A polypeptide composition specifically binding to HLA-A2 and use thereof

Abstract

The present application belongs to the field of medical immunology, and particularly relates to a polypeptide composition specifically binding to HLA-A2 typing and application, the polypeptide composition is selected from one or more polypeptides derived from antigen proteins Survivin, Her2, CEA, hTERT, MAGE-A3, EGFR, gp100 or p53, the polypeptide derived from each antigen protein contains an antigen epitope or a variant of the antigen epitope of each antigen protein.The polypeptide composition is presented and activates specific CD8+ cytotoxic T lymphocytes by loading antigen presenting cells to achieve the targeted toxicity effect on tumor cells.The polypeptide composition of the present application and the tumor vaccine, DC vaccine, pharmaceutical composition derived therefrom can significantly activate immune effector cells, especially T cells, significantly improve the secretion level of the activated related cytokines and the killing level on tumor cells, and have potential clinical value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medical immunology, and particularly relates to a polypeptide composition and application. BACKGROUND

[0002] In recent years, some progress has been made in treating cancer through surgery combined with radiotherapy and chemotherapy, which has improved the survival rate of patients, especially the survival rate of patients with breast, lung, prostate and kidney cancer. However, most of such treatments have significant toxic side effects, which are easy to harm normal cells.

[0003] Tumors can elicit humoral and cellular immune responses in the body. After tumor antigens are processed into peptide segments in cells, they bind to major histocompatibility complex class I molecules on the cell surface and are presented to CD8+ cytotoxic lymphocytes, or they are shed from tumor cells, taken up by antigen-presenting cells, processed into peptide segments, and then bind to major histocompatibility complex class II molecules on the surface and are presented to CD4+ helper lymphocytes, thereby inducing the body's anti-tumor cellular immune response. With the deepening of the understanding of anti-tumor immunity and genetic changes during the progression of malignant tumors, humans can develop more selective and safe treatment methods, using methods that activate the immune system to attack the developing tumor, i.e., tumor vaccines. According to the specific use of tumor vaccines, they can be divided into two categories: prophylactic vaccines and therapeutic vaccines. The main function of prophylactic vaccines 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. Because DCs express a large number of costimulatory molecules and have the ability to effectively sensitize both CD4+ helper T cells (T helper, Th) and CD8+ cytotoxic T cells (Cytotoxic T Lymphocyte, CTL), DCs are different from B lymphocytes and macrophages. DCs generate specific anti-tumor immune responses by loading tumor antigens and inducing mature DCs. Based on this, various anti-tumor vaccines have been developed using DCs, including tumor antigen peptide-loaded DCs, tumor whole cell antigen-loaded DCs, tumor cell RNA-loaded DCs, tumor cell DNA-loaded DCs, exosome-loaded DCs, and cytokine and chemokine gene-modified DCs. At present, DC vaccines have been tried in malignant melanoma, prostate cancer, renal cancer, and other cancers, and some have achieved success. Various forms of DC vaccines have been tried in the immunotherapy of tumors and have shown good efficacy in preliminary clinical trials. Among them, the DC vaccine Provenge produced by the American company Dendreon was approved for marketing by the US Food and Drug Administration in 2010 for use in patients with advanced prostate cancer, especially those who have failed hormone therapy, and the efficacy shows that it can prolong the survival time of patients by more than 4 months (Nature Medicine, 2010, 16(6): 615.).

[0004] Current most clinical trials still use autologous whole tumor lysate to load DCs, which is achieved by repeated freeze-thaw cycles to lyse the patient's own tumor tissue to stimulate DCs (Cancer Immunol Immunother, 2006, 55: 819; medical oncology, 2006, 23: 273.). Freeze-thaw cycles induce tumor cell necrosis, but freeze-thaw induced tumor cell necrosis is not immunogenic and even inhibits TLR-induced DC maturation and normal function (Hatfeld 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 immunotherapy. J Immunother (2008) 31(7):620-632), and patient's tumor tissue is not always readily available. Tumor cell lysate, purified tumor-associated antigens and tumor-derived mRNA have also been shown to be useful as antigen sources to load DCs. Tumor cell lysate can provide multiple antigens for DC loading and induce CD4+ and CD8+ T cell responses and confer different Damage-Associated Molecular Patterns (DAMP) to DCs to ensure DC maturation, but also provides immunomodulatory cytokines to DCs to induce tolerogenic conversion of DCs (Guida M, Pisconte S, Colucci G. Metastatic melanoma: the new era of targeted therapy. Expert Opin Ther Targets 2012; 16 Suppl 2:S61-70); purified tumor-associated antigens load DCs to activate antigen-specific T cell responses and induce CD4+ and CD8+ T cell responses, but the number of different antigens used in a single use is limited.Tumor-derived mRNA can be transduced with tumor-associated antigens and costimulatory molecules, ensuring antigen presentation by class I MHC and obviating the need for cross-presentation (Robbins PF, Morgan RA, Feldman SA, Yang JC, Sherry 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 lymphocytes reactive with NY-ESO-1. J Clin Oncol 2011; 29:917-24), but cannot induce DC maturation and effective CD4+ immune responses, and the number of different antigen species used in a single application is limited.

[0005] When short peptide fragments in tumor-associated antigens are used as antigens to load DCs, the number of antigen types that can be involved in a single use can be effectively increased, and the immune response levels of CD4+ and CD8+ T cells can be improved. However, the HLA haplotype of the subject needs to be determined first, and appropriate peptide segments need to be selected from the selected tumor-associated antigens to verify whether they can bind to the HLA haplotype. HLA alleles have a high degree of polymorphism in different ethnic populations. According to the statistics of the World Health Organization, as of April 2018, the number of HLA alleles of type I has exceeded 13,000, including 4,200 HLA-A alleles, 5,091 HLA-B alleles, and 3,854 HLA-C alleles (http: / / www.hla.alleles.org / nomenclature / stats.html). Among them, the common HLA types in the Asian population are mainly HLA-A2, A3, and A24 (Experimental and Therapeutic Medicine, 2011, 2: 109-117.). HLA-A2, A11, and A24 three types can cover more than 90% of the Chinese population (Immunol Today, 1996; 17: 261.). HLA-A2 belongs to the HLA-A2 superfamily, with the highest frequency of 45.9% in the Chinese population. HLA-A11 belongs to the HLA-A3 superfamily, with the lowest frequency of 37.5% in Caucasians and the highest frequency of 52.7% in the Chinese population. HLA-A24 belongs to the HLA-A24 superfamily, with the lowest frequency of 23.9% in Caucasians, 40.1% in the Chinese population, and 58.6% in the Japanese population (Curr Opinion In Immunol, 1998, 10: 478-482; Immunogenetics, 1999, 50(3-4): 201-212.). At present, there is a lack of immunogenic polypeptide compositions containing peptides that can be effectively presented by antigen-presenting cells and tumor vaccines containing such polypeptide compositions for each HLA type. SUMMARY

[0006] In tumor immunity, the polypeptide fragments of the antigen epitopes of tumor-associated antigens bind to the HLA on the surface of antigen-presenting cells such as DCs to form HLA-tumor antigen epitope peptide complexes that are recognized by TCRs, which are then presented to T cells, so that T cells that can recognize the corresponding tumor antigen epitopes are specifically activated and expanded. The expanded T cells become cytotoxic T lymphocytes (CTLs) that specifically target the tumor-associated antigens, and produce cell-mediated immune killing effects on tumor cells expressing the tumor-associated antigens.

[0007] Senescent cells can activate innate and adaptive immune responses, maintaining tissue homeostasis. In addition, new findings suggest that the programmed induction of cellular senescence can be important in regulating reproductive processes, in part due to immune clearance. The antigens p16, p53, and p21 have a significant connection with senescent cells and tumorigenesis. The currently widely recognized biomarkers of senescent cells, such as β-galactosidase, p16INK4A, include p16, p53, and p21, which have similar effects to p16 in the regulation of the cell cycle, and regulatory mutations or deletions of p16 or p53 can be seen in various tumor cells.

[0008] To improve the cell-mediated tumor immune killing effect, the present application screens an immunogenic polypeptide composition specific to HLA typing (A2) based on tumor-related antigens and cell senescence-related proteins, and obtains cells loaded with the immunogenic polypeptide composition, activated immune effector cells, and a method for initiating an immune response in vivo based on the immunogenic polypeptide composition. The tumor-related antigens are Survivin, Her2, CEA, hTERT, MAGE-A3, EGFR, gp100, and the cell senescence-related proteins are p53. The technical solutions of the present application are as follows:

[0009] Polypeptide composition

[0010] The present application provides a polypeptide composition, which is selected from one or more polypeptides derived from antigen proteins Survivin, Her2, CEA, hTERT, MAGE-A3, EGFR, gp100 or p53, and is a part of each corresponding antigen protein, and has a length equal to or shorter than the corresponding full-length antigen protein; preferably, the polypeptide derived from each antigen protein contains (1) an antigen epitope of each antigen protein, which has immunogenicity; or contains an antigen epitope with the same activity as the antigen epitope in (1) obtained by amino acid substitution, addition or deletion, preferably has more than 75% homology with the amino acid sequence of the antigen epitope in (1), more preferably has more than 85% homology with the amino acid sequence of the antigen epitope in (1), and even more preferably has more than 95% homology with the amino acid sequence of the antigen epitope in (1), i.e. is a variant with equivalent function to the antigen epitope in (1), has an altered amino acid sequence, such as one or more amino acid substitutions in the amino acid sequence of the antigen epitope in (1), or one or more amino acids added to the amino acid sequence of the antigen epitope in (1), or one or more amino acids deleted from the amino acid sequence of the antigen epitope in (1), without affecting the function of the polypeptide containing the antigen epitope in (1) and the polypeptide composition containing the polypeptide containing the antigen epitope in (1). As an embodiment of the present application, 1-5 amino acids, preferably 1-3 amino acids, are added to the N-terminus and / or C-terminus of the amino acid sequence of the antigen epitope in (1). The mass ratio of the polypeptide derived from each antigen protein in the polypeptide composition to each other is 1-5:1, preferably 1-3:1, and more preferably 1:1.

