CN111848733B

CN111848733B - Polypeptide composition and vaccine

Info

Publication number: CN111848733B
Application number: CN201910360198.7A
Authority: CN
Inventors: 金华君; 杨欢; 郝方元; 彭元锴; 钱其军
Original assignee: Shanghai Cell Therapy Research Institute; Shanghai Cell Therapy Group Co Ltd
Current assignee: Shanghai Cell Therapy Research Institute; Shanghai Cell Therapy Group Co Ltd
Priority date: 2019-04-30
Filing date: 2019-04-30
Publication date: 2024-03-12
Anticipated expiration: 2039-04-30
Also published as: CN111848733A

Abstract

The present invention provides a polypeptide composition comprising a polypeptide derived from gp100, EPS8 and p 16. The invention also provides a pharmaceutical composition comprising the polypeptide composition, a tumor vaccine, cells loaded with the polypeptide composition, a preparation method of the cells, a DC vaccine comprising the polypeptide composition, an activated T cell and the use of any one or more of the foregoing in preparing a medicament for preventing and/or treating cancer. The polypeptide composition provided by the invention presents and activates specific CD8+ CTL after loading DC, thereby achieving the targeted toxic effect on tumor cells. The derived tumor vaccine, DC vaccine, pharmaceutical composition and the like can obviously activate immune effector cells and improve the cytokine bleeding level and the killing level of tumor cells related to activation, and have potential clinical value.

Description

Polypeptide composition and vaccine

Technical Field

The invention relates to the field of medical immunology, in particular to a polypeptide composition and a vaccine.

Background

In recent years, some progress has been made in the treatment of cancer by surgery in combination with chemoradiotherapy, and the survival rate of patients, particularly patients with breast, lung, prostate and kidney diffuse cancer, has been improved. However, most of these treatments have significant toxic side effects, which are prone to damage to normal cells.

Tumors are capable of eliciting both humoral and cellular immune responses in the body. The tumor antigen is combined with the major histocompatibility complex I type molecule on the cell surface after being processed into peptide segments in the cell and is presented to CD8+ cytotoxic lymphocytes, or is firstly detached from the tumor cells, then is taken up by antigen presenting cells and is combined with the major histocompatibility complex II type molecule on the surface after being processed into peptide segments and is presented to CD4+ auxiliary lymphocytes, so that the immune response of the tumor cells of the organism is induced. The increased awareness of genetic alterations in anti-tumor immunity and malignant tumor progression has enabled humans to develop more selective and safe treatments that employ methods by activating the immune system to attack the developing tumor, i.e., tumor vaccine. Depending on the specific use of the tumor vaccine, it can be classified into a prophylactic vaccine and a therapeutic vaccine. The main function of the prophylactic vaccine is to control the occurrence of tumors; therapeutic vaccines are based on tumor-associated antigens and are mainly used for adjuvant therapy after chemotherapy. One of the tumor vaccines is a Dendritic Cell (DC) based vaccine. DC cells differ from B lymphocytes and macrophages in that they express costimulatory molecules in large amounts and have the ability to sensitize both cd4+ helper T cells (T helper, th) and cd8+ cytotoxic T cells (Cytotoxic T Lymphocyte, CTL). DCs generate specific anti-tumor immune responses by loading tumor antigens and inducing them into mature DCs. Based on this, a variety of anti-tumor vaccines have been developed with DCs, including tumor antigen peptide-loaded DCs, tumor whole cell antigen-loaded DCs, tumor cell RNA-loaded DCs, tumor cell DNA-loaded DCs, exosome (exosome) -loaded DCs, cytokines, chemokine gene-modified DCs. DC vaccines have been tried among malignant melanoma, prostate cancer, renal cancer, and the like, with some success. Various forms of DC vaccine have been tried in the immunotherapy of tumors and have shown good efficacy in preliminary clinical trials. Wherein DC vaccine profnge produced by Dendreon corporation, usa was approved by the national food and drug administration in 2010 for advanced prostate cancer patients, especially those who failed hormone therapy, the efficacy was shown to extend patient survival by more than 4 months compared to placebo (Nature Medicine,2010,16 (6): 615).

Autologous whole tumor lysate is still used to load DCs in most clinical trials today, specifically by lysing the patient's own tumor tissue through multiple cycles of freeze thawing to stimulate the DC cells with lysate (Cancer Immunol Immunother,2006,55:819;medical oncology,2006,23:273.). The freeze-thaw cycle induces tumor cell necrosis, but freeze-thaw induced tumor cell necrosis is not immune and even inhibits Toll-like receptor (TLR) -induced DC cell maturation and normal function (Hatgeld P, merrick AE, west E, O' Donnell D, selby P, vile R, et al optimization of dendritic cell loading with tumor cell lysates for cancer immunology J. Immunother (2008) 31 (7): 620-32), and tumor tissue in patients is not always readily available. Tumor cell lysates, purified tumor-associated antigens and tumor-derived mRNA have also been demonstrated to be useful as a source of DC-loaded antigens. Tumor cell lysates can provide multiple antigens for DC loading, and can induce CD4+ and CD8+ T cell responses and confer differential Damage-associated molecular patterns to the DCs (Damage-Associated Molecular Patterns, DAMP) to ensure maturation of the DCs, but also provide immunoregulatory cytokines to the DCs, induce tolerance transformation of the DC cells (Guida M, pisconte S, colucci G.Metastatic melana: the new era of targeted therapy.Expert Opin Ther Targets 2012;16Suppl 2:S61-70); purified tumor-associated antigen-loaded DCs are capable of activating antigen-specific T cell responses and inducing cd4+ and cd8+ T cell responses, but have a limited number of different antigen species for single use. Tumor derived mRNA can be transferred into tumor associated antigens and costimulatory molecules, ensuring antigen presentation by MHC class I, and does not require cross presentation (Robbins PF, morgan RA, feldman SA, yang JC, shermy RM, dudley ME, wunderlich JR, nahvi AV, helman LJ, mackall CL, et al Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytesreactive with NY-ESO-1.J Clin Oncol 2011;29:917-24), but does not induce DC cell maturation nor effective CD4+ immune responses, and the number of different antigen species for single use is limited.

When using short peptide fragments of tumor-associated antigens as antigens for loading DCs, the number of antigen species that can be involved in a single use can be effectively increased, increasing the level of immune response in cd4+ and cd8+ T cells, but it is desirable to first determine the subject's HLA haplotype and select the appropriate peptide fragment in the selected tumor-associated antigen to verify that it is able to bind to that HLA haplotype. HLA alleles are highly polymorphic in different ethnic groups. According to world health organization statistics, by month 4 of 2018, the number of HLA class I alleles has exceeded 13000, with 4200 HLA-A alleles, 5091 HLA-B alleles, 3854 HLA-C alleles (http:// www.hla.alleles.org/non-clamp/stats. Html). Among them, HLA types common to Asian population are mostly HLa-A2, A3 and A24 (Experimental and Therapeutic Medicine,2011, 2:109-117.). Three types of HLa-A2, A11 and A24 can cover more than 90% of the Chinese population (Immunol Today,1996; 17:261.). HLA-A2 belongs to the HLA-A2 super type, the highest frequency in Chinese population is 45.9%, HLA-A11 belongs to the HLA-A3 super type, the lowest frequency in Caucasian (Caucasian) is 37.5%, and the highest frequency in Chinese population is 52.7%; HLA-A24 is of the super-type HLA-A24, with a frequency of 23.9% in caucasians, 40.1% in Chinese and 58.6% in Japanese (Curr Opinion In Immunol,1998,10:478-482; immunogenetics,1999,50 (3-4): 201-212). Currently there is a lack of tumor vaccines for each HLA class comprising immunogenic polypeptide compositions that are capable of being efficiently presented by antigen presenting cells and comprising such polypeptide compositions.

Disclosure of Invention

Aiming at the technical problems that HLA-A11 typing with highest frequency in Chinese population still lacks an immunogenic polypeptide composition capable of being effectively presented by antigen presenting cells and a DC vaccine comprising the polypeptide composition, the invention provides a polypeptide composition and a tumor vaccine comprising the polypeptide composition. The polypeptide composition and the tumor vaccine containing the polypeptide composition can effectively induce DC maturation, activate T cells to generate higher tumor cell killing activity, and have larger antitumor potential.

In one aspect, the invention provides a polypeptide composition comprising an isolated polypeptide derived from a polypeptide comprising one or more selected from the group consisting of CypB, WHSC2, SART3, p56, p14, gp100, EPS8, p53, p16, and p 21.

In a preferred embodiment, the polypeptide composition comprises isolated polypeptides derived from SART3, p56 and p 14. In a preferred embodiment, the isolated polypeptides derived from SART3, p56 and p14 are present in a mass ratio of 1:1:1.

In a preferred embodiment, the polypeptide composition comprises isolated polypeptides derived from gp100, EPS8 and p 16. In a preferred embodiment, the isolated polypeptides derived from gp100, EPS8 and p16 are present in a mass ratio of 1:1:1.

In a preferred embodiment, the polypeptide composition comprises isolated polypeptides derived from p14, cypB and WHSC 2. In a preferred embodiment, the isolated polypeptides derived from p14, cypB and WHSC2 are present in a mass ratio of 1:1:1.

In a preferred embodiment, the polypeptide composition comprises isolated polypeptides derived from EPS8, cypB and WHSC 2. In a preferred embodiment, the isolated polypeptides derived from EPS8, cypB and WHSC2 are present in a mass ratio of 1:1:1.

In a preferred embodiment, the polypeptide composition comprises isolated polypeptides derived from SART3, p56, p14, gp100, EPS8 and p 16. In a preferred embodiment, the isolated polypeptides derived from SART3, p56, p14, gp100, EPS8 and p16 are of equal mass.

In a preferred embodiment, the polypeptide composition comprises isolated polypeptides derived from SART3, p56, p14, gp100, EPS8, p16, p53 and p 21. In a preferred embodiment, the isolated polypeptides derived from SART3, p56, p14, gp100, EPS8, p16, p53 and p21 are of equal mass.

In a preferred embodiment, the polypeptide composition comprises isolated polypeptides derived from CypB, WHSC2, SART3, p56, p14, gp100, EPS8, p53, p16 and p 21. In a preferred embodiment, the isolated polypeptides derived from CypB, WHSC2, SART3, p56, p14, gp100, EPS8, p53, p16 and p21 are of equal mass.

In a preferred embodiment, the sequence of the SART 3-derived isolated polypeptide is shown in SEQ ID NO. 1.

In a preferred embodiment, the sequence of the isolated polypeptide derived from p56 is shown in SEQ ID NO. 2.

In a preferred embodiment, the sequence of the isolated polypeptide derived from p14 is shown in SEQ ID NO. 3.

In a preferred embodiment, the sequence of the isolated polypeptide derived from gp100 is shown in SEQ ID NO. 4.

In a preferred embodiment, the sequence of the isolated polypeptide derived from EPS8 is shown in SEQ ID NO. 5.

In a preferred embodiment, the sequence of the isolated polypeptide derived from p16 is shown in SEQ ID NO. 6.

In a preferred embodiment, the sequence of the isolated polypeptide derived from CypB is shown in SEQ ID NO. 7.

In a preferred embodiment, the sequence of the isolated polypeptide derived from WHSC2 is shown in SEQ ID NO. 8.

In a preferred embodiment, the sequence of the isolated polypeptide derived from p53 is shown in SEQ ID NO. 9.

In a preferred embodiment, the sequence of the isolated polypeptide derived from p21 is shown in SEQ ID NO. 10.

In a preferred embodiment, the polypeptide composition comprises an isolated polypeptide having the sequence shown in SEQ ID NO. 1-3.

In a preferred embodiment, the polypeptide composition comprises an isolated polypeptide having the sequence shown in SEQ ID NOS.4-6.

In a preferred embodiment, the polypeptide composition comprises isolated polypeptides having the sequences SEQ ID NO. 3, SEQ ID NO. 7 and SEQ ID NO. 8.

In a preferred embodiment, the polypeptide composition comprises isolated polypeptides having the sequences SEQ ID NO. 5, SEQ ID NO. 7 and SEQ ID NO. 8.

In a preferred embodiment, the polypeptide composition comprises isolated polypeptides having the sequences SEQ ID NO 1-6, SEQ ID NO 9 and SEQ ID NO 10.

In another aspect, the invention provides a pharmaceutical composition comprising the aforementioned polypeptide composition.

In a preferred embodiment, the pharmaceutical composition further comprises an adjuvant and/or a pharmaceutically acceptable salt.

In another aspect, the invention provides a tumor vaccine comprising the aforementioned polypeptide composition.

