueous solution of PVA under high pressure or high shear conditions, with a final concentration of PVA of 0.2-2 mg / mL; (4) dialyzing the PLGA nanoparticles loaded with tumor neoantigens obtained in step (3) with flowing sterile deionized water for 12 hours, and then homogenizing with high pressure or high shear force to obtain tumor neoantigen nanoformulation PLGA nanoparticles. Wherein, the water-soluble tumor neoantigen is the hydrophilic antigen peptide OVA with the sequence SIINFEKL, and the hydrophobic tumor neoantigen is the hydrophobic antigen peptide B-M27 with the sequence LCPGNKYGM and the hydrophobic antigen peptide B-M30 with the sequence DWENVSPELNSTDQ, or The water-soluble tumor neoantigen is a hydrophilic antigen peptide OVA with a sequence of SIINFEKL, and the hydrophobic tumor neoantigen is a hydrophobic antigen peptide 4-M27 with a sequence of FAICFSCLLAHALNLIKLVRGRKPLSW and a hydrophobic antigen peptide 4-M32 with a sequence of SHRSCSHQTSAPSPKALAHNGTPRNAI.

2. The kit according to claim 1, wherein The dendritic cell vaccine is prepared by the following method: Monocytes derived from bone marrow, spleen or peripheral blood are obtained, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) are added to promote their differentiation, and then they are sorted and purified using magnetic bead sorting or flow cytometry to obtain dendritic cell vaccines.

3. The kit according to claim 1, wherein The dendritic cell vaccine is prepared by the following method: (1) After the tumor tissue is prepared into a single cell suspension by a tissue grinder, the cells are counted and centrifuged at 300-500 g for 10-30 minutes at 4°C to remove the supernatant. The suspension is then redispersed into a single cell suspension of 1×10 using a 50-500 μM hypochlorous acid solution. 7 cells / mL and incubate for 0.5-2 hours; (2) The obtained cell suspension was centrifuged at 500-1500 g for 0.5-1 hour at 4°C to remove the supernatant, washed and redispersed with serum-free cell culture medium, and repeatedly frozen and thawed 3-5 times at 20°C to -80°C for use; (3) Obtain the patient's own blood and separate mononuclear cells using lymphocyte separation fluid and gradient density centrifugation; The mononuclear cells were dispersed into 1×10 cells using complete medium containing 10% serum. 6 -1×10 7 / mL and added to the cell culture plate to adhere to the wall for 8-24 hours; then, the suspended cells and supernatant culture medium in each well were aspirated and discarded, and complete cell culture medium with a final concentration of GM-CSF and IL-4 of 10-100 ng / mL was added to the plate for 2-3 days, and the culture conditions were a 37°C cell culture incubator with a carbon dioxide concentration of 5%; then, half of the culture medium in the well was replaced and cultured for another 2 days; on the 5th day, the suspended cells in the well were counted, and autologous tumor cell lysate was added to the well at a ratio of 1:10-1:1 for the number of tumor cells to the number of dendritic cells for co-culture; on the 6th day, lipopolysaccharide LPS at a concentration of 500-5000 ng / mL and IFN-γ at a concentration of 20-200 ng / mL were added to the well and cultured for another 4-16 hours; then, the suspended dendritic cells were aspirated and incubated at 4°C-10°C for 100-500 The cells were centrifuged at 4000 × g for 3-30 minutes and redispersed in physiological saline to form a cell suspension of 1 million to 10 million / mL to obtain a dendritic cell vaccine.

4. The kit according to any one of claims 1 to 3, wherein The organic phase is one or more of ethanol, acetonitrile, N, N-dimethylformamide, N, N-dimethylacetamide, ethyl acetate, dichloromethane, chloroform, and acetone.

5. The kit according to any one of claims 1 to 3, wherein The high pressure and high shear force conditions are: homogenization is performed under the conditions of a high pressure homogenizer, an ultrasonic crusher, a high shear mixer, or a high shear stirrer.

6. Use of the kit according to claim 1 in the preparation of a medicament for preventing or treating malignant melanoma, in, The water-soluble tumor neoantigen is a hydrophilic antigen peptide OVA with a sequence of SIINFEKL, and the hydrophobic tumor neoantigen is a hydrophobic antigen peptide B-M27 with a sequence of LCPGNKYGM and a hydrophobic antigen peptide B-M30 with a sequence of DWENVSPELNSTDQ.

7. Use of the kit according to claim 1 in the preparation of a drug for preventing or treating breast cancer, in, The water-soluble tumor neoantigen is a hydrophilic antigen peptide OVA with a sequence of SIINFEKL, and the hydrophobic tumor neoantigen is a hydrophobic antigen peptide 4-M27 with a sequence of FAICFSCLLAHALNLIKLVRGRKPLSW and a hydrophobic antigen peptide 4-M32 with a sequence of SHRSCSHQTSAPSPKALAHNGTPRNAI.

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