[0011] As one embodiment of the present application, the polypeptide composition is selected from any two or three polypeptides derived from the antigenic proteins Survivin, Her2, CEA, hTERT, MAGE-A3, EGFR, gplOO or p53, and Her2 preferably contains the amino acid sequence shown in SEQ ID NO: 6; specifically, polypeptides derived from the antigenic proteins Survivin, CEA and Her2, and Her2 preferably contains the amino acid sequence shown in SEQ ID NO: 6, or polypeptides derived from Her2, hTERT and MAGE-A3, or polypeptides derived from P53, EGFR and gplOO, or polypeptides derived from Survivin, P53 and Her2, and Her2 preferably contains the amino acid sequence shown in SEQ ID NO: 6, or two different polypeptides derived from Her2 (such as the amino acid sequences shown in SEQ ID NO: 2 and SEQ ID NO: 6) and a polypeptide derived from hTERT; preferably, the polypeptides derived from each antigenic protein contain (1) an antigenic epitope of each antigenic protein, which has immunogenicity, or an antigenic epitope obtained by amino acid substitution, addition or deletion, which has the same activity as the antigenic epitope in (1), and preferably has 75% or more homology with the amino acid sequence of the antigenic epitope in (1), more preferably 85% or more homology, and even more preferably 95% or more homology. The mass ratio of any two polypeptides derived from each antigenic protein in the polypeptide composition is 1-5: 1, preferably 1-3: 1, and more preferably 1: 1. The mass ratio of any three polypeptides derived from each antigenic protein in the polypeptide composition is 1-5: 1-5: 1, preferably 1-3: 1-3: 1, and more preferably 1: 1: 1.

[0012] As an embodiment of the present application, the polypeptide composition is selected from polypeptides derived from the antigenic proteins Survivin, CEA and any one of Her2, hTERT, MAGE-A3, EGFR, gplOO or p53, Her2 preferably having the amino acid sequence shown in SEQ ID NO: 6; specifically polypeptides derived from Survivin, CEA and Her2, Her2 preferably having the amino acid sequence shown in SEQ ID NO: 6, or polypeptides derived from Survivin, CEA and hTERT, or polypeptides derived from Survivin, CEA and hTERT, or polypeptides derived from Survivin, CEA and MAGE-A3, or polypeptides derived from Survivin, CEA and EGFR, or polypeptides derived from Survivin, CEA and gplOO, or polypeptides derived from Survivin, CEA and p53; preferably, the polypeptides derived from each of the antigenic proteins contain (1) an antigenic epitope of each of the antigenic proteins, the antigenic epitope being immunogenic; or an antigenic epitope obtained by amino acid substitution, addition or deletion, which has the same activity as the antigenic epitope in (1), preferably having 75% or more homology with the amino acid sequence of the antigenic epitope in (1), more preferably having 85% or more homology with the amino acid sequence of the antigenic epitope in (1), and even more preferably having 95% or more homology with the amino acid sequence of the antigenic epitope in (1). The mass ratio of the polypeptides derived from Survivin, CEA and any one of Her2, hTERT, MAGE-A3, EGFR, gplOO or p53 is 1-5: 1-5: 1, preferably 1-3: 1-3: 1, and more preferably 1: 1: 1.

[0013] Each of the polypeptides derived from each of the antigenic proteins in the polypeptide composition has a length of 50 amino acids or less; preferably, 30 amino acids or less. Specifically, each of the polypeptides derived from each of the antigenic proteins can have a length of 5-30 amino acids, 8-25 amino acids, 8-15 amino acids or 9-12 amino acids. As an embodiment of the present application, each of the polypeptides derived from each of the antigenic proteins has a length of 9 amino acids.

[0014] The antigenic proteins Survivin, Her2, CEA, hTERT, MAGE-A3, EGFR, gplOO or p53 have the meanings and sequences well known in the art, including the amino acid wild type sequences of each of the antigenic proteins and various known or possible variant sequences.

[0015] The polypeptide derived from the antigen protein Survivin contains an amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence with the same activity obtained by amino acid substitution, addition or deletion, preferably has 75% or more homology with the amino acid sequence shown in SEQ ID NO: 1, more preferably has 85% or more homology with the amino acid sequence shown in SEQ ID NO: 1, and more preferably has 95% or more homology with the amino acid sequence shown in SEQ ID NO: 1.

[0016] The polypeptide derived from the antigen protein Her2 contains an amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence with the same activity obtained by amino acid substitution, addition or deletion, preferably has 75% or more homology with the amino acid sequence shown in SEQ ID NO: 2, more preferably has 85% or more homology with the amino acid sequence shown in SEQ ID NO: 2, and more preferably has 95% or more homology with the amino acid sequence shown in SEQ ID NO: 2; or an amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence with the same activity obtained by amino acid substitution, addition or deletion, preferably has 75% or more homology with the amino acid sequence shown in SEQ ID NO: 6, more preferably has 85% or more homology with the amino acid sequence shown in SEQ ID NO: 6, and more preferably has 95% or more homology with the amino acid sequence shown in SEQ ID NO: 6. The polypeptide derived from the antigen protein Her2 preferably contains an amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence with the same activity obtained by amino acid substitution, addition or deletion.

[0017] The polypeptide derived from the antigen protein CEA contains an amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence with the same activity obtained by amino acid substitution, addition or deletion, preferably has 75% or more homology with the amino acid sequence shown in SEQ ID NO: 3, more preferably has 85% or more homology with the amino acid sequence shown in SEQ ID NO: 3, and more preferably has 95% or more homology with the amino acid sequence shown in SEQ ID NO: 3.

[0018] The polypeptide derived from the antigen protein hTERT contains an amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence with the same activity obtained by amino acid substitution, addition or deletion, preferably has 75% or more homology with the amino acid sequence shown in SEQ ID NO: 4, more preferably has 85% or more homology with the amino acid sequence shown in SEQ ID NO: 4, and more preferably has 95% or more homology with the amino acid sequence shown in SEQ ID NO: 4.

[0019] The polypeptide derived from the antigenic protein MAGE-A3 contains an amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence with the same activity obtained by amino acid substitution, addition or deletion, preferably has 75% or more homology with the amino acid sequence shown in SEQ ID NO: 5, more preferably has 85% or more homology with the amino acid sequence shown in SEQ ID NO: 5, and more preferably has 95% or more homology with the amino acid sequence shown in SEQ ID NO: 5.

[0020] The polypeptide derived from the antigenic protein P53 contains an amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence with the same activity obtained by amino acid substitution, addition or deletion, preferably has 75% or more homology with the amino acid sequence shown in SEQ ID NO: 7, more preferably has 85% or more homology with the amino acid sequence shown in SEQ ID NO: 7, and more preferably has 95% or more homology with the amino acid sequence shown in SEQ ID NO: 7.

[0021] The polypeptide derived from the antigenic protein EGFR contains an amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence with the same activity obtained by amino acid substitution, addition or deletion, preferably has 75% or more homology with the amino acid sequence shown in SEQ ID NO: 8, more preferably has 85% or more homology with the amino acid sequence shown in SEQ ID NO: 8, and more preferably has 95% or more homology with the amino acid sequence shown in SEQ ID NO: 8.

[0022] The polypeptide derived from the antigenic protein gp100 contains an amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence with the same activity obtained by amino acid substitution, addition or deletion, preferably has 75% or more homology with the amino acid sequence shown in SEQ ID NO: 9, more preferably has 85% or more homology with the amino acid sequence shown in SEQ ID NO: 9, and more preferably has 95% or more homology with the amino acid sequence shown in SEQ ID NO: 9.

[0023] The polypeptide composition of the present application can be prepared by conventional methods well known in the art, including but not limited to chemical synthesis by solid phase synthesis followed by separation of the synthesis product from by-products by HPLC, and expression of nucleic acid encoding polypeptide comprising the antigenic fragment in the polypeptide composition of the present application in living cells or translation of the above-mentioned encoding nucleic acid by in vitro cell-free translation system followed by purification to obtain the antigenic peptide fragment in the polypeptide composition of the present application. In addition, the unnecessary small molecules contained in the polypeptide composition of the present application can be removed by sufficient dialysis, and the resulting product can be lyophilized and other carriers or excipients can be added to form the desired preparation. It should also be understood that some amino acids, mutants, chemical modifications, etc. produced in the vaccine component that can be incidental to the polypeptide composition of the present application will not substantially interfere with the recognition of the antigenic epitope sequence by the antibody or TCR.

[0024] The polypeptide composition of the present application binds to HLA (especially HLA-A2 subtype) on the surface of antigen-presenting cells such as DCs, forms HLA-tumor antigen epitope peptide complex recognized by TCR, and is then presented to T cells, enabling T cells capable of recognizing the corresponding tumor antigen epitope to be specifically activated and expanded, and the expanded T cells become cytotoxic T lymphocytes (CTLs) that specifically target the tumor-associated antigen, producing cell-mediated immune killing effect on tumor cells expressing the tumor-associated antigen. That is, the polypeptide composition of the present application can stimulate cell-mediated immune response and / or humoral immune response in vivo, producing a prophylactic and / or therapeutic effect on tumors, and can be used to prepare a drug, or reagent, or kit for detecting, and / or preventing, and / or treating tumors, which includes but is not limited to biological products.

[0025] Cells loaded with the polypeptide composition

[0026] The present application also provides a cell loaded with the polypeptide composition, the cell being an antigen presenting cell (APC) having an HLA type (A2 subtype) matched with the loaded polypeptide. The antigen presenting cell is a professional antigen presenting cell or a non-professional antigen presenting cell. The professional antigen presenting cell is a lymphocyte (B cell), a dendritic cell (DC), a macrophage, an endothelial cell or a stem cell, preferably a dendritic cell, a macrophage or a lymphocyte, more preferably a dendritic cell. The DC can be autologous or allogeneic, and the DC can be a DC cell line isolated from a living individual or artificially constructed to have similar biological characteristics as natural DCs. The artificially constructed DC cell line has similar morphology and / or gene phenotype as natural DCs, such as a DC cell line transduced with a lentiviral vector expressing Tax gene, having negative CD3 expression and positive expression of DC marker molecules such as CD70, CD80, CD83, CD86, CCR7 and HLA-DR, as described in CN108546679A; or a GEN2.2 cell line as described in US20050272151A1, which is a plasmacytoid DC cell line having a phenotype of CD4+, HLA-DR+, CD123+, CD45RA+, CD11c-, CD13-, and the DC can also be differentiated from a DC precursor cell line, such as monocytes differentiated from PBMC or MUTZ-3 cell line, which is a cell line expressing 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. 2008 Dec;84(6):1364-73.).