In a preferred embodiment, the tumor vaccine further comprises an adjuvant. Preferably, the method comprises the steps of, the adjuvant includes a material selected from the group consisting of aluminum adjuvants (e.g., aluminum hydroxide), freund's adjuvants (e.g., complete Freund's adjuvant and incomplete Freund's adjuvant), prostaglandin E2, interferon alpha, corynebacterium parvum, lipopolysaccharide, cytokines, oil-in-water emulsions, water-in-oil emulsions, nanoemulsions, microparticle delivery systems, liposomes, microspheres, biodegradable microspheres, plaque virions, proteoliposomes, proteasomes, immunostimulatory complexes (ISCOMs, ISCOMATRIX), microparticles, nanoparticles, biodegradable nanoparticles, silicon nanoparticles, polymeric micro/nanoparticles, polymeric Lamellar Substrate Particles (PLSP), microparticle resins, nanoliposome polymeric gels (nanonolipogel) synthetic/biodegradable and biocompatible semisynthetic or natural polymers or dendrimers (e.g. PLG, PLGA, PLA, polycaprolactone, silicon polymers, polyesters, polydimethylsiloxane, sodium polystyrene sulfonate, polystyrene benzyl trimethyl ammonium chloride, polystyrene divinylbenzene resins, polyphosphazenes, poly- [ di- (carboxyacetyl phenoxy) phosphazenes (PCPP), poly- (methyl methacrylate), dextran, polyvinylpyrrolidone, hyaluronic acid and derivatives, chitosan and derivatives thereof, polysaccharides, delta inulin polysaccharides, glycolipids (synthetic or natural), lipopolysaccharides, one or more polycationic compounds (e.g. polyamino acids, poly- (gamma-glutamic acid), poly-arginine-HCl, poly-L-lysine, polypeptides, biopolymers), cationic Dimethyl Dioctadecyl Ammonium (DDA), alpha-galactosyl ceramide and derivatives thereof, archaebacteria lipids and derivatives, lactam, bellen, glyceride, phospholipid and one or more of spirochete. One or more of the following.

In another aspect, the invention provides a cell loaded with the aforementioned polypeptide composition.

In a preferred embodiment, the cell is an antigen presenting cell.

In a preferred embodiment, the HLA class expressed by the antigen presenting cells is HLA-A11.

In a preferred embodiment, the antigen presenting cell is one or more selected from the group consisting of macrophages, B cells and Dendritic Cells (DCs); preferably, the antigen presenting cells are DCs.

In a preferred embodiment, the DCs are derived from monocytes in PBMCs. In another preferred embodiment, the DC is a DC cell line or DC precursor cell line that is artificially constructed and capable of immortalizing and culturing in vitro.

In another aspect, the present invention provides a method for preparing a cell loaded with the aforementioned polypeptide composition, comprising:

(1) Contacting a DC precursor cell with a cytokine, differentiating the DC precursor cell into an immature DC;

(2) Contacting the aforementioned polypeptide composition with the immature DC of (1) to effect antigen loading, thereby obtaining a DC-polypeptide mixture;

(3) Contacting the DC-polypeptide mixture of (2) with a DC maturation-promoting factor to further induce DC maturation and obtain cells loaded with the aforementioned polypeptide composition.

In a preferred embodiment, the DC precursor cells in (1) are cd14+ DC precursor cells; preferably, it is monocytes in PBMC in blood.

In another preferred embodiment, the DC precursor cells in (1) are cd34+ DC precursor cells; preferably, hematopoietic progenitor cells.

In a preferred embodiment, the cytokine in (1) comprises IL-4 and GM-CSF. In a preferred embodiment, the IL-4 working concentration is 10-100ng/mL; preferably, 10-50ng/mL; more preferably, 50ng/mL in another preferred embodiment, the working concentration of the GM-CSF factor is from 10 to 100ng/mL; preferably 50-100ng/mL; more preferably 100ng/mL.

In a preferred embodiment, the contacting in (1) is by adding the cytokine to a medium comprising the DC precursor cells, and incubating. In a preferred embodiment, the co-incubation time is 2-5 days; preferably 3-5 days. In a preferred embodiment, the temperature of the co-incubation is 37 ℃; in another preferred embodiment, the CO-incubated CO ₂ The concentration was 5%.

In a preferred embodiment, the polypeptides in the polypeptide composition of (2) are each added separately to the immature DCs of (1).

In a preferred embodiment, the polypeptides in the polypeptide composition of (2) are added to the immature DC of (1) after being homogeneously mixed.

In a preferred embodiment, the contacting in (2) is by adding the aforementioned polypeptide composition to a medium comprising the immature DC and the cytokine in (1), and incubating.

In a preferred embodiment, the co-incubation time is 2-5 days; excellent (excellent)Alternatively, 2 days. In a preferred embodiment, the temperature of the co-incubation is 37 ℃; in another preferred embodiment, the CO-incubated CO ₂ The concentration was 5%, the percentages being by volume.

In a preferred embodiment, the working concentration of each polypeptide in said DC-polypeptide mixture after addition of the aforementioned polypeptide composition of (2) to said immature DC of (1) is from 20 to 60 μg/mL; preferably 40-60. Mu.g/mL, such as 40. Mu.g/mL.

In a preferred embodiment, said contacting in (3) is by adding said DC-factor to said DC-polypeptide mixture in (2), and incubating.

In a preferred embodiment, the co-incubation time is 1-3 days; preferably, 1 day. In a preferred embodiment, the temperature of the co-incubation is 37 ℃; in another preferred embodiment, the CO-incubated CO ₂ The concentration was 5%, the percentages being by volume.

In a preferred embodiment, the DC maturation-promoting factor comprises any one or more selected from the group consisting of TNF-alpha, IL-1β, IL-6 and PGE 2.

In a preferred embodiment, the DC maturation-promoting factor comprises any one or more selected from the group consisting of TNF- α, IL-1β, IL-6, PGE2, IFN- γ, poly (I: C), R848, and ATP; preferably, IFN-gamma, poly (I: C) and R848 are included; more preferably, IFN-gamma, poly (I: C), R848 and ATP are included. In a preferred embodiment, IFN-gamma is administered at a concentration of 10-1000IU/mL; preferably 100-300IU/mL; more preferably 100IU/mL. In another preferred embodiment, the working concentration of poly (I: C) is 1-200. Mu.g/mL, preferably 20-40. Mu.g/mL; more preferably 30. Mu.g/mL. In another preferred embodiment, R848 is present at a working concentration of 0.1 to 50 μg/mL; preferably 1-10. Mu.g/mL; more preferably 5. Mu.g/mL. In another preferred embodiment, the working concentration of ATP is 0.1-10mM; preferably 0.1-5mM; more preferably, it is 1mM.

In a preferred embodiment, the method for preparing a cell carrying the aforementioned polypeptide composition comprises:

(1) Adding IL-4 and GM-CSF to a monocyte culture system comprising a subject;

Preferably, the IL-4 is present at a working concentration of 10-100ng/mL; more preferably, 10-50ng/mL;

preferably, the working concentration of the GM-CSF factor is 10-100ng/mL; more preferably, 50-100ng/mL;

(2) Culturing until day 3, half-changing culture medium, and supplementing IL-4 and GM-CSF factors to their respective initial working concentrations;

(3) Culturing until day 5, adding the polypeptide composition, and continuing culturing, wherein the working concentration of each polypeptide in the polypeptide composition is 20-60 mug/mL; preferably 40. Mu.g/mL;

(4) Culturing until 7 days, adding DC maturation-promoting factors IFN-gamma, poly (I: C) and R848 to working concentrations of 100IU/mL, 30 μg/mL and 5 μg/mL respectively, and culturing for 24h to obtain mature DC.

In a preferred embodiment, the monocytes in (1) are obtained by culturing and isolating PBMCs isolated from peripheral blood. Preferably, it is obtained by the following method: peripheral blood PBMC are obtained, and after culture, suspension cells are separated from adherent cells, wherein the adherent cells are monocytes.

In another aspect, the invention provides a DC vaccine comprising a DC loaded with the aforementioned polypeptide composition.

In a preferred embodiment, the DC carrying the aforementioned polypeptide composition can be produced by the aforementioned method.

In a preferred embodiment, the DC vaccine further comprises an adjuvant. Preferably, the method comprises the steps of, the adjuvant includes a material selected from the group consisting of aluminum adjuvants (e.g., aluminum hydroxide), freund's adjuvants (e.g., complete Freund's adjuvant and incomplete Freund's adjuvant), prostaglandin E2, interferon alpha, corynebacterium parvum, lipopolysaccharide, cytokines, oil-in-water emulsions, water-in-oil emulsions, nanoemulsions, microparticle delivery systems, liposomes, microspheres, biodegradable microspheres, plaque virions, proteoliposomes, proteasomes, immunostimulatory complexes (ISCOMs, ISCOMATRIX), microparticles, nanoparticles, biodegradable nanoparticles, silicon nanoparticles, polymeric micro/nanoparticles, polymeric Lamellar Substrate Particles (PLSP), microparticle resins, nanoliposome polymeric gels (nanonolipogel) synthetic/biodegradable and biocompatible semisynthetic or natural polymers or dendrimers (e.g. PLG, PLGA, PLA, polycaprolactone, silicon polymers, polyesters, polydimethylsiloxane, sodium polystyrene sulfonate, polystyrene benzyl trimethyl ammonium chloride, polystyrene divinylbenzene resins, polyphosphazenes, poly- [ di- (carboxyacetyl phenoxy) phosphazenes (PCPP), poly- (methyl methacrylate), dextran, polyvinylpyrrolidone, hyaluronic acid and derivatives, chitosan and derivatives thereof, polysaccharides, delta inulin polysaccharides, glycolipids (synthetic or natural), lipopolysaccharides, one or more polycationic compounds (e.g. polyamino acids, poly- (gamma-glutamic acid), poly-arginine-HCl, poly-L-lysine, polypeptides, biopolymers), cationic Dimethyl Dioctadecyl Ammonium (DDA), alpha-galactosyl ceramide and derivatives thereof, archaebacteria lipids and derivatives, lactam, bellen, glyceride, phospholipid and one or more of spirochete.

In another aspect, the invention provides an activated T cell that is activated by contacting an inactivated T cell with the DC vaccine described above.

In a preferred embodiment, the contacting is co-incubation. In a preferred embodiment, the temperature of the co-incubation is 37 ℃. In a preferred embodiment, the co-incubation time is 2-48 hours, preferably 24-48 hours; more preferably 24 hours. In a preferred embodiment, the CO-incubated CO ₂ The concentration was 5%. In a preferred embodiment, the co-incubation medium is AIM-V medium comprising 2% V/vFBS; preferably, the medium used for the co-incubation further comprises IL-2 at a working concentration of 100U/mL.

In a preferred embodiment, the ratio of the number of said non-activated T cells to DC in said DC vaccine is from 10:1 to 50:1; preferably, the ratio is 20:1-50:1; more preferably, it is 20:1.

In a preferred embodiment, the non-activated T cells are derived from the same or different individual as the DCs in the DC vaccine.

In a preferred embodiment, the non-activated T cells are cd8+ T cells.

In another aspect, the invention provides a method of eliciting an immune response in a subject comprising:

(1) Obtaining DC precursor cells of a subject;

(2) The DC vaccine is prepared by the preparation method of the DC vaccine by using the DC precursor cells of the tested individual in the step (1), and is returned into the body of the tested individual in the step (1).

In a preferred embodiment, the monocytes are obtained by the following method: and (3) extracting peripheral blood of the tested individual, separating by a density gradient centrifugation method to obtain PBMC, and separating suspension cells from adherent cells, wherein the adherent cells are monocytes after culture.

In a preferred embodiment, the culture medium is AIM-V medium without serum.

In a preferred embodiment, the time of the incubation is 1-5 hours, preferably 2 hours.

In a preferred embodiment, the incubation time is overnight.

In a preferred embodiment, the temperature of the culture is 37 ℃.

In a preferred embodiment, the cultured CO ₂ The concentration was 5%.

In another aspect, the invention provides the use of one or more of the aforementioned polypeptide composition, pharmaceutical composition, tumor vaccine, cells loaded with the aforementioned polypeptide composition, DC vaccine and activated T cells in the manufacture of a medicament for the prevention and/or treatment of cancer.

In a preferred embodiment of the present invention, in a preferred embodiment, the cancer is selected from lung cancer, non-small cell lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, renal cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancy, head and neck cancer, glioma, mesothelioma, carcinoma of large intestine, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine fibroid and osteosarcoma, bone cancer, pancreatic cancer, renal cell carcinoma, skin cancer, prostate cancer, cutaneous or intraocular malignant melanoma, uterine cancer, anal region cancer, testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulval cancer, hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, cholangiocarcinoma thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urinary tract cancer, urothelial cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia), childhood solid tumors, lymphoblastic lymphoma, renal or ureteral cancer, renal pelvis cancer, central Nervous System (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brain stem glioma, pituitary adenoma, kaposi's sarcoma, hodgkin's lymphoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including asbestos-induced cancers and any one or more of various leukemia and lymphoma types and various precancerous lesions.

The invention has the following positive effects: the polypeptide composition provided by the invention achieves the targeted toxic effect on tumor cells by presenting and activating specific CD8+ cytotoxic T lymphocytes (cytotoxic T lymphocyte, CTL) after DC loading. The polypeptide composition and the tumor vaccine, the DC vaccine and the pharmaceutical composition derived from the polypeptide composition can obviously activate immune effector cells, particularly T cells, obviously improve the secretion level of cytokines related to activation and the killing level of tumor cells, and have potential clinical value.

Drawings

Fig. 1a: inducing secretion levels of DC secreted IL-12 after loading of polypeptide composition 1;

fig. 1b: inducing secretion levels of DC secreted IL-12 upon loading of polypeptide composition 2;

fig. 2a: positive rate of DC surface CD80 and HLA-ABC after loading of polypeptide composition 1;

fig. 2b: positive rate of DC surface CD80 and HLA-ABC after loading of polypeptide composition 2;

fig. 3a: the ratio of CD4+ cells to CD8+ cells of DC-CTL loaded with polypeptide composition 1 to control T cells;

fig. 3b: ratio of DC-CTL loaded with polypeptide composition 1 to cd3+cd107a cells of T cells of the control group;

fig. 3c: IFN-gamma secretion levels of DC-CTL loaded with polypeptide composition 1 and control T cells;

Fig. 4a: the ratio of DC-CTL loaded with polypeptide composition 2 to cd4+ cells to cd8+ cells of T cells of the control group;

fig. 4b: ratio of DC-CTL loaded with polypeptide composition 2 to cd3+cd107a cells of T cells of the control group;

fig. 4c: IFN-gamma secretion levels of DC-CTL loaded with polypeptide composition 2 and control T cells;

fig. 5a: comparison of the level of T cell proliferation under DC stimulation loaded with polypeptide composition 1 with the level of proliferation of control T cells;

fig. 5b: comparison of the level of T cell proliferation under DC stimulation loaded with polypeptide composition 2 with the level of proliferation of control T cells;

fig. 6a: a killing effect profile of DC-CTLs loaded with polypeptide composition 1 and peptides 1, 2 and 3, respectively, on PANC-1 cells;

fig. 6b: a killing effect profile of DC-CTLs loaded with polypeptide composition 2 and peptides 4, 5 and 6, respectively, on PANC-1 cells;

fig. 6c: a killing effect profile of DC-CTL loaded with polypeptide composition 2 on PANC-1 cells;

fig. 7: curves of killing effect of DC-CTLs loaded with polypeptide compositions 5, 6, 7, 8 and peptide 2, respectively, on PANC-1 cells;

fig. 8a: a killing effect profile of DC-CTL loaded with polypeptide composition 1 and peptides 1, 2 and 3, respectively, on SKOV-3 cells;

fig. 8b: a killing effect profile of DC-CTL loaded with polypeptide composition 2 and peptides 4, 5 and 6, respectively, on SKOV-3 cells;

Fig. 9a: a killing effect profile of DC-CTL loaded with polypeptide composition 1 and polypeptide composition 3, respectively, on PANC-1 cells;

fig. 9b: a killing effect profile of DC-CTL loaded with polypeptide composition 2 and polypeptide composition 4, respectively, on PANC-1 cells;

the "control T cell group" noted in the above figures refers to a T cell experimental group stimulated with DC that is not loaded with any polypeptide.

Detailed Description

In the present invention, the CypB, WHSC2, SART3, p56, p14, gp100, EPS8, p53, p16 and p21 include tumor associated antigens CypB, WHSC2, SART3, p56, p14, gp100, EPS8 and cell senescence associated proteins p53, p16 and p21. In tumor immunity, a polypeptide fragment of an epitope of a tumor-associated antigen binds to an HLA of an antigen presenting cell, such as a DC surface, and forms an HLA-tumor epitope peptide complex that is recognized by the TCR and is then presented to T cells, such that T cells capable of recognizing the corresponding tumor epitope are specifically activated and expanded. The expanded T cells become cytotoxic T lymphocytes (Cytotoxic T Lymphocyte, CTL) that specifically target the tumor-associated antigen, producing cell-mediated immune killing of tumor cells expressing the tumor-associated antigen. Senescent cells can activate both innate and adaptive immune responses, maintaining tissue homeostasis. Furthermore, new findings suggest that programmatically induced cellular senescence may be important in regulating reproductive processes, in part due to immune clearance. Antigens p16, p53 and p21 have a significant link to the development of senescent cells and tumors. Biomarkers of senescent cells (e.g., β -galactosidase, p16.sup.INK4A) are now widely accepted, including p16, and p53 and p21 have similar effects in the regulation of the cell cycle as p16, while regulatory mutations or deletions of either p16 or p53 are seen in a variety of tumor cells. Therefore, the invention activates specific CTL by loading aging cell short peptide by DC, thereby achieving the targeted toxic effect on tumor cells.

In particular, the invention provides a polypeptide composition comprising an isolated polypeptide derived from a polypeptide comprising one or more selected from the group consisting of CypB, WHSC2, SART3, p56, p14, gp100, EPS8, p53, p16 and p 21. In one embodiment, the isolated polypeptide of the polypeptide composition is associated with antigen presenting cells and may be used to elicit an immune response. The polypeptide composition may be administered to a subject as an agent for the prevention and treatment of an ongoing cancer.

The invention also provides a tumor vaccine comprising the polypeptide composition. The tumor vaccine is capable of eliciting an immune response in vivo. In one embodiment, the immune response may be a humoral immune response. In another embodiment, the immune response may be a cell-mediated immune response.

The invention also provides a DC vaccine comprising the polypeptide composition. The DC vaccine comprises a mature DC loaded with one or more isolated polypeptides of the polypeptide compositions described above. The invention also provides a method for preparing the DC vaccine, which comprises contacting immature DC with the polypeptide composition, so that the DC loads one or more isolated polypeptides in the polypeptide composition. The loading of the polypeptide composition enables the transformation of immature DCs into mature DCs to be significantly achieved, thereby obtaining mature DCs comprising tumor antigen polypeptides and/or senescence cell associated antigen polypeptides loaded in the polypeptide composition and the DC vaccine.

The invention also provides a method of eliciting an immune response in a subject comprising obtaining PBMCs from the subject, isolating monocytes after culturing, inducing the resulting immature DCs to carry one or more isolated polypeptides of the polypeptide composition, allowing the DCs to mature and returning the DCs carrying the polypeptide composition to the subject. In particular, the invention also provides a DC loaded with the polypeptide composition.

The invention also provides an activated T cell that is activated by exposure to the DC vaccine described above.

The invention also provides a method of preventing and/or treating cancer comprising reinfusion of one or more of the above polypeptide composition, tumor vaccine and DC vaccine to an individual suffering from cancer. In a preferred embodiment, the tumor-associated antigen having an elevated level of expression of the surface of cancer cells in the individual suffering from cancer comprises one or more of the epitopes represented by the isolated polypeptides in the above-described polypeptide composition.

The following is a description of some of the terms involved in the present invention. Unless otherwise defined below, the terms herein are used in the manner commonly used in the art.

In the present invention, the term "polypeptide" refers to a molecule composed of monomers (amino acids) that are linearly linked by amide bonds (also referred to as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of a product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains" or any other term used to refer to one or more chains having two or more amino acids are included in the definition of "polypeptide", and the term "polypeptide" may be used in place of, or interchangeably with, any of these terms. The term "polypeptide" is also intended to refer to products of modification of a polypeptide after expression, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-standard amino acids. The polypeptide may be derived from a natural biological source or produced by recombinant techniques, but is not necessarily translated from a specified nucleic acid sequence. It can be produced in any manner, including by chemical synthesis.

The term "variant" refers to a peptide or polypeptide that has been altered in amino acid sequence by insertion, deletion, or conservative substitution of amino acids, but retains at least one biological activity. A variant may also refer to a polypeptide having an amino acid sequence that is substantially identical to the sequence of a reference polypeptide. The reference polypeptide has an amino acid sequence that retains at least one biological activity. Conservative substitutions of amino acids, i.e., substitution of an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree of dotted region, and distribution), are generally recognized in the art as involving minor changes. These minor changes can be identified in part by considering the hydropathic index of amino acids.

The term "wild-type" has its meaning as understood in the art, which refers to an entity having a structure and/or activity as found in nature in a "normal" (as opposed to mutants, diseased persons, altered persons, etc.) state or condition. Those of skill in the art will appreciate that wild-type genes and polypeptides typically exist in a variety of different forms (e.g., alleles).

The term "tumor-associated antigen" or "TAA" refers to an antigen that is specifically expressed by tumor cells or expressed by tumor cells at a higher frequency or density than non-tumor cells of the same tissue type. The tumor-associated antigen may be an antigen that is not normally expressed by the host; they may be displayed abnormally by mutation, truncation, misfolding, or other means of the molecule normally expressed by the host; they may be identical to normally expressed molecules but expressed at abnormally high levels; or they may be expressed in an abnormal situation or environment. The tumor-associated antigen may be, for example, a protein or protein fragment, complex carbohydrate, ganglioside, hapten, nucleic acid, or a combination of these or other biomolecules.

The term "vaccine" refers to an immunogenic composition for administration to a mammal for eliciting an immune response in the mammal against a specific antigen. Vaccines typically comprise an agent (known as an "antigen" or "immunogen") that is similar to or derived from a target of the immune response, such as a disease causing microorganism or tumor cell. Vaccines intended for the treatment of tumors, such as cancers, typically comprise an antigen derived from a tumor-associated antigen found on the tumor of interest and capable of eliciting immunogenicity to the tumor-associated antigen on the tumor of interest.

The term "CypB" refers to the expression product of the cypB gene, cyclophilin B protein, which may be a human cyclophilin B protein or a non-human mammalian cyclophilin B protein.

The term "WHSC2" refers to the expression product WHSC2 protein of the "WHSC2" gene, which may be a human WHSC2 protein or a non-human mammalian WHSC2 protein.

The term "SART3" refers to SART3 protein, which is an expression product of the SART3 gene, and may be a SART3 protein or a non-human mammalian SART3 protein.

The term "p56" refers to the p56 protein, which is the expression product of the p56 gene, and may be a human p56 protein or a non-human mammalian p56 protein.

The term "p14" refers to the p14 protein, which is the expression product of the p14 gene, and may be a human p14 protein or a non-human mammalian p14 protein.

The term "gp100" refers to the gp100 protein, which is the expression product of the gp100 gene, and may be a human gp100 protein or a non-human mammalian gp100 protein.

The term "EPS8" refers to the expression product EPS8 protein of the EPS8 gene, which may be a human EPS8 protein or a non-human mammalian EPS8 protein.

The term "p53" refers to the p53 protein, which is the expression product of the p53 gene, and may be a human p53 protein or a non-human mammalian p53 protein.

The term "p16" refers to the p16 protein, which is the expression product of the p16 gene, and may be a human p16 protein or a non-human mammalian p16 protein.

The term "p21" refers to the p21 protein, which is the expression product of the p21 gene, and may be a human p21 protein or a non-human mammalian p21 protein.

The terms "cancer," "tumor," and "malignancy" refer to or describe a physiological condition in a mammal that is typically characterized by uncontrolled cell growth. Examples of cancers include, but are not limited to, epithelial cancers, including adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small-cell lung carcinoma, non-small cell lung carcinoma, gastrointestinal carcinoma, hodgkin's lymphoma and non-hodgkin's lymphoma, pancreatic carcinoma, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer (such as liver cancer and hepatoma), bladder cancer, breast cancer (including hormone-mediated breast cancer), colon cancer, colorectal cancer, endometrial cancer, myeloma (such as multiple myeloma), salivary gland cancer, renal cancer (such as renal cell carcinoma and wilms ' tumor), basal cell carcinoma, melanoma, prostate cancer, vulval cancer, thyroid cancer, testicular cancer, esophageal cancer, blood cancers (including but not limited to Acute Myelogenous Leukemia (AML) and Multiple Myeloma (MM)), various types of head and neck cancer (including but not limited to squamous cell carcinoma), and cancers of mucous origin (such as mucinous ovarian cancer), cholangiocarcinoma (liver), and papillary renal carcinoma. In certain embodiments, the blood cancer is selected from the group consisting of: hodgkin's lymphoma, non-hodgkin's lymphoma, multiple myeloma, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, and chronic myelogenous leukemia.