[0027] The skilled person is aware and understands that the antigen presenting cells (APC) are "pulsed" or loaded with the polypeptide composition described above in a manner that exposes the APC to the polypeptide composition for a time sufficient to allow the polypeptide composition to be presented on the surface of the APC to obtain polypeptide composition loaded APC; the "exposure" is achieved by contacting the APC with the polypeptide composition, such as co-incubation, or the like. In particular, the APC is exposed to the antigen in the form of the plurality of short polypeptide fragments, i.e. to the antigenic peptides, which are directly loaded onto the surface of the APC. In addition to the short polypeptide fragments, the APC can be incubated with large fragments derived from the antigenic protein, the whole antigenic protein, or particles comprising the antigenic protein, which can be taken up into the APC by endocytosis or the like, and then processed by lysosomes or proteasomes into short polypeptide fragments and finally transported and presented on the surface of the APC to form an antigen presenting complex with HLA on the surface of the APC.

[0028] The polypeptide composition loaded APC can be prepared by contacting the APC with the polypeptide composition described above in vitro or in vivo. When the APC is loaded with the antigenic peptides of the polypeptide composition described above in vitro, the APC can be plated on a culture dish or a well plate, and then exposed to a sufficient amount of the polypeptide composition comprising the antigenic peptides for a sufficient time to allow the antigenic peptides to bind to the APC. The amount and the time required for the antigenic peptides to bind to the APC can be determined by detection methods known in the art. Other methods known to the skilled person, such as immunodetection or binding detection, can be used to detect whether the APC is loaded with the antigenic peptides after being exposed to the polypeptide composition comprising the antigenic peptides.

[0029] The present application provides a method for preparing a cell loaded with the polypeptide composition described above, which comprises contacting the polypeptide composition described above with an antigen presenting cell, and loading the polypeptide composition on the antigen presenting cell to obtain a polypeptide composition loaded antigen presenting cell.

[0030] The contacting of the polypeptide composition described above with the antigen presenting cell is co-incubation, and the culture medium used for the co-incubation is any conventional culture medium suitable for culturing immune cells in the art, such as any one or more of AIM-V, DMEM or RPMI-1640, preferably AIM-V culture medium, and more preferably serum-free AIM-V culture medium; the time for the co-incubation is 1-2 days, preferably 2 days, as judged by the ability of the antigenic peptides in the polypeptide composition of the present application to bind to the HLA (A11 subtype) on the surface of the antigen presenting cell to form an HLA-antigenic peptide complex; the temperature for the co-incubation is room temperature to 37°C; and the concentration of each polypeptide in the polypeptide composition described above during the co-incubation is 10-100 μg / mL, preferably 20-80 μg / mL, and more preferably 40 μg / mL.

[0031] The antigen presenting cells are contacted with the polypeptide composition, which has a certain promoting effect on the maturation of the antigen presenting cells, but other maturation promoting factors are still needed to be added to contact with the antigen presenting cells loaded with the polypeptide composition to further promote the maturation of the antigen presenting cells. When the antigen presenting cells loaded with the polypeptide composition are still in an immature state, the antigen presenting cells loaded with the polypeptide composition are also contacted with the maturation promoting factors to induce the maturation of the antigen presenting cells, so as to obtain the antigen presenting cells loaded with the polypeptide composition in a mature state.

[0032] The antigen presenting cells loaded with the polypeptide composition are contacted with the maturation-promoting factor(s) in a co-incubation, the medium used for the co-incubation is any conventional medium suitable for culturing immune cells in the art, such as any one or more of AIM-V, DMEM or RPMI-1640, preferably AIM-V medium, more preferably AIM-V medium without any serum; the time of the co-incubation is 8-72 hours, preferably 24-48 hours, more preferably 24 hours; the temperature of the co-incubation is room temperature to 37°C; the maturation-promoting factor(s) is selected from one or more of TNF-α, IL-1β, IL-6, PGE2, IFN-γ, poly(I:C), R848 or ATP, preferably TNF-α, IL-1β, IL-6 and PGE2, the working concentration of TNF-α is 5-50 ng / mL, preferably 10-30 ng / mL; the working concentration of IL-1β is 5-50 ng / mL, preferably 10-30 ng / mL; the working concentration of IL-6 is 800-1500 U / mL, preferably 800-1200 U / mL; the working concentration of PGE2 is 0.5-3 μg / mL, preferably 0.5-1.5 μg / mL; or preferably TNF-α, IL-1β, IL-6 and PGE2, the working concentration of TNF-α is 5-50 ng / mL, preferably 10-30 ng / mL; the working concentration of IL-1β is 5-50 ng / mL, preferably 10-30 ng / mL; the working concentration of IL-6 is 800-1500 U / mL, preferably 800-1200 U / mL; the working concentration of PGE2 is 0.5-3 μg / mL, preferably 0.5-1.5 μg / mL; or preferably IFN-γ, poly(I:C) and R848, or preferably IFN-γ, poly(I:C), R848 and ATP, the working concentration of IFN-γ is 10-1000 IU / mL, preferably 100-300 IU / mL, more preferably 100 IU / mL; the working concentration of poly(I:C) is 1-200 μg / mL, preferably 20-40 μg / mL, more preferably 30 μg / mL; the working concentration of R848 is 0.1-50 μg / mL, preferably 1-10 μg / mL, more preferably 5 μg / mL; the working concentration of ATP is 0.1-10 mM, preferably 0.1-5 mM, more preferably 1 mM; preferably IFN-γ, poly(I:C), R848 and ATP, the working concentration of IFN-γ is 100 IU / mL, the working concentration of poly(I:C) is 30 μg / mL, the working concentration of R848 is 5 μg / mL.

[0033] The antigen-presenting cells include professional antigen-presenting cells or non-professional antigen-presenting cells. The professional antigen-presenting cells are lymphocytes (B cells), dendritic cells (DCs), macrophages, endothelial cells, stem cells, etc., preferably dendritic cells, macrophages or lymphocytes, and more preferably dendritic cells. The non-professional antigen-presenting cells are antigen-presenting cells expressing HLA type I.

[0034] The antigen-presenting cells can be differentiated from precursor cells of the antigen-presenting cells, and the preparation includes contacting the precursor cells of the antigen-presenting cells with cytokines to induce differentiation into the antigen-presenting cells.

[0035] As an embodiment of the present application, the method for preparing the cells loaded with the polypeptide composition includes: (1) contacting the polypeptide composition with immature dendritic cells, and loading the polypeptide composition on the immature dendritic cells to obtain immature dendritic cells loaded with the polypeptide composition;

[0036] (2) contacting the immature dendritic cells loaded with the polypeptide composition with dendritic cell maturation-promoting factors to induce maturation of the immature dendritic cells, and obtaining mature dendritic cells loaded with the polypeptide composition.

[0037] The contacting in step (1) is co-incubation, and the medium used for the co-incubation is any conventional medium suitable for culturing immune cells in the art, such as any one or more of AIM-V, DMEM or RPMI-1640, preferably AIM-V medium, and more preferably AIM-V medium without any serum. The time for the co-incubation is 1-2 days, preferably 2 days, as judged by the ability of the antigen peptides in the polypeptide composition of the present application to bind to HLA (A2 subtype) on the surface of the antigen-presenting cells to form HLA-antigen peptide complexes. The temperature for the co-incubation is room temperature to 37°C. The concentration of each polypeptide in the polypeptide composition during the co-incubation is 10-100 μg / mL, preferably 20-80 μg / mL, and more preferably 40 μg / mL.

[0038] The contacting in step (2) is co-incubation, the medium used for co-incubation is any conventional medium suitable for culturing immune cells in the art, such as any one or more of AIM-V, DMEM or RPMI-1640, preferably AIM-V medium, more preferably AIM-V medium without any serum; the time for co-incubation is 8-72 hours, preferably 24-48 hours, more preferably 24 hours; the temperature for co-incubation is room temperature to 37°C; the dendritic cell maturation-promoting factor is selected from one or more of TNF-a, IL-1β, IL-6, PGE2, IFN-γ, poly(I:C), R848 or ATP, preferably TNF-a, IL-1β, IL-6 and PGE2, the working concentration of TNF-a is 5-50 ng / mL, preferably 10-30 ng / mL; the working concentration of IL-1β is 5-50 ng / mL, preferably 10-30 ng / mL; the working concentration of IL-6 is 800-1500 U / mL, preferably 800-1200 U / mL; the working concentration of PGE2 is 0.5-3 μg / mL, preferably 0.5-1.5 μg / mL; or preferably TNF-a, IL-1β, IL-6 and PGE2, the working concentration of TNF-a is 5-50 ng / mL, preferably 10-30 ng / mL; the working concentration of IL-1β is 5-50 ng / mL, preferably 10-30 ng / mL; the working concentration of IL-6 is 800-1500 U / mL, preferably 800-1200 U / mL; the working concentration of PGE2 is 0.5-3 μg / mL, preferably 0.5-1.5 μg / mL; or preferably IFN-γ, poly(I:C) and R848, or preferably IFN-γ, poly(I:C), R848 and ATP, the working concentration of IFN-γ is 10-1000 IU / mL, preferably 100-300 IU / mL, more preferably 100 IU / mL; the working concentration of poly(I:C) is 1-200 μg / mL, preferably 20-40 μg / mL, more preferably 30 μg / mL; the working concentration of R848 is 0.1-50 μg / mL, preferably 1-10 μg / mL, more preferably 5 μg / mL; the working concentration of ATP is 0.1-10 mM, preferably 0.1-5 mM, more preferably 1 mM; preferably IFN-γ, poly(I:C), R848 and ATP, the working concentration of IFN-γ is 100 IU / mL, the working concentration of poly(I:C) is 30 μg / mL, the working concentration of R848 is 5 μg / mL.

[0039] In step (2), the mature dendritic cells are verified by detecting the molecular markers expressed on the surface of the mature DCs and / or the cytokines secreted by the mature DCs, including but not limited to CD80, CD83, CD86, CCR7, HLA-ABC, HLA-DR or IL-6. The detection means used in the verification method can be any detection means capable of detecting the above-mentioned molecular markers and / or cytokines in the art, including but not limited to ELISA, Western blot or flow cytometry detection.