The term "immunopotentiator", as used herein, means a substance that when mixed with an immunogen is capable of eliciting a stronger immune response than when the immunogen alone is present. For example, an immunopotentiator can enhance immunogenicity and provide an excellent immune response. For another example, an immunopotentiator can function by increasing the expression of co-stimulatory factors on macrophages and other antigen presenting cells.

The term "adjuvant" refers to a non-specific immunopotentiator that, when delivered with an antigen or pre-delivered into an organism, can enhance the organism's immune response to the antigen or alter the type of immune response. There are many adjuvants including, but not limited to, aluminum adjuvants (e.g., aluminum hydroxide), freund's adjuvants (e.g., complete Freund's adjuvant and incomplete Freund's adjuvant), corynebacterium parvum, lipopolysaccharide, cytokines, and the like. Freund's adjuvant is the most commonly used adjuvant in current animal trials. Aluminum hydroxide adjuvants are used more in clinical trials.

The term "DC maturation-promoting factor" refers to any protein, nucleic acid, polypeptide, complex, extract, isolate, or combination thereof capable of promoting the conversion of immature DC to mature DC by contact with the immature DC. Verification of mature DCs can be by detection of molecular markers expressed on the surface of mature DCs and/or cytokines secreted by mature DCs as known in the art, including, but not limited to, CD80, CD83, CD86, CCR7, HLA-ABC, HLa-DR, and IL-6.

The term "isolated" refers to a state in which naturally occurring biological macromolecules such as proteins, polypeptides, nucleic acids, antibodies, or complexes thereof formed therein, are isolated, purified, or de novo synthesized in vitro by artificial means from a natural state in vivo.

The term "working concentration" refers to the actual concentration of an agent or active ingredient as it functions in a solution system. Usually, a certain reagent or a certain effective component is prepared into mother solution or storage solution with higher concentration before use, and then is added into a final reaction system according to a certain proportion to be diluted when in use, and the final concentration obtained after dilution is usually the working concentration.

The term "loaded" refers to the binding of a protein, polypeptide or nucleic acid molecule directly to a receptor on the surface of a cell, forming a complex of the cell and the protein, polypeptide or nucleic acid molecule. The binding may be covalent or non-covalent, including receptor-ligand interactions. For example, the antigen peptide-loaded DC is a DC-antigen peptide complex formed by combining an antigen peptide with an HLA molecule which is expressed on the surface of the DC and matched with HLA type of the antigen peptide.

The term "DC-cytotoxic T lymphocyte" or "DC-CTL" refers to a cytotoxic T lymphocyte (Cytotoxic T Lymphocyte, CTL) activated by mature DCs loaded with an antigen peptide, which is capable of specifically binding to an antigen expressing an antigen containing an antigen peptide loaded by said mature DCs and producing a cell killing effect on cells expressing the antigen, which is the main executor of cellular immunity mediated by DCs.

The term "antigen presenting cells" (APC) refers to a class of cells capable of expressing Major Histocompatibility Complex (MHC) type I or II, and capable of forming MHC-antigen peptide complexes by MHC binding to antigen peptides and further binding to receptors on the surface of T cells, thereby activating T cells, including but not limited to Dendritic Cells (DCs), monocytes/macrophages, B cells, langerhans cells.

The term "antigen-loaded antigen-presenting cell" includes APCs that have been exposed to and activated by an antigen. For example, APCs can be loaded with antigen in vitro (e.g., during culture in the presence of antigen). APCs can also be loaded in vivo by exposure to antigen. "antigen-loaded APCs" are typically prepared in one of two ways: (1) Small fragments called antigenic peptides are "pulsed" directly outside of the APC to bind to MHC molecules; (2) The APC is incubated with a large fragment of the polypeptide, the complete protein or the protein particle, and then the large fragment of the polypeptide, the complete protein or the protein particle is taken up by the APC. These large fragments of polypeptides or protein molecules are digested into small peptide fragments by the APC and eventually transported and presented on the APC surface. In addition, antigen-loaded APCs can also be produced by introducing a polynucleotide encoding an antigen into a cell. Polypeptide composition

The present invention provides a polypeptide composition comprising an isolated polypeptide selected from one or more of the group consisting of CypB, WHSC2, SART3, p56, p14, gp100, EPS8, p53, p16 and p 21. In one embodiment, the isolated polypeptides comprised in the polypeptide composition are epitopes of their respective corresponding antigenic proteins. In one embodiment, the epitope corresponding to the isolated polypeptide is immunogenic. In one embodiment, the isolated polypeptide may be part of a corresponding antigenic protein that is shorter in length than the full length antigenic protein. The invention also provides methods of stimulating an immune response in a subject individual and methods of preventing and/or treating cancer in a subject individual using the polypeptide compositions. The invention also provides a vaccine comprising the polypeptide composition, and the vaccine has the purpose of treating and/or preventing cancers.

In the present invention, cypB, WHSC2, SART3, p56, p14, gp100, EPS8, p53, p16 and p21 represent their respective antigenic proteins, with the meanings and sequences known in the art, including the amino acid wild-type sequences and various known or possible variant sequences of their respective molecules.

In a preferred embodiment, the isolated polypeptide comprises an epitope of the corresponding antigenic protein described above. In a preferred embodiment, the epitopes are each capable of eliciting an immune response, either individually or together. In a preferred embodiment, the immune response may be a cell-mediated immune response and/or a humoral immune response. In a preferred embodiment, one or more of the epitopes or the polypeptides of the polypeptide composition are capable of providing a prophylactic and/or therapeutic effect on cancer.

In a preferred embodiment, the isolated polypeptide is within 50 amino acids in length; preferably within 30 amino acids. For example, the isolated polypeptide may be 5-30 amino acids, 8-25 amino acids, 8-15 amino acids, or 9-12 amino acids in length. For example, the isolated polypeptide may be 9 amino acids in length.

In a preferred embodiment, the isolated polypeptide in the polypeptide composition may be a variant comprising an epitope of the isolated polypeptide. The variants are functional equivalents of the isolated polypeptide having an altered sequence that has one or more amino acid substitutions in an epitope sequence corresponding to that contained in the isolated polypeptide, or has one or more amino acids added to the epitope sequence, or has one or more amino acids deleted from the epitope sequence without affecting the function of the isolated polypeptide comprising the epitope and the polypeptide composition comprising the isolated polypeptide. In a preferred embodiment, 1-5 amino acids, preferably 1-3 amino acids, are added to the N-and/or C-terminus of the epitope sequence.

In a preferred embodiment, the isolated polypeptide derived from SART3 is an epitope of SART 3. Preferably, it comprises the sequence shown in SEQ ID NO. 1.

In a preferred embodiment, the isolated polypeptide derived from p56 is an epitope of p 56. Preferably, it comprises the sequence shown in SEQ ID NO. 2.

In a preferred embodiment, the isolated polypeptide derived from p14 is an epitope of p 14. Preferably, it comprises the sequence shown in SEQ ID NO. 3.

In a preferred embodiment, the isolated polypeptide derived from gp100 is an epitope of gp 100. Preferably, it comprises the sequence shown in SEQ ID NO. 4.

In a preferred embodiment, the isolated polypeptide derived from EPS8 is an epitope of EPS 8. Preferably, it comprises the sequence shown in SEQ ID NO. 5.

In a preferred embodiment, the isolated polypeptide derived from p16 is an epitope of p 16. Preferably, it comprises the sequence shown in SEQ ID NO. 6.

In a preferred embodiment, the isolated polypeptide derived from CypB is an epitope of CypB. Preferably, it comprises the sequence shown in SEQ ID NO. 7.

In a preferred embodiment, the isolated polypeptide derived from WHSC2 is an epitope of WHSC 2. Preferably, it comprises the sequence shown in SEQ ID NO. 8.

In a preferred embodiment, the isolated polypeptide derived from p53 is an epitope of p 53. Preferably, it comprises the sequence shown in SEQ ID NO. 9.

In a preferred embodiment, the isolated polypeptide derived from p21 is an epitope of p 21. Preferably, it comprises the sequence shown in SEQ ID NO. 10.

In a preferred embodiment, the amino acid substitutions may be non-conservative amino acid substitutions or conservative amino acid substitutions. Conservative amino acid substitutions refer to substitutions of amino acids having similar structure and chemical properties as the corresponding amino acids in the wild-type isolated polypeptide. For example, conservative amino acid substitutions may include an aliphatic or hydrophobic amino acid substitution, such as an alanine, valine, leucine, and isoleucine substitution; substitutions comprising hydroxy amino acids, such as serine and threonine; also possible are the exchanges between acidic amino acids, such as aspartic acid and glutamine; also, aromatic amino acid exchanges, such as exchanges between phenylalanine and tyrosine; basic amino acid exchanges, such as exchanges between lysine, arginine and histidine; small amino acid exchanges, such as exchanges between alanine, serine, threonine, methionine and glycine, are also possible.

The polypeptide composition may comprise a suitable carrier or excipient. Such a carrier or excipient should not have any substantial effect on the survival and biological function of the DCs. The polypeptide composition may be in a form acceptable in the art, such as a lyophilized powder. Alternatively, the polypeptide composition may be in the form of a solution, such as comprising sterile injectable water or an organic solvent, such as DMSO.

The dosage ratio of each polypeptide in the polypeptide composition is not particularly limited, and the weight ratio of any two polypeptides can be in the range of 1:3 to 3:1. Generally, the polypeptides are used in the same amount, i.e., in a weight ratio of 1, in the polypeptide composition.

It will be appreciated that the polypeptide compositions of the invention may be prepared by conventional methods well known in the art, including, but not limited to, chemical synthesis by solid phase synthesis followed by HPLC separation of the synthesized product from the byproducts, and expression of nucleic acids encoding polypeptides comprising the antigenic fragments in the polypeptide compositions of the invention in living cells or purification after translation of the above-described encoding nucleic acids by an in vitro cell-free translation system to obtain the antigenic peptide fragments in the polypeptide compositions of the invention. In addition, unwanted small molecules contained in the polypeptide compositions of the present invention can be removed by extensive dialysis, and the resulting product can be lyophilized and then added with other excipients to form the desired formulation. It will also be appreciated that some of the amino acids, mutants, chemical modifications etc. which may be attached to the polypeptide composition of the invention that are produced in the vaccine component do not substantially interfere with the recognition of the epitope sequence by the antibody or TCR.

Cells

The invention also provides a cell loaded with the polypeptide composition. In a preferred embodiment, the cells are antigen presenting cells (antigen presenting cell, APC) with HLA typing matched to the polypeptide being loaded. The antigen presenting cells may be professional antigen presenting cells (professional antigen presenting cell) or non-professional antigen presenting cells (non-professional antigen presenting cell). In a preferred embodiment, the professional antigen presenting cell is a DC, macrophage or B cell, preferably a DC. The non-professional antigen presenting cells are antigen presenting cells that express HLA class I. The antigen presenting cells are loaded with the polypeptide composition by exposure to the polypeptide composition, e.g., by incubation with the polypeptide composition, resulting in antigen presenting cells loaded with the polypeptide composition.

Those skilled in the art know and understand that APCs are "pulsed" or loaded with fragments comprising epitopes of the antigen in a manner that they are exposed to the antigen, the exposure time being long enough to enable fragments comprising epitopes of the antigen to be presented to the surface of the APC. In a preferred embodiment, the APC may be exposed to an antigen in the form of a plurality of short polypeptide fragments, i.e. to an antigenic peptide, which is directly loaded onto the APC surface. In addition to short polypeptide fragments, APCs can also be incubated with large fragments derived from antigen proteins, whole antigen whole proteins, or particles comprising antigen proteins. The large fragments derived from the antigen protein, the whole antigen protein or the particles comprising the antigen protein may be engulfed by the APC by endocytosis or the like, and then processed into short polypeptide fragments by lysosomes or proteasomes and finally carried and presented to the surface of the APC, binding to HLA at the surface of the APC to form an antigen presenting complex.

In a preferred embodiment, antigen presenting cells loaded with the polypeptide composition may be prepared by contacting an APC with one or more isolated polypeptides in the above polypeptide composition, either in vitro (in vitro) or in vivo (in vivo). When the APC is loaded with the antigenic peptide of the polypeptide composition in vitro, the APC may be plated on a petri dish or well plate and then exposed to a sufficient amount of the polypeptide composition comprising the antigenic peptide and contacted therewith for a sufficient period of time to allow the antigenic peptide to bind to the APC. The amount of antigen peptide required to bind to APC and the time of binding can be determined by detection methods well known in the art. Other methods known to those skilled in the art, such as immunoassays or binding assays, can also be used to detect whether an APC is loaded with an antigenic peptide after exposure to a polypeptide composition comprising the antigenic peptide.

In a preferred embodiment, the APC is a DC, and the source of the DC may be autologous or allogeneic. In a preferred embodiment, the DCs may be isolated from a subject. In another preferred embodiment, the DCs may be artificially constructed DC cell lines with similar biological properties to natural DCs, which are similar to natural DCs in cell morphology and/or gene phenotype, such as DC cells transduced with lentiviral vectors expressing the Tax gene described in CN201810368646.3, which are negative for CD3 expression, and which express DC marker molecules such as CD70, CD80, CD83, CD86, CCR7 and HLA-DR; also as described in US20050272151A1 is a GEN2.2 cell line, which is a plasmacytoid DC cell line, having a cd4+, HLA-dr+, cd123+, cd45ra+, CD11c-, CD 13-phenotype. In another preferred embodiment, the DCs may be differentiated from a DC precursor cell line, such as from the MUTZ-3 cell line, which is a cell line expressing the monocyte marker molecule monocyte-specific esterase and CD14 (Santegoets SJ, van den Eertwegh AJ, van de Loosdrecht AA, scheper RJ, de Gruijl TD. Human dendritic cell line models for DC differentiation and clinical DC vaccination, studies. J Leukoc biol. 20088 Dec;84 (6): 1364-73.).

In a preferred embodiment, the antigen presenting cells loaded with the polypeptide composition of the invention are DCs, preferably the DCs may be derived from monocytes. For example, the DCs may be obtained by isolating PBMC from the blood of a subject, isolating monocytes therefrom, and then adding appropriate cytokines such as GM-CSF and IL-4 to the monocytes to induce the differentiation of the monocytes toward DCs. For another example, the DCs may be obtained by adding the above cytokines to the cell line of immortalized monocytes and inducing their differentiation into DCs. The DCs may also be obtained directly from an immortalized DC cell line, such as the DC cell line described in CN201810368646.3 transduced with a lentiviral vector expressing the Tax gene. In a preferred embodiment, the working concentration of GM-CSF is 50-500ng/mL, preferably 50-100ng/mL, more preferably 100ng/mL; IL-4 is present at a working concentration of 5-100ng/mL, preferably 10-50ng/mL, more preferably 50ng/mL.

In a preferred embodiment, the monocytes are induced to differentiate into immature DCs at day 5 after the addition of the cytokine, at which time the polypeptide composition of the invention is contacted, e.g., co-incubated, with the immature DCs to load the polypeptide composition of the invention with the immature DCs. In a preferred embodiment, the working concentration of each polypeptide in the polypeptide composition of the invention is 10-100. Mu.g/mL; preferably 20-80. Mu.g/mL; more preferably 40. Mu.g/mL. In a preferred embodiment, the immature DC and the polypeptide composition can be contacted for 1-2 days, preferably 2 days. In a preferred embodiment, the immature DC is still not fully mature after 1-2 days of contact with the polypeptide composition, i.e., 6-7 days after cytokine addition. At this time, a DC maturation-promoting factor, such as one or more selected from the group consisting of TNF-alpha, IL-1β, IL-6, PGE2, IFN- γ, poly (I: C), R848 and ATP, is added to a mixture comprising an immature DC and a polypeptide composition of the present invention, and incubated for 8 to 48 hours, preferably 24 hours, to allow the DC to mature completely, thereby obtaining a DC loaded with the polypeptide composition of the present invention. In a preferred embodiment, the DC maturation-promoting factors include TNF-alpha, IL-1β, IL-6 and PGE2, preferably TNF-alpha at a working concentration of 5-50ng/mL, such as 10-30ng/mL, IL-1β at a working concentration of 5-50ng/mL, such as 10-30ng/mL, IL-6 at a working concentration of 800-1500U/mL, such as 800-1200U/mL, and PGE2 at a working concentration of 0.5-3 μg/mL, such as 0.5-1.5 μg/mL. In a preferred embodiment, the DC maturation-promoting factors include IFN-gamma, poly (I: C), and R848. In another preferred embodiment, the DC maturation-promoting factors include IFN-gamma, poly (I: C), R848, and ATP. In a preferred embodiment, IFN-gamma is administered at a concentration of 10-1000IU/mL; preferably 100-300IU/mL; more preferably, 100IU/mL; the working concentration of poly (I: C) is 1-200. Mu.g/mL, preferably 20-40. Mu.g/mL; more preferably 30. Mu.g/mL; the working concentration of R848 is 0.1-50 mug/mL; preferably 1-10. Mu.g/mL; more preferably, 5. Mu.g/mL; the working concentration of ATP is 0.1-10mM; preferably 0.1-5mM; more preferably, it is 1mM. In a preferred embodiment, the DC maturation-promoting factors include IFN-gamma, poly (I: C), R848, and ATP, wherein the IFN-gamma has a working concentration of 100IU/mL, the poly (I: C) has a working concentration of 30 μg/mL, and R848 has a working concentration of 5 μg/mL.

The present invention thus also provides a method of preparing a cell carrying a polypeptide composition comprising (1) contacting the aforementioned polypeptide composition with immature DC for antigen loading to obtain a DC-polypeptide mixture; (2) Contacting the DC-polypeptide mixture of (1) with a DC maturation-promoting factor to further induce DC maturation and obtain cells loaded with the aforementioned polypeptide composition.

In a preferred embodiment, the immature DCs can be differentiated from DC precursor cells. In a preferred embodiment, the mature DCs can be differentiated from monocytes. The monocytes may be capable of inducing differentiation of immature DCs in vitro upon contact with the cytokine GM-CSF and IL-4 as is well known in the art. The amount of GM-CSF and IL-4 may be any of the amounts already reported as known in the art. Preferably, the working concentration of GM-CSF is 50-500ng/mL, preferably 50-100ng/mL, more preferably 100ng/mL; IL-4 is present at a working concentration of 5-100ng/mL, preferably 10-50ng/mL, more preferably 50ng/mL. In a preferred embodiment, the monocytes may be obtained by isolation of PBMCs from the individual followed by culturing. The separation method of the PBMCs may be a method well known in the art, such as drawing blood from an individual and separating by density gradient centrifugation. The isolated PBMC were cultured and the adherent cells were essentially monocytes. The time for culturing the PBMCs is preferably 2-8 hours, more preferably 8 hours.

In a preferred embodiment, the precursor cells of the immature DC can be CD34 derived from hematopoietic stem cell lineages ⁺ DC precursor cells. In a preferred embodiment, the CD34+ DC precursor cells are isolated from cord blood and may be subjected to substantial expansion and then contacted with the cytokines GM-CSF and IL-4 to induce differentiation into immature DCs. CD34 ⁺ Methods for the bulk expansion of DC precursor cells may be those known in the art, see in particular the methods disclosed in WO2010055900 A1. In another preferred embodiment, the precursor cells of the immature DCs may be immortalized DC precursor cell lines. The MUTZ-3 cell line as described above, which is a cell line expressing the monocyte marker molecules monocyte-specific esterase and CD 14.

In a preferred embodiment, the immature DC cells can be cell lines that have been transgenic to have the ability to immortalize unlimited expansion in vitro. A DC cell line transduced with a lentiviral vector expressing the Tax gene as described in CN 201810368646.3.

In a preferred embodiment, the cytokines GM-CSF and IL-4 are contacted with the precursor cells of said immature DCs by co-incubation in a culture medium. The duration of the co-incubation is a time known in the art, and is standard to enable differentiation of immature DC precursor cells into immature DCs. In a preferred embodiment, the duration of the co-incubation is 3-6 days; preferably, 3 to 5 days; more preferably, it is 5 days. The medium in which the cytokines GM-CSF and IL-4 are incubated with the precursor cells of the immature DCs may be any medium conventional in the art suitable for culturing immune cells. In a preferred embodiment, the medium may be any one or more selected from AIM-V, DMEM and RPMI-1640, preferably AIM-V medium. In a preferred embodiment, the AIM-V medium is a serum-free medium.

The working concentrations of the cytokines GM-CSF and IL-4 incubated with the precursor cells of the immature DCs may be concentrations well known in the art, as standard to be able to achieve differentiation of precursor cells, such as monocytes, of the immature DCs into the immature DCs. For example, the working concentrations of GM-CSF and IL-4 described in Chinese patent application CN201610522851.1 can be used. In a preferred embodiment, the working concentration of GM-CSF may be in the range of 5 to 300ng/mL; preferably, 10-100ng/mL; more preferably, 50-100ng/mL; even more preferably 100ng/mL. In a preferred embodiment, IL-4 working concentration of 5-100ng/mL; preferably, 10-100ng/mL; more preferably, 10-50ng/mL; even more preferably 10ng/mL.

The contacting of the polypeptide composition of the invention with the immature DC may be co-incubation in a medium. The duration of the co-incubation may be that of loading of the DC with the polypeptide as known in the art, subject to the ability of the antigenic peptide in the polypeptide composition of the invention to bind to the HLA of the DC surface to form an HLA-antigenic peptide complex. In a preferred embodiment, the duration of the co-incubation is 1-2 days, preferably 2 days. In a preferred embodiment, the medium used for incubation may be any medium conventional in the art suitable for culturing immune cells. In a preferred embodiment, the medium may be any one or more selected from AIM-V, DMEM and RPMI-1640, preferably AIM-V medium. In a preferred embodiment, the AIM-V medium is a serum-free medium.

Following contact of an immature DC with a polypeptide composition of the invention, the polypeptide composition of the invention has a certain promoting effect on maturation of the DC, but still requires the addition of additional DC maturation-promoting factors and contact with the mixture of DC and polypeptide composition. Additional DC maturation-promoting factors that may be added may be factors known or disclosed in the art that promote DC maturation, including, but not limited to, any one or more of TNF- α, IL-1β, IL-6, PGE2, IFN- γ, poly (I: C), R848, and ATP. In a preferred embodiment, the DC maturation-promoting factor is one or more selected from the group consisting of TNF-alpha, IL-1 beta, IL-6, PGE2, IFN-gamma, poly (I: C), R848, and ATP. In another preferred embodiment, the DC maturation-promoting factors added include TNF- α, IL-1β, IL-6 and PGE2; preferably, the working concentration of TNF- α is 5-50ng/mL, such as 10-30ng/mL; IL-1β working concentrations of 5-50ng/mL, such as 10-30ng/mL; IL-6 working concentration is 800-1500U/mL, such as 800-1200U/mL; PGE2 may be used at a working concentration of 0.5-3. Mu.g/mL, such as 0.5-1.5. Mu.g/mL. In another preferred embodiment, the DC maturation-promoting factors include IFN-gamma, poly (I: C), and R848. In another preferred embodiment, the DC maturation-promoting factors include IFN-gamma, poly (I: C), R848, and ATP. In a preferred embodiment, IFN-gamma is administered at a concentration of 10-1000IU/mL; preferably 100-300IU/mL; more preferably, 100IU/mL; the working concentration of poly (I: C) is 1-200. Mu.g/mL, preferably 20-40. Mu.g/mL; more preferably 30. Mu.g/mL; the working concentration of R848 is 0.1-50 mug/mL; preferably 1-10. Mu.g/mL; more preferably, 5. Mu.g/mL; the working concentration of ATP is 0.1-10mM; preferably 0.1-5mM; more preferably, it is 1mM. In a preferred embodiment, the DC maturation-promoting factors include IFN-gamma, poly (I: C) and R848, wherein IFN-gamma has a working concentration of 100IU/mL, poly (I: C) has a working concentration of 30 μg/mL, and R848 has a working concentration of 5 μg/mL.

In a preferred embodiment, the contacting of the DC-polypeptide mixture with the DC-maturation-promoting factor is co-incubation in a medium. The duration of the co-incubation may be of a duration known in the art, normalized to the ability of the DC maturation-promoting factor to induce immature DC to mature. In a preferred embodiment, the duration of the co-incubation may be 24-72 hours; preferably 24-48 hours; more preferably 24 hours. In a preferred embodiment, the medium used for incubation may be any medium conventional in the art suitable for culturing immune cells. In a preferred embodiment, the medium may be any one or more selected from AIM-V, DMEM and RPMI-1640, preferably AIM-V medium. In a preferred embodiment, the AIM-V medium is a serum-free medium.