[0040] In step (1), the immature dendritic cells are differentiated from the precursor cells of dendritic cells, and the step comprises: contacting the precursor cells of dendritic cells with cytokines to induce differentiation into immature dendritic cells.

[0041] The contacting of the precursor cells of dendritic cells with the cytokines is co-incubation, and the culture medium used in the co-incubation is any conventional culture medium suitable for culturing immune cells in the art, such as any one or more of AIM-V, DMEM and RPMI-1640, preferably AIM-V culture medium, more preferably AIM-V culture medium without any serum; the temperature of the co-incubation is room temperature to 37°C; the length of the co-incubation is 2-6 days, preferably 3-5 days, more preferably 5 days.

[0042] The precursor cells of dendritic cells are CD14+DC precursor cells, preferably monocytes of PBMC in blood; the cytokines include GM-CSF and IL-4, the working concentration of GM-CSF is 50-500 ng / mL, preferably 50-100 ng / mL, more preferably 100 ng / mL; the working concentration of IL-4 is 5-100 ng / mL, preferably 10-50 ng / mL, more preferably 50 ng / mL.

[0043] The monocytes are obtained by culturing the PBMC isolated from an individual. The method for isolating the PBMC can be any method known in the art, for example, extracting blood from an individual and separating by density gradient centrifugation. After culturing the isolated PBMC, the adherent cells are basically monocytes. The time for culturing the PBMC is preferably 2-8 hours, more preferably 8 hours.

[0044] The precursor cells of dendritic cells can also be CD34+DC precursor cells derived from the hematopoietic stem cell lineage, and the cytokines include GM-CSF and IL-4. The CD34+DC precursor cells are isolated from umbilical cord blood, expanded in large quantities, and then contacted with the cytokines GM-CSF and IL-4 to induce differentiation into immature DCs. The method for expanding the CD34+DC precursor cells in large quantities can be any method known in the art, and specific methods can be found in WO2010055900A1.

[0045] The precursor cell of the immature DC can also be an immortalized DC precursor cell line, such as the MUTZ-3 cell line described above, which is a cell line expressing the monocyte marker molecule monocyte-specific esterase and CD14.

[0046] The cell surface of the polypeptide composition loaded cell of the present application forms an HLA-tumor antigen epitope peptide complex, which is presented to a T cell via TCR recognition, so that a T cell capable of recognizing the corresponding tumor antigen epitope is specifically activated and expanded, and the expanded T cell becomes a cytotoxic T lymphocyte (CTL) that specifically targets the tumor-associated antigen, producing a cell-mediated immune killing effect on tumor cells expressing the tumor-associated antigen. That is, the polypeptide composition loaded cell of the present application can stimulate a cell-mediated immune response and / or a humoral immune response in vivo, producing a prophylactic and / or therapeutic effect on tumors, and can be used to prepare a medicament, or a reagent, or a kit for detecting, and / or preventing, and / or treating tumors, which includes but is not limited to a biological product.

[0047] Activated immune effector cell

[0048] The present application also provides an activated immune effector cell, which is prepared by contacting the polypeptide composition loaded cell of the present application with an unactivated immune effector cell. The polypeptide composition loaded cell of the present application, such as a DC, expresses a certain subtype of HLA, such as A2, on its surface. After an antigen peptide, such as a tumor antigen peptide, that matches the certain specific subtype of HLA binds to the polypeptide composition loaded cell, an HLA-antigen peptide complex is formed, which is then recognized and bound by the T cell surface-specific receptor TCR. The T cell expressing the TCR is thus stimulated and begins to proliferate.

[0049] The immune effector cell is selected from a T cell or an NK cell, preferably a T cell. The polypeptide composition loaded cell of the present application is selected from a lymphocyte (B cell), a dendritic cell DC, a macrophage, an endothelial cell, or a stem cell, preferably a polypeptide composition loaded lymphocyte, macrophage, or dendritic cell, more preferably a polypeptide composition loaded dendritic cell. The immune effector cell and the polypeptide composition loaded cell of the present application can be from the same individual or different individuals, preferably from the same individual. The ratio of the polypeptide composition loaded cell to the immune effector cell can be any ratio known in the art, as long as the polypeptide composition loaded cell can effectively activate the immune effector cell that recognizes the HLA-antigen peptide complex on the surface of the polypeptide composition loaded cell. The ratio of the polypeptide composition loaded cell to the immune effector cell is preferably 1:10-50, more preferably 1:10-30, and even more preferably 1:10.

[0050] The contacting of the polypeptide composition-loaded cells with the immune effector cells is co-incubation. The co-incubation is for 2-48 hours, preferably for 24-48 hours, more preferably for 24 hours. The co-incubation is in a culture medium, which is AIM-V, DMEM or RPMI1640, preferably AIM-V medium, more preferably 2% v / v FBS AIM-V medium. As a preferred embodiment, the AIM-V medium further comprises IL-2, and the working concentration of IL-2 is 10-100 U / mL, preferably 100 U / mL.

[0051] The activated immune effector cells of the present application are cytotoxic T lymphocytes (CTLs) that specifically target tumor-associated antigens, and produce cell-mediated immune killing effect on tumor cells expressing tumor-associated antigens. That is, the activated immune effector cells of the present application can stimulate cell-mediated immune response and / or humoral immune response in vivo, and produce prophylactic and / or therapeutic effect on tumors, and can be used for preparing a medicament, or an agent, or a kit for detecting, and / or preventing, and / or treating tumors, which includes but is not limited to biological products.

[0052] Medicament, agent or kit

[0053] The medicament, agent or kit has activity of detecting, and / or preventing, and / or treating tumors, and includes but is not limited to biological products, and contains the polypeptide composition of the present application, and / or the polypeptide composition-loaded cells, and / or the activated immune effector cells.

[0054] The medicament, agent or kit further contains an adjuvant and / or an immunomodulator. The adjuvant and / or the immunomodulator are well known in the art, and are small molecule compounds, biological macromolecules, compositions, complexes or extracts that can enhance the effect of immune response and / or reduce the toxic side effects of immune response. The adjuvant and / or the immunomodulator can be delivered to target cells or target organisms before, together with, or after the polypeptide composition of the present application, to enhance the immune response of the cells or organisms to antigens or change the type of immune response, and reduce the toxic side effects of immune response.

[0055] The adjuvant or immunomodulator is selected from the group consisting of an aluminum adjuvant (e.g. aluminum hydroxide, aluminum salts), Freund's adjuvant (e.g. complete Freund's adjuvant and incomplete Freund's adjuvant), prostaglandin E2, alpha interferon, Corynebacterium parvum, lipopolysaccharide, cytokine, oil-in-water emulsion, water-in-oil emulsion, nanoemulsion, microparticulate delivery system, liposome, microsphere, biodegradable microsphere, plaque virion, proteoliposome, proteosome, immunostimulating complex (ISCOMs, ISCOMATRIX), microparticle, nanoparticle, biodegradable nanoparticle, silicon nanoparticle, polymeric micro / nanoparticle, polymeric lamellar substrate particle (PLSP), microparticulate resin, nanolipogel, synthetic / biodegradable and biocompatible semi-synthetic or natural polymers or dendrimers (e.g. PLG, PLGA, PLA, polycaprolactone, silicon polymers, polyesters, dimethicone, polystyrene sulfonate sodium, polystyrene benzyl trimethylammonium chloride, polystyrene divinyl benzene resin, polyphosphazene, poly-[di-(carboxyacetphenoxy)phosphazene (PCPP), poly-(methyl methacrylate), dextran, polyvinylpyrrolidone, hyaluronic acid and derivatives, chitosan and derivatives thereof, polysaccharide, inulin polysaccharide, glycolipid (synthetic or natural), lipopolysaccharide, one or more polycationic compounds (e.g. polyamino acid, poly-(gamma-glutamic acid)), poly-arginine-HCl, poly-L-lysine, polypeptide, biopolymer), cationic dimethyl dioctadecyl ammonium (DDA), alpha-galactosylceramide and derivatives thereof, archaeal lipid and derivatives, lactam, gallen, glyceride, phospholipid and spirochete.

[0056] The medicament, agent or kit further comprises a pharmaceutically acceptable carrier and / or excipient, which does not substantially affect the properties and biological functions of the polypeptide composition, and / or the cells loaded with the polypeptide composition, and / or the activated immune effector cells.

[0057] The medicament, agent or kit can be prepared into any dosage form of a pharmaceutically acceptable agent with a detecting, and / or preventing, and / or therapeutic effect, such as a lyophilized powder or a liquid preparation, which comprises sterile water for injection or an organic solvent, such as DMSO.

[0058] The medicament or agent comprising the polypeptide composition can be injected into a subject, and form HLA-antigen peptide complexes by binding to the corresponding subtype of HLA recognizing the antigen peptide in the tumor vaccine on the antigen presenting cells (e.g. DCs) loaded into the subject, which bind to the corresponding specific TCR, and activate the T cells expressing the specific TCR.

[0059] The antigen presenting cells can also be obtained by differentiating precursor cells of antigen presenting cells (such as DC precursor cells) isolated from autologous blood, such as DCs differentiated from CD34+ hematopoietic precursor cells derived from umbilical cord blood or monocytes derived from CD14+ peripheral blood. The antigen presenting cells (especially mature antigen presenting cells loaded with the polypeptide composition and in a mature state) obtained by in vitro incubation of the polypeptide composition of the present application with the antigen presenting cells can be introduced into the body to activate specific T cell responses by presenting the antigen peptides loaded by the antigen presenting cells. The agent for reducing immune rejection can be administered simultaneously with the introduction of the antigen presenting cells into the body.

[0060] The administration of the agent or the drug can occur before or after surgical removal of the tumor, or before or after chemotherapy treatment of the tumor. The agent or the drug can be administered to the patient together with or in combination with other compositions or pharmaceutical products. It should be understood that the agent of the present application can be administered not only to individuals who have suffered from a tumor, but also to individuals who have not suffered from a tumor but are at risk of suffering from a tumor.