The invention accordingly also provides an activated immune effector cell obtained by contacting an inactivated immune effector cell with a cell carrying a polypeptide composition of the invention as described above. Cells such as DCs capable of supporting the polypeptide composition provided by the invention express a subtype of HLA such as a11 on their surface, and an antigen peptide such as a tumor antigen peptide that matches the HLA of the particular subtype binds to the subtype to form an HLA-antigen peptide complex, which is then recognized and bound by a receptor TCR whose T cell surface specifically recognizes the complex, and T cells expressing the TCR are thereby stimulated and begin to proliferate.

In a preferred embodiment, the immune effector cell is selected from T cells or NK cells, preferably T cells. In a preferred embodiment, the cells loaded with the polypeptide composition of the invention are DCs. In a preferred embodiment, the immune effector cell and the cell carrying the polypeptide composition of the invention are from the same individual or from different individuals; preferably from the same individual. In a preferred embodiment, the cells loaded with the polypeptide composition are DCs and the immune effector cells are T cells. The ratio of the number of DCs to the T cells of the supported polypeptide composition may be any ratio known in the art as long as the DCs of the supported polypeptide composition are effective to activate T cells that recognize their surface HLA-antigen peptide complexes, preferably 1:10-1:50, more preferably 1:20-1:50, even more preferably 1:20. In a preferred embodiment, the contacting of the DC of the supported polypeptide composition with T cells is co-incubation. Preferably, the duration of the co-incubation is 2-48 hours; more preferably 24-48 hours; even more preferably, 24 hours. In a preferred embodiment, the co-incubation is performed in a medium, which is AIM-V, DMEM or RPMI1640; preferably, the co-incubation is performed in AIM-V medium; more preferably, the AIM-V medium comprises 2% V/V fbs. In a preferred embodiment, the AIM-V medium further comprises IL-2. Preferably, the IL-2 is present at a working concentration of 10-100U/mL, such as 100U/mL.

Vaccine

The invention also provides a vaccine suitable for immunotherapy. In a preferred embodiment, the vaccine is a tumor vaccine comprising the aforementioned polypeptide composition. In a preferred embodiment, the tumor vaccine may be injected into a subject and HLA-antigen peptide complexes are formed by binding to antigen presenting cells such as DCs in the subject to HLA recognizing the corresponding subtype of antigen peptide in the tumor vaccine, which bind to the corresponding specific TCR and activate T cells expressing the specific TCR.

In another preferred embodiment, the vaccine is a DC vaccine comprising a DC loaded with a polypeptide composition of the invention as described above. In a preferred embodiment, the DCs may be DCs differentiated from DC precursor cells isolated from autologous blood derived from a subject, hematopoietic precursor cells such as cd34+ derived from umbilical cord blood, or monocytes derived from cd14+ from peripheral blood. After co-incubation of the polypeptide composition of the present invention with autologous DCs obtained after separation, culture, expansion and differentiation in a subject, a cell mixture preparation comprising the polypeptide composition of the present invention and mature DCs loaded with the antigenic peptides in the polypeptide composition is obtained. The method of differentiating the DC precursor cells into DC may be a method known in the art or any other method capable of differentiating the DC precursor cells into DC, such as differentiation culture by adding the cytokines GM-CSF and IL-4 to a medium. The cell mixture preparation is returned to the subject as a DC vaccine, the antigenic peptide in the polypeptide composition is presented via the subject's autologous mature DC, and specific T cells are activated to elicit an immune response in vivo against the epitope comprised by the antigenic peptide in the polypeptide composition. In another preferred embodiment, the DCs may be cells obtained from an immortalized DC precursor cell line that has been subjected to in vitro expansion culture followed by differentiation culture. The immortalized DC precursor cell line may be a cell line known in the art or publicly reported, such as the MUTZ3 cell line, or an immortalized DC precursor cell line prepared by the method described in CN 201810368646.3. The immortalized DC precursor cell line may be expanded in vitro in large amounts and then subjected to a differentiation culture to form DCs, which may be the method described above. The polypeptide composition of the present invention is incubated with a DC obtained by subjecting the immortalized DC precursor cell line to differentiation culture after amplification to obtain a cell mixture preparation comprising the polypeptide composition of the present invention and a mature DC loaded with an antigen peptide of the polypeptide composition, and the cell mixture preparation is introduced into a subject as a DC vaccine, and the loaded antigen peptide is presented by the DC to activate a specific T cell response. In a preferred embodiment, the vaccine comprising DC differentiated from the immortalized DC precursor cell line may be administered to the subject individual concurrently with an agent capable of reducing immune rejection, such as an inhibitor of endogenous TCR expression.

Verification of mature DCs can be by detection of molecular markers expressed on the surface of mature DCs and/or cytokines secreted by mature DCs as known in the art, including, but not limited to, CD80, CD83, CD86, CCR7, HLA-ABC, HLa-DR, and IL-6. The detection means employed in the validation method may be any detection means known in the art capable of detecting the molecular markers and/or cytokines described above, including, but not limited to ELISA, western hybridization and flow cytometer detection.

In a preferred embodiment, the vaccine further comprises an adjuvant. The adjuvant may be a small molecule, a biological macromolecule, a composition, a complex or an extract of a compound known in the art to be capable of enhancing the effect of an immune response. In a preferred embodiment, the adjuvant comprises a material selected from the group consisting of aluminum adjuvants (e.g., aluminum hydroxide), freund's adjuvants (e.g., complete Freund's adjuvant and incomplete Freund's adjuvant), prostaglandin E2, interferon-alpha, corynebacterium parvum, lipopolysaccharide, cytokines, oil-in-water emulsions, water-in-oil emulsions, nanoemulsions, microparticle delivery systems, liposomes, microspheres, biodegradable microspheres, plaque virions, proteoliposomes, proteasomes, immunostimulatory complexes (ISCOMs, ISCOMATRIX), microparticles, nanoparticles, biodegradable nanoparticles, silicon nanoparticles, polymeric microparticles/nanoparticles, polymeric Lamellar Substrate Particles (PLSP), microparticulate resins, nanoliposome polymeric gels (nanolog), synthetic/biodegradable and biocompatible semisynthetic or natural polymers or dendrimers (e.g., PLG, PLGA, PLA, polycaprolactone, silicone polymers, polyesters, polydimethylsiloxane, sodium polystyrene sulfonate, polystyrene benzyl trimethyl ammonium chloride, polystyrene divinylbenzene resins, polyphosphazenes, poly- [ di- (carboxyacetyl phenoxy) phosphazenes), poly- (PCP), poly- (methylnitrile) methyl methacrylate, poly- (vinyl ether), poly- (L-co-vinyl polysaccharide, poly- (L-glutamic acid), poly- (alphA-Amino-polysaccharide, poly- (alpha-polysaccharide, poly-L-polysaccharide, poly- (alpha-polysaccharide, poly-polysaccharide, poly-L-polysaccharide, polysaccharide derivatives thereof, and derivatives thereof, biopolymer), cationic Dimethyl Dioctadecyl Ammonium (DDA), alpha-galactosyl ceramide and derivatives thereof, archaebacteria lipids and derivatives, lactam, gallin, glyceride, phospholipid and spirochete.

In a preferred embodiment, the vaccine is used for the prevention or treatment of cancer. In a preferred embodiment, the administration of the vaccine to a patient according to the invention may occur before or after surgical removal of the tumor, or before or after treatment of the cancer with chemotherapy. In another preferred embodiment, the vaccine may be administered to an individual suffering from cancer, either together with other compositions or pharmaceutical products or in combination. It will be appreciated that the vaccine of the invention may be administered to individuals who are not suffering from cancer but who are at risk of suffering from cancer, in addition to individuals who are already suffering from cancer.

The vaccine prepared according to the present invention may be widely applied to the treatment or prevention of cancer, depending in part on the choice of antigen-forming portion of the vaccine. Cancers that can be treated or prevented in accordance with the practice of the invention include, but are not limited to, lung cancer, non-small cell lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, renal cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancy, head and neck cancer, glioma, mesothelioma, carcinoma of the large intestine, gastric cancer, nasopharyngeal carcinoma, laryngeal carcinoma, cervical cancer, uterine fibroids and osteosarcomas, bone cancer, pancreatic cancer, renal cell carcinoma, skin cancer, prostate cancer, cutaneous or intraocular malignant melanoma, uterine cancer, anal region cancer, testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulval cancer, hodgkin's disease, non-hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, biliary tract cancer, thyroid cancer, parathyroid cancer, adrenal tissue sarcoma, urinary tract cancer, urothelial carcinoma, penile carcinoma, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia), childhood solid leukemia, renal cell carcinoma, lymphoblastic carcinoma, renal cell carcinoma, carcinoma of the human tumor, lymphoblastic carcinoma of the human tumor, lymphomas of the CNS, lymphomas of the CNS, lymphomas, tumors of the nervous system, lymphomas, tumors of the CNS, lymphomas, including asbestos-induced cancers and various leukemia and lymphoma and various precancerous lesions

In another preferred embodiment, the vaccine may be administered to the inguinal segment by intra-segment injection. Alternatively, depending on the target of the vaccine, the vaccine may be administered subcutaneously or intradermally to the extremities of a patient suffering from cancer that is being treated. Other routes of administration, such as intramuscular injection or blood injection, may also be employed.

In addition, the vaccine may also be administered with adjuvants and/or immunomodulators to enhance its activity in the immune response of the patient. The adjuvant can be selected from any one or more of the above adjuvants, and can be selected and combined differently according to the specific situation. The immunomodulator may be small molecules, biological macromolecules, extracts, pharmaceutical compositions and/or complexes known in the art to have immunomodulating activity, and may be obtained in papers, textbooks, conference notes, etc. which have been published in the art.

In a preferred embodiment, depending on the type of vaccine prepared, the production scale of the vaccine can be expanded if necessary by culturing the cells in a bioreactor or fermenter or similar container and apparatus suitable for cell mass growth. In a preferred embodiment, the device or composition comprising the vaccine or antigen produced or recovered according to the invention is adapted for sustained or intermittent release, and may be implanted in the body or applied topically at the corresponding location in the body, to achieve a slow and timed release of these materials into the body.

Method for treating diseases

The invention also provides a method of treating and/or preventing cancer comprising administering to a subject an effective dose of one or more of the foregoing polypeptide compositions, pharmaceutical compositions, cells loaded with polypeptide compositions, and vaccines.

In a preferred embodiment of the present invention, in a preferred embodiment, such cancers include, but are not limited to, lung cancer, non-small cell lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, renal cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancy, head and neck cancer, glioma, mesothelioma, large intestine cancer, stomach cancer, nasopharyngeal carcinoma, laryngeal carcinoma, cervical cancer, uterine body and osteosarcoma, bone cancer, pancreatic cancer, renal cell carcinoma, skin cancer, prostate cancer, cutaneous or intraocular malignant melanoma, uterine cancer, anal region cancer, testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulval cancer, hodgkin's disease, non-hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, bile duct cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urinary tract cancer, urothelial cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia), solid tumors, childhood lymphoma, renal or ureteral carcinoma, renal cell carcinoma, carcinoma of the human tumor, cancer of the renal system, cancer of the human tumor, tumor of the CNS, tumor of the human nervous system, tumor of the human tumor, tumor of the brain, tumor of the nervous system, tumor of the human system, tumor of the brain, tumor of the nervous system, tumor, including asbestos-induced cancers and various leukemia and lymphoma and various precancerous lesions.

In a preferred embodiment, the method comprises the effect of at least one of treating and preventing. In a preferred embodiment, the methods of the invention are for prophylactic purposes, and one or more of the polypeptide compositions, pharmaceutical compositions, cells loaded with the polypeptide compositions, and vaccines of the invention are administered to a subject individual prior to the occurrence of a cancer or precancerous condition. In certain instances, the vaccine is administered to the subject individual after the onset of one or more of the cancers described above, with the aim of preventing further symptoms from occurring or further exacerbation of symptoms that have occurred. Prophylactic administration of one or more of the polypeptide compositions, pharmaceutical compositions, cells loaded with the polypeptide compositions, and vaccines of the present invention is intended to prevent or alleviate any subsequent symptoms. In another preferred embodiment, the methods of the invention are for therapeutic purposes, and one or more of the polypeptide compositions, pharmaceutical compositions, cells loaded with the polypeptide compositions, and vaccines of the invention are administered to a subject individual at or after the onset of cancer, with the aim of alleviating the symptoms of the cancer that has developed.