[0061] The agent or the drug can be administered to the inguinal nodes by intra- nodal injection, or can be administered subcutaneously or intradermally to the hands and feet of the cancer patient to be treated, or other administration routes, such as intramuscular injection or blood injection.

[0062] The tumors that can be treated and / or prevented by the administration of the agent or the drug include, but are not limited to, lung cancer, non-small cell lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematologic malignancy, head and neck cancer, glioma, mesothelioma, large bowel cancer, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body tumor and osteosarcoma, bone cancer, pancreatic cancer, renal cell carcinoma, skin cancer, prostate cancer, cutaneous or intraocular malignant melanoma, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, endocrine system cancer, bile duct cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, urothelial cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), pediatric solid tumor, lymphocytic lymphoma, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumor, primary CNS lymphoma, tumor angiogenesis, spinal column tumor, brain stem neuroglioma, pituitary adenoma, Kaposi's sarcoma, Hodgkin's lymphoma, epidermoid cancer, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including asbestos-induced cancers, and various types of leukemia and lymphocarcinoma, and various types of precancerous lesions.

[0063] Method for eliciting an immune response in vivo

[0064] To achieve the prophylactic and / or therapeutic effects on tumors, the present application also provides a method of eliciting an immune response in vivo, which comprises administering to the body an effective amount of one or more of the polypeptide composition, the polypeptide composition-loaded cells and the activated immune effector cells.

[0065] To achieve the prophylactic effects, one or more of the polypeptide composition, the polypeptide composition-loaded cells and the activated immune effector cells of the present application are administered to the subject before the occurrence of a tumor or a tumor lesion. In some cases, the drug is administered to the subject after the occurrence of one or more of the above-mentioned tumors, aiming at preventing the appearance of further symptoms or the further aggravation of the symptoms that have already occurred. The prophylactic administration of one or more of the polypeptide composition, the polypeptide composition-loaded cells and the activated immune effector cells of the present application aims at preventing or alleviating any subsequent symptoms. To achieve the therapeutic effects, one or more of the polypeptide composition, the polypeptide composition-loaded cells and the activated immune effector cells of the present application are administered to the subject when or after the occurrence of a cancer, aiming at alleviating the symptoms of the cancer that has already occurred.

[0066] The effective amount for any particular therapeutic application depends on various factors, such as the type of cancer, the extent of the occurrence of the cancer, the condition of the subject, such as age, sex, weight, levels of various physical indicators, etc., and the components of the specific agent to be administered, as well as the specific mode of administration, for example, the subject can be administered by one or more of the means including, but not limited to, intravenous, intramuscular, intradermal, transdermal, intraarterial, intraperitoneal, intralesional, intracranial, intraarticular, intraprostatic, intrapleural, intratracheal, intrathecal, intranasal, intravaginal, intrarectal, parenterally, systemically, topically, intratumorally, peritoneally, intracerebroventricularly, subcutaneously, subconjunctivaly, transmucosally, transdermally, pulmonarily, by inhalation, by injection, by implantation, by reinfusion, by continuous reinfusion, by local perfusion, by catheter, by lavage, by emulsion and by liposome composition. For the effective amount of the drug to be administered comprising one or more of the polypeptide composition, the polypeptide composition-loaded cells and the activated immune effector cells of the present application, the person skilled in the art can determine empirically according to the specific components contained in the drug without the need for additional unnecessary experiments.

[0067] The polypeptide composition provided by the present application can present and activate specific CD8+ cytotoxic T lymphocytes (CTL) after loading DCs, thereby achieving targeted toxicity on tumor cells. The polypeptide composition of the present application and the tumor vaccine, DC vaccine and pharmaceutical composition derived therefrom can significantly activate immune effector cells, especially T cells, significantly increase the secretion level of activation-related cytokines and the killing level on tumor cells, and have potential clinical value.

[0068] The following explains some terms related to the present application. Unless otherwise defined below, the terms herein are used in the manner generally used in the art.

[0069] In the present application, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly connected by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any one or more chains of two or more amino acids, and does not refer to a specific length of the product. Thus, a peptide, dipeptide, tripeptide, oligopeptide, "protein", "amino acid chain", or any other term used to refer to one or more chains of two or more amino acids is included in the definition of "polypeptide", and the term "polypeptide" can be used instead of, or interchangeably with, any of these terms. The term "polypeptide" is also intended to refer to products produced by post-expression modifications of a polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic processing, or other modifications known from standard reference works and

[0070] The term "polypeptide derived from" refers to a full-length, fragment, modification or mutant of a protein or polypeptide. The term "fragment" when referring to a polypeptide can be a polypeptide of any length that retains the antigenicity of the full-length protein or polypeptide. In one or more embodiments, the fragment is an antigenic epitope of the full-length protein. In one or more embodiments, the fragment comprises 50, 40, 30 or fewer amino acids. For example, the fragment can be 5-30 amino acids, 8-25 amino acids, 8-15 amino acids, or 9-12 amino acids in length, or any range formed by any combination of the above endpoints, such as 9-15 amino acids. In one or more embodiments, the fragment can be 9 amino acids in length.

[0071] The term "variant" or "mutant" refers to a peptide or polypeptide whose amino acid sequence has been changed by the insertion, deletion, or substitution (replacement) of one or more amino acids compared to a reference sequence, while retaining at least one biological activity. In one or more embodiments, the variant or mutant comprises an amino acid sequence having at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity to a reference sequence (e.g., each of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9 fragments as described herein) and retains the biological activity of the reference sequence (e.g., as an antigenic epitope). The substitution can be a non-conservative amino acid substitution or a conservative amino acid substitution. A conservative amino acid substitution refers to the substitution of an amino acid with a corresponding amino acid that has similar structural and chemical properties, similar performance, or similar functionality, that does not change the function of the protein or polypeptide. For example, conservative amino acid substitutions include families of amino acid residues with similar side chains; these families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), uncharged small side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, replacement of one or more sites in a polypeptide of the application with another amino acid residue from the same side chain class will not materially affect its activity.

[0072] The term "wild type" has its art-understood meaning and refers to an entity having a structure and / or activity as found in nature in a "normal" (e.g., as contrasted with mutant, diseased, altered, etc.) state or condition. Those skilled in the art will appreciate that wild type genes and polypeptides generally exist in a variety of different forms (e.g., alleles).

[0073] The term "tumor-associated antigen" or "TAA" refers to an antigen that is specifically expressed by tumor cells or expressed at a higher frequency or density by tumor cells as compared to non-tumor cells of the same tissue type. Tumor-associated antigens can be antigens that are not normally expressed by a host; they can be aberrantly presented, mutated, truncated, misfolded, or otherwise abnormally presented molecules that are normally expressed by a host; they can be identical to normally expressed molecules but expressed at abnormally high levels; or they can be expressed in an abnormal context or environment. Tumor-associated antigens can be, for example, proteins or protein fragments, complex carbohydrates, gangliosides, haptens, nucleic acids, or combinations of these or other biological molecules.

[0074] The term "vaccine" refers to an immunogenic composition for administration to a mammal that is used to elicit an immune response in the mammal against a particular antigen. A vaccine typically contains an agent (known as an "antigen" or "immunogen") that is similar to or derived from the target of the immune response, such as a microorganism that causes disease or a tumor cell. A vaccine intended for the treatment of a tumor, such as a cancer, typically contains an antigen that is derived from a tumor-associated antigen found on the target tumor and is capable of eliciting immunogenicity to the tumor-associated antigen on the target tumor.

[0075] The term "Survivin" refers to the expression product of the Survivin gene, which can be a human Survivin or a non-human mammalian Survivin.

[0076] The term "Her2" refers to the expression product of the Her2 gene, which is human epidermal growth factor receptor 2.

[0077] The term "CEA" refers to the expression product of the CEA gene, which is carcinoembryonic antigen protein.

[0078] The term "hTERT" refers to the expression product of the hTERT gene, which is human telomerase reverse transcriptase protein.

[0079] The term "MAGE-A3" refers to the expression product of the MAGE-A3 gene, which is melanoma antigen protein.

[0080] The term "EGFR" refers to the expression product of the EGFR gene, which is epidermal growth factor protein.

[0081] The term "gp100" refers to the expression product of the gp100 gene, which can be a human gp100 protein or a non-human mammalian gp100 protein.

[0082] The term "p53" refers to the expression product of the p53 gene, which can be a human p53 protein or a non-human mammalian p53 protein.

[0083] The terms "cancer," "tumor," and "malignant" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinomas, including adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's and non-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer (such as hepatoma and hepatocellular carcinoma), bladder cancer, breast cancer (including hormone mediated breast cancer), colon cancer, colorectal cancer, endometrial cancer, myeloma (such as multiple myeloma), salivary gland carcinoma, kidney cancer (such as renal cell carcinoma and Wilms' tumor), basal cell carcinoma, melanoma, prostate cancer, vulvar cancer, thyroid cancer, testicular cancer, esophageal cancer, blood cancers (including but not limited to acute myeloid leukemia (AML) and multiple myeloma (MM)), various types of head and neck cancers (including but not limited to squamous cell carcinoma), and cancers of mucous origin (such as mucinous ovarian cancer), cholangiocarcinoma (liver), and renal papillary 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.

[0084] The term "immunomodulator" means a substance that, when mixed with an immunogen, is capable of eliciting a stronger immune response than the immunogen alone, and / or reducing immunotoxic side effects. For example, an immune enhancer can increase immunogenicity and provide an excellent immune response. For another example, an immune enhancer can work by increasing the expression of costimulatory factors on macrophages and other antigen presenting cells.

[0085] The term "adjuvant" refers to a non-specific immune enhancer that, when delivered to the body with or prior to an antigen, can enhance the immune response to the antigen or change the type of immune response, and / or reduce immunotoxic side effects. There are many types of 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, etc. Freund's adjuvant is the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvant is more commonly used in clinical experiments.

[0086] The term "DC maturation-promoting factor" refers to any protein, nucleic acid, polypeptide, complex, extract, isolate, or combination thereof, which is capable of promoting the conversion of immature DC to mature DC, which can be achieved by contacting the immature DC with the factor. The verification of mature DC can be achieved by detecting the molecular markers expressed on the surface of mature DC and / or the cytokines secreted by mature DC, including but not limited to CD80, CD83, CD86, CCR7, HLA-ABC, HLA-DR, and IL-6.

[0087] The term "isolated" refers to a state of a naturally occurring biological macromolecule, such as a protein, polypeptide, nucleic acid, antibody, or complex formed therefrom, which is removed from the natural state and isolated by the hand of man, is purified and is in a state other than in vivo.

[0088] The term "working concentration" refers to the actual concentration of a reagent or an active ingredient in a solution system when it is in use. Usually, a reagent or an active ingredient is configured into a mother liquor or a stock solution with a higher concentration before use, and is diluted into the final reaction system according to a certain proportion when used, and the final concentration obtained after dilution is usually the working concentration.

[0089] The term "loading" refers to the direct combination of a protein, polypeptide, or nucleic acid molecule with a receptor on the surface of a cell, forming a complex of the cell and the protein, polypeptide, or nucleic acid molecule. The combination can be covalent or non-covalent, including the mutual combination of a receptor and a ligand. For example, a DC loaded with an antigen peptide is a DC-antigen peptide complex formed by the combination of the antigen peptide with the HLA molecule expressed on the surface of the DC, which matches the HLA type of the antigen peptide.

[0090] The term "DC-cytotoxic T lymphocyte" or "DC-CTL" refers to a cytotoxic T lymphocyte (CTL) activated by a mature DC loaded with an antigen peptide, which is capable of specifically binding to an antigen containing the antigen peptide loaded by the mature DC, and producing cell killing effect on cells expressing the antigen, and is the main executor of cell-mediated immunity mediated by DC.

[0091] The term "antigen presenting cell" (APC) refers to a type of cell capable of expressing major histocompatibility complex (MHC) type I or type II, capable of forming MHC-antigen peptide complex by MHC binding antigen peptide and further binding to the receptor on the surface of T cells to activate T cells, including but not limited to dendritic cells (DC), monocytes / macrophages, B cells, Langerhans cells.

[0092] The term "antigen-loaded antigen-presenting cell" includes APCs that have been exposed to and activated by antigens. For example, APCs can be loaded with antigens in vitro (e.g., during culture in the presence of antigens). APCs can also be loaded in vivo by exposure to antigens. "Antigen-loaded APCs" are typically prepared in one of two ways: (1) a small fragment called an antigenic peptide is directly "pulsed" to the exterior of the APC and binds to an MHC molecule; (2) the APC is incubated with a large peptide fragment, a complete protein, or a protein particle, which is then taken up by the APC. These large peptide fragments or protein molecules are digested by the APC into smaller peptides and are ultimately transported and presented on the surface of the APC. In addition, antigen-loaded APCs can also be produced by introducing polynucleotides encoding antigens into the cell. Attached Figure Description

[0093] Figure 1 This diagram shows the proportions of maturation markers on the surface of different dendritic cells (DCs) loaded with different peptide compositions after cytokine-induced culture in Example 1. The horizontal diagram, from left to right, shows the proportions of maturation markers CD80, CD83, and CD86 in each DC group loaded with different peptide compositions. The control group refers to DCs without any peptide loading.

[0094] Figure 2 Example 2 shows the flow cytometry detection of the proportions of DC-CTL surface markers loaded with different peptide compositions. The detection results for each activation marker detection group (CD3 / CD4, CD3 / CD8, CD25, CD69, CD107a, CD137) are, from left to right: control group, peptide-loaded composition 1, peptide-loaded composition 2, and peptide-loaded composition 3.

[0095] Figure 3 The ratio of the DC-CTL surface marker CD3+ / CD137 loaded with different peptide compositions in Example 2 is shown.

[0096] Figure 4 The ratio of the DC-CTL surface marker CD3+ / CD107α loaded with different peptide compositions in Example 2 is shown.

[0097] Figure 5 Example 2 shows the intracellular IFN-γ secretion levels of DC-CTLs loaded with different polypeptide compositions.

[0098] Figure 6 This is a flow cytometry diagram showing the intracellular IFN-γ secretion level of DC-CTLs loaded with different peptide compositions in Example 2.

[0099] Figure 7 Example 3 illustrates the killing effect of DC-CTL loaded with different polypeptide compositions on pancreatic cancer tumor cells PANC-1.

[0100] Figure 8 TNF-α secretion level in supernatant after killing of pancreatic cancer tumor cell PANC-1 by DC-CTL loaded with different polypeptide compositions of Example 3.

[0101] Figure 9 IFN-γ secretion level in supernatant after killing of pancreatic cancer tumor cell PANC-1 by DC-CTL loaded with different polypeptide compositions of Example 3.

[0102] Figure 10 Killing curve of tumor cell line PANC-1 by DC-CTL loaded with HLA-A2 binding polypeptide composition 1 (preferred peptide combination), peptide replacement group 1 and peptide replacement group 2 of Comparative Example.

[0103] Figure 11 TNF-α secretion level in supernatant after killing of tumor cell line PANC-1 by DC-CTL loaded with HLA-A2 binding polypeptide composition 1, peptide replacement group 1 and peptide replacement group 2 of Comparative Example.

[0104] Figure 12 IFN-γ secretion level in supernatant after killing of tumor cell line PANC-1 by DC-CTL loaded with HLA-A2 binding polypeptide composition 1, peptide replacement group 1 and peptide replacement group 2 of Comparative Example.

[0105] "Control group or T cell group" marked in the above figures refers to experimental group of T cells stimulated by DC not loaded with any polypeptide, unless otherwise specified.

[0106] "Pep combination 1 group, combination 1, loaded polypeptide combination 1, pep combination 1" marked in the above figures refers to experimental group loaded with polypeptide composition 1; "pep combination 2 group, combination 2, loaded polypeptide combination 2, pep combination 2" refers to experimental group loaded with polypeptide composition 2; "pep combination 3 group, combination 3, loaded polypeptide combination 3, pep combination 3" refers to experimental group loaded with polypeptide composition 3. DETAILED DESCRIPTION

[0107] The present application is further illustrated by the following examples without limiting the present application to the described examples. The experimental methods in the following examples, unless otherwise specified, are carried out according to conventional methods and conditions, or according to the instructions of the commercial product.

[0108] "Room temperature" in the examples refers to the temperature in the operation room where the experiment is carried out, generally 25°C.

[0109] "Overnight" in the examples refers to more than 8 hours.

[0110] The following examples are directed to the HLA-A2 type, which is a relatively high proportion of HLA typing in the Chinese population, and provide a plurality of polypeptide compositions comprising a plurality of tumor antigen peptides, each of which includes at least three tumor-associated antigen epitope polypeptides for HLA-A2 typing. The polypeptides used in the following examples are shown in Table 1 below:

[0111] Table 1

[0112]

[0113]

[0114] The polypeptide compositions used in the following examples are shown in Table 2 below:

[0115] Table 2

[0116]

[0117] The DC precursor cells and T cells in the following examples were isolated from the blood PBMC of a donor subject, and the specific steps are as follows: the blood of an HLA-A2 donor subject was mixed with an equal volume of physiological saline, the total volume was 35 mL, and was slowly added to a centrifuge tube containing 15 mL of Ficoll along the wall. The blood and Ficoll were clearly layered, and were centrifuged at 800 g for 20 min. After centrifugation, the white cell layer was aspirated and transferred to another centrifuge tube, and physiological saline was added, and centrifuged at 1500 rpm for 10 min. The supernatant was discarded, and the cells were washed once more with physiological saline. The washed cells were transferred to a culture bottle and cultured overnight, and the suspended cells were collected, counted, and frozen. The remaining adherent cells were mononuclear cells (DC precursor cells) that could be induced to differentiate into DCs.

[0118] In the following examples, unless otherwise specified, the working concentration of each polypeptide in the polypeptide composition used is 40 μg / mL.

[0119] Example 1 Effect of different HLA-A2 binding type polypeptide compositions on the induction of DC maturation

[0120] Operation steps:

[0121] (1) Culture of immature DC cells: DC precursor cells were added to serum-free AIM-V medium (purchased from Gibco), and IL-4 and GM-CSF were added to a working concentration of 50 ng / mL and 100 ng / mL, respectively, and then the culture was continued. Half of the medium was replaced on the third day, and IL-4 and GM-CSF were added to a working concentration of 50 ng / mL and 100 ng / mL, respectively.

[0122] (2) Peptide loading of immature DC cells: After day 5, DC precursor cells were divided into 4 groups: control group, group 1, group 2, and group 3. Group 1 was loaded with peptide composition 1 mixture, group 2 with peptide composition 2 mixture, and group 3 with peptide composition 3 mixture. The working concentration of each peptide was 40 μg / mL. No antigenic peptides were added to the cells in the control group.

[0123] (3) DC maturation and detection: On day 7, IFN-γ, poly(I:C), and R848 were added according to the labeled dosages. The working concentrations of IFN-γ were 100 IU / mL, poly(I:C) were 30 μg / mL, and R848 were 5 μg / mL. The cells were cultured for another 24 hours to further stimulate and activate the DCs. The cultured DCs were then detected using flow cytometry antibodies against CD80, CD83, and CD86.

[0124] result:

[0125] like Figure 1 As shown, DC cells stimulated with cytokines, including the control group, peptide composition group 1, peptide composition group 2, and peptide composition group 3, all exhibited high positive rates of CD80, CD83, and CD86 (all greater than 99%), indicating good DC maturation after induction. There was no significant difference between the control group and the experimental groups loaded with different peptide compositions.

[0126] Example 2: Detection of cell activation phenotype in T cells induced by different polypeptide compositions with DC activation

[0127] Operating steps:

[0128] (1) Co-incubation of DCs loaded with antigenic peptides with T cells: DCs loaded with different antigenic peptides from Example 1 were co-incubated with T cells of the corresponding donor at a ratio of 1:10 for 72 hours to form DC-CTLs. The culture conditions were AIM-V medium supplemented with 2% FBS and 100 U / mL IL-2 factor, with the control group consisting of DCs without antigen loading.

[0129] (2) Flow cytometry phenotype: DC-CTLs were labeled with CD3, CD4 and CD8 antibodies, and the proportions of CD3CD4+ and CD3CD8+ cells in different experimental groups were analyzed by flow cytometry. DC-CTLs were labeled with CD3 and CD137, CD107a, CD25, CD69 and IFN-γ antibodies, and the corresponding cell phenotype proportions were analyzed.