In a preferred embodiment, the effective dose for any particular therapeutic application in the method may be determined depending on different factors, such as the type of cancer, the extent of the cancer onset, the condition of the subject individual, such as age, sex, weight, level of various body indicators, etc., as well as the composition of the particular agent being administered and the particular mode of administration. For an effective dose of an agent comprising one or more of the polypeptide composition, pharmaceutical composition, cells loaded with the polypeptide composition, and vaccine of the present invention to be administered, one skilled in the art can empirically determine the specific components contained in the agent without performing additional unnecessary experimentation.

In a preferred embodiment, the specific manner of administration of one or more of the polypeptide composition, pharmaceutical composition, cells loaded with the polypeptide composition, and vaccine may be determined by one of skill in the art, depending on the type of cancer, the extent of the cancer onset, the condition of the subject individual, the subject individual's condition such as age, sex, weight, the level of each physical indicator, etc., and the composition of the particular agent administered, and may be determined, for example, by means including but not limited to intravenous, intramuscular, intradermal, transdermal, intra-arterial, intraperitoneal, intralesional, intracranial, intra-articular, intraprostatic, intrapleural, intratracheal, intrathecal, intranasal, intravaginal, intrarectal, parenteral, systemic, local, intratumoral, intraperitoneal, subcutaneous, subconjunctival, transmucosal, intracardiac, intraorbital, oral, transdermal, intrapulmonary, inhaled, injectable, implantable, retrograde, continuous retrograde, topical, transcatheter, via catheter, via lavage, and liposomal administration to the subject by one or more of the subject compositions.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute a preferred technical solution.

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

The term "room temperature" as used in the examples refers to the temperature between the operations in which the test is conducted, typically 25 ℃.

The term "overnight" as used in the examples means more than 8 hours.

The following examples provide a number of polypeptide compositions comprising a plurality of tumor antigen peptides for higher proportion of HLA typing-A11 in the Chinese population, each polypeptide composition species comprising at least 3 tumor associated epitope polypeptides for HLA-A11 typing. The polypeptides used in the examples below are shown in table 1 below:

TABLE 1

The polypeptide compositions used in the examples below are shown in Table 2 below:

TABLE 2

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The DC precursor cells and T cells in the following examples were isolated from blood PBMCs of donor subjects, as follows: mixing blood of HLA-A11 type donor subject with equal volume of physiological saline, slowly adding the mixture into a centrifuge tube containing 15mL of Ficoll along the tube wall, obviously layering the blood and the Ficoll, centrifuging for 20min at 800g, sucking a white cell layer after centrifuging, transferring to another centrifuge tube, adding physiological saline, 1500rpm for 10min, centrifuging for cleaning, discarding waste liquid, and adding physiological saline for centrifuging and cleaning once. Transferring the cleaned cells into a culture flask for culturing and adhering overnight, collecting suspension cells, namely T cells, counting, freezing, and taking the remaining adhering cells as mononuclear cells (DC precursor cells) which can be induced and differentiated into DC.

The working concentration of each polypeptide in the practical polypeptide composition was 40. Mu.g/mL, unless otherwise specified in the examples below.

Example 1 polypeptide composition induces DC maturation and activation

DC precursor cells were added to serum-free AIM-V medium (available from Gibco), IL-4 and GM-CSF were added to working concentrations of 50ng/mL and 100ng/mL, respectively, and culture was continued. Half-volume liquid exchange is carried out until the third day, and IL-4 and GM-CSF are added to the working concentrations of 50ng/mL and 100ng/mL respectively. And (3) culturing until the fifth day, respectively adding the polypeptide compositions 1 and 2 into different groups of DC precursor cells, wherein the specific adding method is that half of the culture medium is replaced, then each peptide stock solution is taken out from a refrigerator at the temperature of minus 80 ℃, naturally melted at room temperature, uniformly mixed by vortex, and added into a culture bottle until the working concentration of each peptide in the bottle is 40 mug/mL. As a control, DC not loaded with the polypeptide composition was additionally set. And the repeated freezing and thawing of the polypeptide stock solution are avoided in the use process. And (3) culturing until the seventh day, adding IFN-gamma, poly (I: C) and R848 according to the marked dosage, wherein the working concentration of the IFN-gamma is 100IU/mL, the working concentration of the poly (I: C) is 30 mug/mL, and the working concentration of the R848 is 5 mug/mL, and continuously culturing for 24 hours to further stimulate and activate DC. Cell culture supernatants were collected, IL-12 content was detected by ELISA, the collected DCs were gently flicked, incubated with FITC-labeled CD80 antibody and APC-labeled HLA-ABC antibody, respectively, for 30 minutes in the dark from the collected DCs, and cells were loaded to a flow cytometer for detection of CD80 and HLA-ABC positive rates. CD80 is one of the surface markers of mature DCs, HLA-ABC can represent the expression of major MHC class I molecules, whose expression is closely related to antigen presentation.

The results are shown in FIGS. 1-2. FIGS. 1a and 1b show that the IL-12 content of the cell culture supernatant after treatment with polypeptide composition 1 or 2, respectively, was about 600pg/mL, which is much higher than the IL-12 secretion of DC without the polypeptide composition. FIGS. 2a and 2b show that the CD80 and HLA-ABC positives of DC after stimulation and activation by polypeptide compositions 1 or 2, respectively, are both higher, indicating higher DC maturity and greater antigen presenting capacity, as shown in FIGS. 2a and 2 b.

Example 2 polypeptide composition induces cell activation phenotype detection of DC activated T cells

Taking DC activated by the stimulation of the polypeptide composition 1 or 2 in the example 1, incubating the DC and T cells of a corresponding donor subject for 24 hours according to the ratio of 1:20, adopting AIM-V culture medium containing 2% FBS, adding IL-2 until the working concentration reaches 100U/mL, and culturing for 3 days to obtain DC-CTL. After co-incubation with fluorescent antibodies against CD3, CD4 and CD8 with the above-described DC-CTL and control T cells (T cells not co-incubated with DC), respectively, the ratio of cd3+cd4+ cells to cd3+cd8+ cells in the DC-CTL cells and control T cells was tested by flow cytometry.

After incubating the above DC-CTLs overnight with stimulation cocktail (plus protein transport inhibitors) (available from eBioscience; cat# 00-4975-93), the fraction of CD3+CD1080a+ cells was measured by flow cytometry by incubating DC-CTLs and control T cells with fluorescent antibodies against CD3 and CD107a, respectively.

The secretion level of IFN-gamma is detected by flow cytometry, and the specific operation steps are shown in Slagter-JG, raney a, lewis WE, debanedette MA, nicolette CA, tcherepanova iy. Evaluation of RNA Amplification Methods to Improve DC Immunotherapy Antigen Presentation and Immune response. Mol Ther Nucleic acids.2013may7; 2 or Tsing-Lee Tang-Huau, paul Guegue, christel Goudet, melanie Durand, myl re Bohec, sylvain Baulode, benoit Pasquier, sebastian Amigorena, elodie Segura Human in vivo-generated monocyte-derived dendritic cells and macrophages cross-present antigens through a vacuolar path. Nature Communications 9,Article number:2570 (2018)

The results are shown in FIGS. 3-4. FIGS. 3a and 4a show that after co-incubation with polypeptide composition 1 (FIG. 3 a) and polypeptide composition 2 (FIG. 4 a), the DC-CTL has no significant change in the ratio of CD3+CD4T cells to CD3+CD8T cells compared to control T cells.

FIGS. 3b and 4b show that, following co-incubation with polypeptide composition 1 (FIG. 3 b) and polypeptide composition 2 (FIG. 4 b), the positive proportion of CD3+CD7a cells was significantly increased in DC-CTL compared to control T cells;

FIGS. 3c and 4c show that after co-incubation with polypeptide composition 1 (FIG. 3 c) and polypeptide composition 2 (FIG. 4 c), the levels of intracellular IFN- γ were significantly higher in DC-CTL compared to control T cells.

The results show that the DC-CTL CD4+/CD8+ ratio obtained after the polypeptide composition is treated is not affected, but the cell activation degree is higher, and the cell factor IFN-gamma with higher level can be secreted, so that the killing function on tumor cells is indirectly reflected to be stronger.

Example 3 detection of the cell proliferation Capacity of polypeptide compositions to induce DC activated T cells

Taking the DC and phase of example 1 after stimulation activation with polypeptide composition 1 or 2Donor subject T cells were incubated at a ratio of 1:20 for 24 hours, and 1X 10 was reserved prior to DC incubation with T cells ⁶ And centrifuging to wash out the culture medium, and freezing at-80 ℃ in a refrigerator to prepare the T cell proliferation standard curve. Incubating the non-supported polypeptide composition with DC and donor T cells loaded with polypeptide composition 1 or 2 at a ratio of 1:20, and culturing with AIM-V medium containing 2% FBS, and adding IL-2 to a working concentration of 100U/mL; after incubation for 24 hours, blowing and evenly mixing cells in each hole by using a liquid transfer device, respectively taking 50 mu L of cell suspension from each culture hole, centrifuging to wash out a culture medium, freezing the culture medium at-80 ℃ in a refrigerator, and using the culture medium for testing T cell proliferation for 24 hours; after incubation for 48 hours, blowing and evenly mixing cells in each hole by using a liquid transfer device, respectively taking 50 mu L of cell suspension from each culture hole, centrifuging to wash out a culture medium, and freezing the culture medium at-80 ℃ in a refrigerator for testing the proliferation of T cells for 48 hours; after incubation for 72 hours, the well cells were blow-mixed with a pipette, 50. Mu.L of cell suspension was taken from each culture well, the culture medium was washed off by centrifugation, and frozen at-80℃for the test point for 72 hours of T cell proliferation. Specific test methods were as per Invitrogen Cell Proliferation Assay Kit instructions, each set of proliferation curves was generated based on the test results.

The results are shown in FIG. 5. FIGS. 5a and 5b show the proliferation curves of DC-CTL obtained after incubation of DC loaded with polypeptide composition 1 and DC loaded with polypeptide composition 2 with T cells, respectively. Figures 5a and 5b show that the proliferation levels of the DCs loaded with polypeptide compositions 1 and 2 were significantly increased after incubation with T cells compared to the cells obtained after incubation with T cells without any of the loaded DCs, indicating that the DCs loaded with polypeptide compositions 1 or 2 could effectively expand T cells. The expansion after T cell activation is also an important link for exerting the anti-tumor immunity effect, so that the polypeptide composition 1 or 2 has better potential anti-tumor effect.

EXAMPLE 4 Effect of the polypeptide composition on inducing killing of DC-activated T cells on pancreatic cancer cells PANC-1 (HLA-A 2, A11)

The killing effect of DC-activated T cells obtained as described in example 2 (without stimulation cocktail stimulation, otherwise in the same manner as described in example 2) by single peptides of polypeptide composition 1 and its constituent peptides 1, 2, and 3, respectively, on pancreatic cancer cells PANC-1 in vitro was examined using a real-time label-free cell function analyzer. Specifically, the real-time label-free cell function analyzer (RTCA) from the company eisen was used to detect the in vitro killing activity of the DC-CTLs obtained by loading DCs with the peptides 1, 2, and 3 and then activating the loaded DCs as in example 2, as follows:

(1) Zeroing: adding 50 mu L of DMEM culture solution into each hole, placing into an instrument, selecting step 1, and zeroing;

(2) Target cell plating: pancreatic cancer cells PANC-1 (purchased from American type culture Collection ATCC) at 10 per well ⁴ Spreading the cells/50 μl in a plate containing detection electrodes, standing for several minutes, placing into an instrument after the cells are stable, and starting step 2 to culture the cells;

(3) Adding effector cells: after culturing the target cells for 24 hours, step 2 was suspended, DC-CTL obtained by loading each of the peptides of polypeptide composition 1 and peptides 1, 2 and 3 as described above in example 2 was added at a target ratio of 50. Mu.l per well of 10:1, and step 3 was started using T cells from the same donor subject not loaded with any polypeptide composition or single peptide as a control, and after further co-culturing for 24 hours, the cell function curve recorded by the apparatus was observed.

The experimental procedure for killing PANC-1 by DC-loaded peptide 4, peptide 5 and peptide 6, which are separate components of polypeptide composition 2, was the same as that described above for polypeptide composition 1 and peptides 1-3.

The results are shown in FIGS. 6a, 6b and 6 c. FIGS. 6a and 6b, 6c show the killing curves of DC-CTL obtained by loading DC post-treated T cells with polypeptide composition 1 and its component peptides 1-3 and polypeptide composition 2 and its component peptides 4-6, respectively, against target cell PANC-1. Fig. 6b and 6c represent the results of two replicates of the same sample. Compared with the cell growth curve of PANC-1 cells, the curve of each other sample is reduced after the effector cells are added for 24 hours, which indicates that the DC-CTL treated by the polypeptide composition 1 or 2 and the component monopeptides thereof has growth inhibition effect on the HLA matched PANC-1 cells (HLA-A 2, A11). In particular, polypeptide compositions 1 and 2 have the strongest inhibitory effect on PANC-1 cells relative to their respective component monopeptides, and have significantly better killing effect on target cells PANC-1 than the respective monopeptides.