[0130] result:

[0131] Flow cytometry results Figure 2The results showed that after co-incubation with different peptide compositions 1, 2, and 3, the proportions of CD3+CD4 T cells and CD3+CD8 T cells in DC-CTLs did not change significantly compared to the control group T cells. However, some activation-related marker detection results showed that, except for CD69, there was no significant difference in other activation markers such as CD25 and CD137. Figure 3 ) and CD107α ( Figure 4 In the study, the peptide-loaded group showed a significant increase compared to the unloaded group, and peptide composition 1 showed higher levels than the other two peptide-loaded groups. Further flow cytometry analysis of intracellular IFN-γ expression in CD3+ cells revealed that the intracellular IFN-γ level in the peptide-loaded group was higher than that in the control group. Figure 5 , Figure 6 The intracellular IFN-γ in the experimental group loaded with peptide composition 1 was also higher than that in the experimental group loaded with peptide composition 2 and the experimental group loaded with peptide composition 3. Figure 5 , Figure 6 ).

[0132] The results showed that DC-CTLs obtained after loading the peptide composition had little effect on the CD4+ / CD8+ ratio, but some T cell activation markers were significantly upregulated, indicating that DCs loaded with the peptide composition played an important role in T cell functional activation. Higher levels of the intracellular cytokine IFN-γ suggest that activated cells may have better tumor-killing capabilities.

[0133] Example 3: Effects of different polypeptide compositions on the specific killing effect of DC-activated T cells on pancreatic cancer cells PANC-1.

[0134] Operating steps:

[0135] (1) Tumor cell line killing experiment:

[0136] DC-CTLs from different experimental groups in Example 2 were used to detect their killing effect on pancreatic cancer tumor cells PANC-1 using a real-time label-free cell function analyzer (RTCA). The pancreatic cancer tumor cells PANC-1 were HLA-A2 type, consistent with the patient's HLA typing and the HLA typing of the tumor antigen peptide used. Specific steps:

[0137] (a) Zeroing: Add 50 μL of DMEM culture medium to each well, place the instrument in the instrument, select step 1, and zero the instrument;

[0138] (b) Target cell plating: Pancreatic cancer cells PANC-1 (purchased from the American Type Culture Collection Center ATCC) were plated at 104 cells / 50μl per well in a plate containing the detection electrode. After a few minutes of incubation and once the cells had stabilized, the plate was placed in the instrument to begin step 2, cell culture.

[0139] (c) Add effector cells: After 24h of culturing target cells, suspend step 2. Add DC-CTLs obtained by polypeptide composition 1, polypeptide composition 2 and polypeptide composition 3 according to the method of Example 2, 50ul per well, and set the effector-target ratio to 10:1. Use T cells from the same donor subject without loading any polypeptide composition as a control. Start step 3, and continue co-culturing for 24h. Then, observe the cell function curve recorded by the instrument.

[0140] (2) Detection of cytokines in supernatant after killing

[0141] Use the cytokine kit to detect the contents of IL-2, IL-4, IL-6, IL-10, TNF-a and IFN-γ cytokines in the supernatant after killing.

[0142] Results:

[0143] As shown in Figure 7 , the killing curve of DC-CTLs obtained by treating T cells with DC loaded with polypeptide composition 1, polypeptide composition 2 and polypeptide composition 3 respectively against target cells PANC-1. Compared with the DC-CTL group without loading polypeptide composition, each experimental group loaded with polypeptide composition has stronger killing effect after adding effector cells for 24h, indicating that the loaded HLA-A2 antigen peptide can be effectively presented to T cells and enhance the killing ability of T cells. Further analysis shows that the killing curve of the experimental group loaded with polypeptide composition 1 is lower than that of polypeptide composition 2 and polypeptide composition 3 group, showing better killing ability to PANC-1 cells.

[0144] Flow detection of TNF-a cytokine concentration in PANC-1 cell supernatant after killing Figure 8 As shown in Figure 8 , the experimental group loaded with peptide composition 1 has the highest TNF-a cytokine concentration in the supernatant, which is 314.55pg / mL, followed by the experimental group loaded with peptide composition 3 and the experimental group loaded with peptide composition 2, which are 252.84pg / mL and 206.90pg / mL respectively.

[0145] Flow detection of IFN-γ factor concentration in PANC-1 cell supernatant after killing Figure 9 ), the experimental group loaded with peptide composition 1 has the highest IFN-γ cytokine concentration in the supernatant, which is 1999.53pg / mL, followed by the experimental group loaded with peptide composition 3 and the experimental group loaded with peptide composition 2, which are 1471.55pg / mL and 1339.53pg / mL respectively, which is similar to the detection result of TNF-a factor.

[0146] The above results show that among the three groups of HLA-A2 antigen peptides, the DC-CTLs loaded with the antigen peptide combination 1 have stronger killing ability on the pancreatic cancer cell PANC-1.

[0147] Example 1 Peptide replacement of polypeptide composition 1 on the specific killing effect of DC-activated T cells on pancreatic cancer cell PANC-1

[0148] Operation steps:

[0149] Culture of immature DC cells: DC precursor cells are added to serum-free AIM-V medium (purchased from Gibco) and cultured after adding IL-4 and GM-CSF to working concentrations of 50 ng / mL and 100 ng / mL, respectively, and half-volume replacement is performed on the third day.

[0150] (1) Polypeptide loading of immature DC cells: After the fifth day, the DC precursor cells are divided into four groups, namely the control group, the polypeptide composition 1 group, the peptide replacement group 1, and the peptide replacement group 2. In the polypeptide composition 1 group, the polypeptide composition 1 mixture is added; in the peptide replacement group 1, the peptide 3 in the polypeptide composition 1 is replaced by the peptide 7, and the rest remains unchanged; in the peptide replacement group 2, the peptide 1 in the polypeptide composition 1 is replaced by the peptide 2, and the peptide 3 is replaced by the peptide 4, and the rest remains unchanged; the working concentration of each peptide is 40 μg / mL.

[0151] (2) Cultured to the seventh day, IFN-γ, poly(I:C) and R848 are added according to the indicated amount, the working concentration of IFN-γ is 100 IU / mL, the working concentration of poly(I:C) is 30 μg / mL, and the working concentration of R848 is 5 μg / mL, and cultured for 24 h to further stimulate activated DC.

[0152] Co-incubation of antigen peptide-loaded DCs with T cells: The DCs loaded with different antigen peptides in Example 1 are co-incubated with the corresponding donor T cells at a ratio of 1:10 for 72 h to form DC-CTLs. The culture conditions are 2% FBS and 100 U / mL IL-2 factor added to AIM-V medium, and the control group is DC without antigen loading.

[0153] Tumor cell line PANC-1 cell killing experiment: Take the DC-CTLs of different experimental groups, and use real-time label-free cell function analyzer (RTCA) to detect their killing effect on pancreatic cancer tumor cells PANC-1. The specific steps are as follows:

[0154] (a) Zero setting: Add 50 μL of DMEM medium to each well, place it in the instrument, select step 1, and set zero.

[0155] (b) Target cell plating: Pancreatic cancer cell PANC-1 (purchased from American Type Culture Collection, ATCC) was plated at 104 cells per well in 50 μl in the plate containing the detection electrode, and placed for a few minutes, and then placed in the instrument after the cells were stable, and step 2 was started to culture the cells;

[0156] (c) Addition of effector cells: After the target cells were cultured for 24 h, step 2 was paused. DC-CTLs obtained by using polypeptide composition 1, polypeptide composition 2, and polypeptide composition 3 according to the method of Example 2 were added at 50 μl per well, and the effector-to-target ratio was set at 10:1. T cells from the same donor subject without loading any polypeptide composition were used as a control, and step 3 was started to continue the co-culture for 24 h. The cell function curve recorded by the instrument was observed.

[0157] Supernatant cytokine detection after killing: The supernatant after killing was detected for IL-2, IL-4, IL-6, IL-10, TNF-a, and IFN-γ cytokine content using a cytokine kit.

[0158] Results:

[0159] As shown in Figure 10 , the killing curve of the DC-CTLs obtained by treating T cells using polypeptide composition 1, peptide replacement group 1, and peptide replacement group 2 after loading DCs against target cells PANC-1. Compared with the DC-CTL group loaded with polypeptide composition 1, the killing ability of the DC-CTLs of peptide replacement group 2 and peptide replacement group 1 against pancreatic cancer cells PANC-1 gradually weakened after the replacement of antigen peptide 3 and antigen peptide 1 with other antigens, indicating that antigen peptide 3 and antigen peptide 1 play an important role in polypeptide composition 1.

[0160] The flow detection of the TNF-a cytokine concentration in the supernatant of PANC-1 cells after killing is shown in Figure 11 , and the TNF-a secretion amount of polypeptide composition 1 was the highest, followed by peptide replacement group 1 and peptide replacement group 2, and the TNF-a content of each group was 385.42 pg / mL, 266.55 pg / mL, and 205.42 pg / mL, respectively. Figure 12 The flow detection of the IFN-γ factor concentration in the supernatant of PANC-1 cells after killing is shown in , and polypeptide composition 1 had a better cytokine secretion amount of 1882.77 pg / mL, which was higher than that of peptide replacement group 1 and peptide replacement group 2, which were 1413.85 pg / mL and 1244.90 pg / mL, respectively.

[0161] Figures 10-12The results show that the polypeptide composition 1 (preferably the peptide combination) has stronger killing effect on pancreatic cancer PANC-1 cells than the peptide replacement group 1 and the peptide replacement group 2, and the secretion of cytokines including TNF-α and IFN-γ is also higher. In addition, whether it is the killing effect on pancreatic cancer PANC-1 cells or the corresponding cytokine secretion, the peptide replacement group 1 is better than the peptide replacement group 2.

[0162] The above results show that in the killing of pancreatic cancer PANC-1 cells, antigen peptide 1 and antigen peptide 3 play a key role in the preferred HLA-A2 type antigen peptide combination and have strong irreplaceability.

[0163] All the documents mentioned in the present application are cited as references in the present application, as if each document is cited as a reference individually. In addition, it should be understood that after reading the above content of the present application, those skilled in the art can make various modifications or changes to the present application, and these equivalent forms also fall within the scope defined by the claims attached to the present application. SEQUENCE LISTING <110> Shanghai Cell Therapy Group Co., Ltd. <120> A polypeptide composition specifically binding to HLA-A2 typing and application <130> 19A768 <160> 9 <170> SIPOSequenceListing 1.0 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence (Artificial Sequence) <400> 1 Leu Thr Leu Gly Glu Phe Leu Lys Leu 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence (Artificial Sequence) <400> 2 Arg Leu Leu Gln Glu Thr Glu Leu Val 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <400> 3 Tyr Leu Ser Gly Ala Asn Leu Asn Leu 1 5 <210> 4 <211> 9 <212> PRT <213> Artificial Sequence <400> 4 Ile Leu Ala Lys Phe Leu His Trp Leu 1 5 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <400> 5 Lys Val Ala Glu Leu Val His Phe Leu 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <400> 6 Lys Ile Phe Gly Ser Leu Ala Phe Leu 1 5 <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <400> 7 Leu Leu Gly Arg Asn Ser Phe Glu Val 1 5 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <400> 8 Ile Met Gin Leu Met Pro Phe Gly Cys 1 5 <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <400> 9 Ile Met Asp Gin Val Pro Phe Ser Val 1 5

Claims

1. A polypeptide composition that specifically binds to HLA-A2 typing, characterized in that, The composition comprises polypeptides derived from antigen proteins Survivin, CEA, and Her2, wherein the polypeptide derived from the antigen protein Survivin is shown in SEQ ID NO:1, the polypeptide derived from the antigen protein CEA is shown in SEQ ID NO:3, and the polypeptide derived from the antigen protein Her2 is shown in SEQ ID NO:

6.

2. The polypeptide composition according to claim 1, characterized in that, The mass ratio of the polypeptides derived from the antigen proteins Survivin, CEA, and Her2 is 1–5:1–5:

1.

3. The polypeptide composition according to claim 1, characterized in that, The mass ratio of the polypeptides derived from the antigen proteins Survivin, CEA, and Her2 is 1–3:1–3:

1.

4. The polypeptide composition according to claim 1, characterized in that, The mass ratio of the polypeptides derived from the antigen proteins Survivin, CEA, and Her2 is 1:1:

1.

5. A cell loaded with the polypeptide composition of any one of claims 1-4.

6. The cell according to claim 5, characterized in that, The cells are antigen-presenting cells, including professional antigen-presenting cells or non-professional antigen-presenting cells.

7. The cell according to claim 6, characterized in that, The professional antigen-presenting cells include lymphocytes, dendritic cells (DCs), macrophages, endothelial cells, or stem cells.

8. The cell according to claim 5, characterized in that, The cells are dendritic cells, macrophages, or lymphocytes.

9. The cell according to claim 5, characterized in that, The cells in question are dendritic cells.

10. The cell according to claim 6, characterized in that, The non-professional antigen-presenting cells are antigen-presenting cells expressing type I HLA.

11. The method for preparing cells according to claim 5, characterized in that, step include: The polypeptide composition according to any one of claims 1-4 is contacted with antigen-presenting cells, and the polypeptide composition is loaded onto the antigen-presenting cells to obtain antigen-presenting cells loaded with the polypeptide composition.

12. The preparation method according to claim 11, characterized in that, The contact is a co-incubation, and the culture medium used for co-incubation is any one or more of AIM-V, DMEM, or RPMI-1640; the co-incubation time is 1-2 days; the co-incubation temperature is room temperature to 37°C; during the co-incubation process, the concentration of each polypeptide in the polypeptide composition of any one of claims 1-4 is 10-100 μg / mL.

13. The preparation method according to claim 12, characterized in that, The culture medium is AIM-V medium.

14. The preparation method according to claim 12, characterized in that, The culture medium is AIM-V medium that does not contain any serum.

15. The preparation method according to claim 12, characterized in that, The co-incubation period is 2 days.

16. The preparation method according to claim 12, characterized in that, During co-incubation, the concentration of each polypeptide in the polypeptide composition according to any one of claims 1-4 is 20-80 μg / mL.

17. The preparation method according to claim 12, characterized in that, During co-incubation, the concentration of each polypeptide in the polypeptide composition according to any one of claims 1-4 is 40 μg / mL.

18. The preparation method according to claim 11, characterized in that, It also includes contacting antigen-presenting cells loaded with peptide compositions with maturation-promoting factors to induce the antigen-presenting cells to mature, thereby obtaining mature antigen-presenting cells loaded with peptide compositions.

19. The preparation method according to claim 18, characterized in that, The contact is co-incubation, and the culture medium used for co-incubation is any one or more of AIM-V, DMEM, or RPMI-1640; the co-incubation time is 8-72 hours; the co-incubation temperature is room temperature to 37°C; the maturation-promoting factor is selected from one or more of TNF-α, IL-1β, IL-6, PGE2, IFN-γ, poly(I:C), R848, or ATP.

20. The preparation method according to claim 19, characterized in that, The culture medium used for the co-incubation was AIM-V medium.

21. The preparation method according to claim 19, characterized in that, The culture medium used for the co-incubation was AIM-V medium that did not contain any serum.

22. The preparation method according to claim 19, wherein the maturation-promoting factors are TNF-α, IL-1β, IL-6 and PGE2, wherein the working concentration of TNF-α is 5-50 ng / mL; the working concentration of IL-1β is 5-50 ng / mL; the working concentration of IL-6 is 800-1500 U / mL; and the working concentration of PGE2 is 0.5-3 µg / mL.

23. The preparation method according to claim 22, wherein the working concentration of TNF-α is 10-30 ng / mL.

24. The preparation method according to claim 22, wherein the working concentration of IL-1β is 10-30 ng / mL.

25. The preparation method according to claim 22, wherein the working concentration of IL-6 is 800-1200 U / mL.

26. According to the preparation method of claim 22, the working concentration of PGE2 is 0.5-1.5 µg / mL.

27. The preparation method according to claim 19, characterized in that, The maturation-promoting factors are IFN-γ, poly(I:C), and R848. The working concentration of IFN-γ is 10-1000 IU / mL, the working concentration of poly(I:C) is 1-200 μg / mL, and the working concentration of R848 is 0.1-50 μg / mL.

28. The preparation method according to claim 19, characterized in that, The maturation-promoting factors are IFN-γ, poly(I:C), R848, and ATP. The working concentration of IFN-γ is 10-1000 IU / mL, the working concentration of poly(I:C) is 1-200 μg / mL, the working concentration of R848 is 0.1-50 μg / mL, and the working concentration of ATP is 0.1-10 mM.

29. The preparation method according to claim 27 or 28, characterized in that, The working concentration of IFN-γ is 100-300 IU / mL.

30. The preparation method according to claim 27 or 28, characterized in that, The working concentration of poly(I:C) is 20-40 μg / mL.

31. The preparation method according to claim 27 or 28, characterized in that, The working concentration of R848 is 1-10 μg / mL.

32. The preparation method according to claim 28, characterized in that, The working concentration of ATP is 0.1-5 mM.

33. The preparation method according to claim 27 or 28, characterized in that, The working concentration of IFN-γ is 100 IU / mL.

34. The preparation method according to claim 27 or 28, characterized in that, The working concentration of poly(I:C) is 30 μg / mL.

35. The preparation method according to claim 27 or 28, characterized in that, The working concentration of R848 is 5 μg / mL.

36. The preparation method according to claim 27, characterized in that, The working concentration of ATP is 1 mM.

37. An activated immune cell obtained by contacting the cell of any one of claims 5-10 with an unactivated immune effector cell.

38. The immune cells according to claim 37, characterized in that, The contact is a co-incubation, the co-incubation time is 2-48 hours; the co-incubation culture medium is AIM-V, DMEM or RPMI 1640; the ratio of cells to immune effector cells as described in claims 5-8 is 1:10-50.

39. The immune cells according to claim 38, characterized in that, The co-incubation period is 24-48 hours.

40. The immune cells according to claim 38, characterized in that, The co-incubation period is 24 hours.

41. The immune cells according to claim 38, characterized in that, The medium used for co-incubation was AIM-V medium.

42. The immune cells according to claim 38, characterized in that, The culture medium for co-incubation was AIM-V medium containing 2% v / v FBS.

43. The immune cells according to claim 38, characterized in that, The ratio of cells to immune effector cells according to any one of claims 5-8 is 1:10-30.

44. The immune cells according to claim 38, characterized in that, The ratio of cells to immune effector cells according to any one of claims 5-8 is 1:

10.

45. The immune cells according to claim 38, characterized in that, The AIM-V culture medium also contains IL-2, with a working concentration of 10-100 U / mL.

46. ​​The immune cell according to claim 43, characterized in that, The working concentration of IL-2 is 100 U / mL.

47. Use of the polypeptide composition of any one of claims 1-4, and / or the cells of claims 5-10, and / or the immune cells of any one of claims 37-46 in the preparation of a reagent or kit for detecting tumors, said reagent or kit comprising biological products, said tumor being pancreatic cancer.

48. Use of the polypeptide composition of any one of claims 1-4, and / or the cells of claims 5-10, and / or the immune cells of any one of claims 37-46 in the preparation of a medicament for the prevention and / or treatment of tumors, said medicament comprising a biological product, said tumor being pancreatic cancer.

49. A drug for the prevention and / or treatment of tumors, characterized in that, The tumor is pancreatic cancer and contains a polypeptide composition according to any one of claims 1-4, and / or cells according to any one of claims 5-10, and / or immune cells according to any one of claims 37-46.

50. A reagent or kit for detecting tumors, characterized in that, The tumor is pancreatic cancer and contains a polypeptide composition according to any one of claims 1-4, and / or cells according to any one of claims 5-10, and / or immune cells according to any one of claims 37-46.

51. The drug according to claim 49 or the reagent or kit according to claim 50, characterized in that, It also contains adjuvants and / or carriers.

52. The drug according to claim 49 or the reagent or kit according to claim 50, characterized in that, It also contains immunomodulators.

53. The drug according to claim 49 or the reagent or kit according to claim 50, characterized in that, It also contains excipients.

54. Use of the polypeptide composition of any one of claims 1-4, and / or the cells of any one of claims 5-10, and / or the immune cells of any one of claims 37-46 in the preparation of a medicament for the prevention or treatment of pancreatic cancer by inducing an immune response in vivo.

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