Example 5 polypeptide composition induces specific killing of DC-activated T cells against cancer cell PANC-1

The killing effect of DC-activated T cells obtained as described in example 2 (without stimulation cocktail stimulation, otherwise identical to the method described in example 2) on pancreatic cancer cells PANC-1 in vitro was examined using a real-time label-free cell function analyzer using polypeptide composition 5, polypeptide composition 6, polypeptide composition 7, polypeptide composition 8 and peptide 2, respectively. Peptide 7 in polypeptide compositions 5 and 8 is the cyclophilin B-derived antigenic peptide disclosed in SEQ ID No. 27 of chinese patent CN1318447C "tumor antigenic peptide derived from cyclophilin B"; peptide 8 is an antigenic peptide derived from WHSC2 as disclosed in SEQ ID NO. 12 of Chinese patent CN101854945B "CTL inducer composition". Wherein specifically, the in vitro killing activity of DC-CTL obtained after DC loading and activation of polypeptide compositions 5, 6, 7, 8 and peptide 2 by the method of example 2 was measured using a real-time label-free cell function analyzer (RTCA) from Aisen company, and the steps were as follows:

(3) Adding effector cells: after 24h of target cell culture, step 2 was suspended, DC-CTL obtained by loading each of the above-described polypeptide compositions 5, 6, 7, 8 and peptide 2 in accordance with the method of example 2 was added, 50. Mu.L per well, and the effective target ratio was set to 10:1, respectively, to start step 3 using T cells from the same donor subject not loaded with any polypeptide composition or single peptide as a control, and after continuing co-culture for 24h, the cell function curve recorded by the instrument was observed.

The results are shown in FIG. 7. FIG. 7 shows the killing curves of DC-CTL obtained by loading DC post-treatment T cells with polypeptide compositions 5, 6, 7, 8 and peptide 2, respectively, against target cell PANC-1. Compared to the cell growth curve with PANC-1 cells alone, the curve was decreased after 24h of effector cell addition for the remaining samples, indicating that DC-CTL treated with polypeptide compositions 5, 6, 7, 8 and peptide 2, respectively, had a growth inhibitory effect on HLA-matched PANC-1 cells (HLA-A 2, A11).

Further, in comparison to the T cell sample group of the subject not loaded with any polypeptide, the killing effect of the polypeptide compositions 5, 6, 8 and peptide 2 on PANC-1 cells after loading the subject DC is at the same level as compared to T cells not loaded with the polypeptide. The killing power of the polypeptide composition 7 on PANC-1 cells is remarkably higher compared with that of T cells without the loaded polypeptide, and the killing power of the polypeptide composition is remarkably superior to that of the polypeptide compositions 5, 6 and 8 and peptide 2 monopeptides.

EXAMPLE 6 polypeptide composition induces nonspecific killing of DC-activated T cells against ovarian adenocarcinoma cells SKOV-3

The killing effect of DC-activated T cells obtained in example 2 (without stimulation cocktail stimulation, otherwise identical to the method described in example 2) induced by each of the single peptides of polypeptide composition 1 and its constituent peptides 1, 2, and 3 on ovarian adenocarcinoma cells SKOV-3 in vitro was examined using a real-time label-free cell function analyzer. Specifically, the in vitro killing activity of the DC-CTL obtained by loading DC and then activating each of the single peptides of the polypeptide composition 1, the peptide 2 and the peptide 3 by the method of example 2 was measured by using a real-time label-free cell function analyzer (RTCA) of the Eisen company, and the steps are as follows:

(2) Target cell plating: ovarian adenocarcinoma cells SKOV-3 (purchased from American type culture Collection ATCC) at 10 per well ⁴ Spreading the cells/50 mu L in a plate containing a detection electrode, standing for a plurality of minutes, putting the cells into an instrument after the cells are stabilized, and starting step 2 to culture the cells;

The killing experimental procedure of the individual components peptide 4, peptide 5 and peptide 6 of the polypeptide composition 2 loaded DC on SKOV-3 was the same as that of the above-described polypeptide composition 1 and peptides 1-3.

The results are shown in FIG. 8. FIGS. 8a and 8b show the killing curves of DC-CTL obtained by loading DC post-treatment T cells with polypeptide composition 1 and its component peptides 1-3 and polypeptide composition 2 and its component peptides 4-6, respectively, against target cells SKOV-3. Compared with the cell growth curve of SKOV-3 cells only, the curve of each of the other groups of samples did not significantly decrease after 24h of effector cell addition. HLA typing of SKOV-3 cells is A3, and FIGS. 8a and 8b show that DC-CTL obtained by treating the polypeptide composition and the component monopeptides thereof have no nonspecific killing effect on HLA-3 cells (HLa-A3) with mismatched HLA types, and prove that the killing effect of the polypeptide composition on tumor cells caused by induction of DC-activated T cells is limited by MHC typing.

Comparison of the killing effect of DC-CTL obtained by treating polypeptide compositions 3 and 4 with the tumor cells by DC-CTL obtained by treating polypeptide compositions 1 and 2, respectively.

The components of polypeptide compositions 3 and 4 are shown in Table 2. In polypeptide composition 3, peptides 7 and 8 replace peptides 1 and 2 in polypeptide composition 1; in polypeptide composition 4, peptides 7 and 8 replaced peptides 4 and 6 in polypeptide composition 2. The killing effect of polypeptide compositions 1 and 3, and polypeptide compositions 2 and 4 on PANC-1 cells was compared, respectively, as in example 4.

The results are shown in FIG. 9. FIG. 9a shows that the killing effect of DC-CTL obtained after treatment with polypeptide composition 1 on target cell PANC-1 is significantly stronger than that obtained after treatment with polypeptide composition 3. FIG. 9b shows that the killing effect of DC-CTL obtained after treatment with polypeptide composition 2 on target cell PANC-1 is more pronounced than that obtained after treatment with polypeptide composition 4.

The results in fig. 9a and 9b demonstrate that polypeptide compositions 1 and 2 are better able to activate T cells after loading with DCs than polypeptide compositions 3 and 4, respectively, have a stronger ability to stimulate T cells to kill tumor cells and thus have a stronger potential anti-tumor effect.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be understood that various changes and modifications may be made by those skilled in the art after reading the foregoing description of the invention, and such equivalents are intended to fall within the scope of the claims appended hereto.

Sequence listing

<110> Shanghai cell therapy group Co., ltd

SHANGHAI CELL THERAPY Research Institute

<120> a polypeptide composition and vaccine

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<213> Artificial sequence (Artificial Sequence)

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Trp Leu Glu Tyr Tyr Asn Leu Glu Arg

1 5

<210> 2

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 2

Ile Gln Asn Leu Glu Arg Gly Tyr Arg

1 5

<210> 3

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 3

Ala Val Cys Pro Trp Thr Trp Leu Arg

1 5

<210> 4

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 4

Ala Val Gly Ala Thr Lys Val Pro Arg

1 5

<210> 5

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 5

Ser Val Leu Ser Pro Leu Leu Asn Lys

1 5

<210> 6

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 6

Gln Val Met Met Met Gly Ser Ala Arg

1 5

<210> 7

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 7

Lys Leu Lys His Tyr Gly Pro Gly Trp Val

1 5 10

<210> 8

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 8

Ala Ser Leu Asp Ser Asp Pro Trp Val

1 5

<210> 9

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 9

Cys Thr Tyr Ser Pro Ala Leu Asn Lys

1 5

<210> 10

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 10

Ser Met Thr Asp Phe Tyr His Ser Lys

1 5

Claims

1. A polypeptide composition is characterized in that the polypeptide composition consists of isolated polypeptides shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.

2. The polypeptide composition of claim 1, wherein the mass ratio of the isolated polypeptides represented by the sequences SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 is 1:1:1.

3. A pharmaceutical composition comprising a polypeptide composition according to claim 1 or 2.

4. A pharmaceutical composition according to claim 3, further comprising an adjuvant and/or a pharmaceutically acceptable salt.

5. A tumor vaccine comprising the polypeptide composition of claim 1 or 2.

6. The tumor vaccine of claim 5, further comprising an adjuvant.

7. A cell loaded with the polypeptide composition of claim 1 or 2.

8. The cell of claim 7, which is an antigen presenting cell.

9. The cell of claim 8, wherein the HLA class expressed by the antigen presenting cell is HLA-a11.

10. The cell of claim 9, wherein the antigen presenting cell is a dendritic cell derived from a monocyte in PBMC in blood.

11. A method for preparing a cell according to any one of claims 7 to 10, comprising the steps of:

(1) Contacting dendritic cell precursors with a cytokine, differentiating the dendritic cell precursors into immature dendritic cells;

(2) Contacting the polypeptide composition of claim 1 or 2 with the immature dendritic cells of (1) for antigen loading to obtain a dendritic cell-polypeptide mixture;

(3) Contacting the dendritic cell-polypeptide mixture of (2) with a dendritic cell maturation-promoting factor to further induce maturation of the dendritic cell to obtain a cell loaded with the polypeptide composition of claim 1 or 2.

12. The method of claim 11, wherein the dendritic cell precursor cells of (1) are monocytes in PBMCs in blood; and/or the number of the groups of groups,

(1) Wherein said cytokines comprise IL-4 and GM-CSF, said IL-4 having a working concentration of 50ng/mL and said GM-CSF having a working concentration of 100ng/mL; and/or the number of the groups of groups,

(1) Wherein said contacting is by adding said cytokine to a culture medium comprising said dendritic cell precursor cells, incubating for a period of 2-5 days; and/or the number of the groups of groups,

the temperature of the co-incubation is 37 ℃; and/or the number of the groups of groups,

the CO-incubated CO ₂ The concentration was 5%, the percentages being by volume.

13. The method of claim 11, wherein the polypeptides in the polypeptide composition of (2) are each added separately or mixed uniformly and then added to the immature dendritic cells of (1); and/or the number of the groups of groups,

(2) Wherein said contacting is by adding the polypeptide composition of any one of claims 1 or 2 to a medium comprising said immature dendritic cells and said cytokine of (1), and incubating for a period of 2 to 5 days; and/or the number of the groups of groups,

the CO-incubated CO ₂ The concentration is 5%, and the percentage is volume percentage; and/or the number of the groups of groups,

(2) The polypeptide composition of claim 1 or 2, wherein each polypeptide in the dendritic cell-polypeptide mixture has a working concentration of 40 μg/mL after addition of the immature dendritic cells of (1).

14. The method of claim 11, wherein said contacting in (3) is by adding said dendritic cell maturation-promoting factor to said dendritic cell-polypeptide mixture in (2), and incubating for a period of 1-3 days; and/or the number of the groups of groups,

the dendritic cell maturation-promoting factors comprise IFN-gamma, poly (I: C) and R848, wherein the working concentration of the IFN-gamma is 100IU/mL, the working concentration of the poly (I: C) is 30 mug/mL, and the working concentration of the R848 is 5 mug/mL.

15. A dendritic cell vaccine comprising dendritic cells loaded with the polypeptide composition of claim 1 or 2.

16. The dendritic cell vaccine of claim 15, wherein said dendritic cell loaded with the polypeptide composition of claim 1 or 2 is obtainable by the method of any one of claims 11-14.

17. The dendritic cell vaccine of claim 15 or 16, further comprising an adjuvant.

18. An activated T cell, characterized in that it is activated by contacting an inactivated T cell with the dendritic cell vaccine of any one of claims 15-17.

19. The activated T cell of claim 18, wherein the contacting is co-incubation at 37 ℃ for 6 hours using AIM-V medium comprising 2% fbs, the percentage being by volume, and the medium further comprising IL-2 at a working concentration of 100U/mL.

20. The activated T cell according to claim 18 or 19, wherein the number ratio of the non-activated T cells to the dendritic cells in the dendritic cell vaccine according to any one of claims 15 to 17 is 20:1.

21. Use of one or more of the polypeptide composition of claim 1 or 2, the pharmaceutical composition of claim 3 or 4, the tumor vaccine of claim 5 or 6, the cell of any one of claims 7-10, the dendritic cell vaccine of any one of claims 15-17 and the activated T cell of any one of claims 18-20 for the preparation of a medicament for the prevention and/or treatment of cancer, the cancer cells of which are pancreatic cancer cells having an HLA-matched HLA-a2, a 11.