阅读说明：本技术 一种用于预防和/或治疗***的药物组合物及其制备方法和其应用 (Pharmaceutical composition for preventing and/or treating cervical cancer and preparation method and application thereof ) 是由 闫小君 罗进 于 2020-06-15 设计创作，主要内容包括：一种用于预防和/或治疗宫颈癌的药物组合物及其制备方法和其应用。本发明提供了HPV单表位T细胞抗原肽、由该抗原肽制备得到的多能T细胞表位肽,以及负载所述多能T细胞表位肽的DC细胞。所述抗原肽与HLA-A2结合特异性更高,能够更有效地刺激IFN-γ的产生,从而诱导更强的体内抗病毒效应和清除宫颈上皮细胞存在的HPV,可用于宫颈癌的预防和/或治疗。(A pharmaceutical composition for preventing and/or treating cervical cancer, and its preparation method and application are provided. The invention provides an HPV single epitope T cell antigen peptide, a pluripotent T cell epitope peptide prepared from the antigen peptide, and a DC cell loaded with the pluripotent T cell epitope peptide. The antigen peptide has higher binding specificity with HLA-A2, can more effectively stimulate the generation of IFN-gamma, thereby inducing stronger in vivo antiviral effect and eliminating HPV existing in cervical epithelial cells, and can be used for preventing and/or treating cervical cancer.)

1. A single epitope T cell antigen peptide, which is selected from any one of LLLKIFGGV, TLADLLLKI, IPLEELPSV, ILFEYDNPA or a combination thereof.

2. A pluripotent T cell epitope peptide is characterized by having a structure shown in a formula I,

wherein X is a basic amino acid, and Z is a single epitope T cell antigen peptide, preferably any one of LLLKIFGGV, TLADLLLKI, IPLEELPSV, ILFEYDNPA or a combination thereof.

3. A pluripotent T cell epitope peptide having a structure represented by formula II,

wherein X is a basic amino acid, and Z is a single epitope T cell antigen peptide, preferably any one of LLLKIFGGV, TLADLLLKI, IPLEELPSV, ILFEYDNPA or a combination thereof.

4. The pluripotent T-cell epitope peptide according to claim 2 or 3, wherein the N-terminus of the pluripotent T-cell epitope peptide is modified with palmitoylserine.

5. The pluripotent T-cell epitope peptide according to claim 4, wherein the palmitoylserine is modified by means of an-AAA-flexible linker.

6. A method of inducing generation of DC cells loaded with said pluripotent T cell epitope peptide, comprising the steps of:

1) isolating the DC cells;

2) incubating the DC cells prepared in step 1) with GM-CSF and IL-4 to differentiate into mature DC cells;

3) (ii) shocking the mature DC cells produced in step 2) with a pluripotent T-cell epitope peptide according to any of claims 2 to 5.

7. DC cells loaded with said pluripotent T cell epitope peptide obtained according to the method of claim 6.

8. A pharmaceutical composition comprising a mono-epitope T-cell antigen peptide according to claim 1, a pluripotent T-cell epitope peptide according to any one of claims 2 to 5, and/or a DC cell according to claim 7.

9. Use of a mono-epitope T-cell antigen peptide according to claim 1, a pluripotent T-cell epitope peptide according to any one of claims 2 to 5, and/or a DC cell according to claim 7 for the preparation of a medicament for the prevention and/or treatment of cervical cancer.

10. Use of a pharmaceutical composition according to claim 8 for the preparation of a medicament for the prevention and/or treatment of cervical cancer.

Technical Field

The invention relates to the field of medicines, in particular to a pharmaceutical composition for preventing and/or treating cervical cancer, a preparation method and application thereof.

Background

Cervical cancer is the most common malignancy of the reproductive tract in women and seriously threatens the health and life of women. China is a high-incidence state of cervical cancer. In recent years, young cervical cancer patients have a significantly rising trend, with an increasing rate of incidence of 2% -3% per year. Human Papillomaviruses (HPV) can cause benign and malignant hyperplasia and are closely associated with high-risk HPV and cervical cancer. At present, cervical cancer is prevented and treated by adopting modes of operation, chemotherapy, radiotherapy, biological treatment, Chinese and western medicine combination therapy and the like. However, these therapies have the defects of lack of treatment specificity and selectivity, etc., thereby causing the treatment effect to be not ideal, and seriously damaging normal tissue cells, thereby having the defects of great toxic and side effects, etc., influencing the life cycle and the life quality of patients and increasing the medical expense of the patients.

Cellular immunotherapy is a safe and effective treatment means, and its preventive and therapeutic effects in clinical treatment are becoming prominent.

CN104998260A discloses a DC cell-based HPV virus vaccine, which co-cultures HPV16 chimeric virus-like particles and immature DC cells to realize antigen loading, and promotes the maturation of the immature DC cells loaded with antigens, thereby obtaining the DC cell vaccine with obviously improved CD80 and CD83 expression capacity and greatly increased IL-12 secretion capacity.

CN109456945A discloses a DC vaccine against HPV, which transfects DC cells with modified HPV16mRNA molecules, resulting in genetically modified DC cells that induce IFN, TNF and IL2 production in patients, thereby eliminating HPV16 in vivo.

CN106084008A discloses a four-branched poly-antigen peptide, which can be applied to sensitizing dendritic cells cultured in vitro to make the dendritic cells carry antigen information and is used for treating cervical cancer infected by high-risk human papilloma virus.

On the basis of the prior art, the antigen peptide for sensitizing dendritic cells is optimized, the binding specificity and the reaction sensitivity of the antigen peptide are increased, and the action quantity and the action efficiency of CTL are obviously improved.

Disclosure of Invention

The invention aims to provide a single epitope T cell antigen peptide of HPV, which is selected from any one or combination of LLLKIFGGV, TLADLLLKI, IPLEELPSV, ILFEYDNPA.

Another object of the present invention is to provide a pluripotent T cell epitope peptide having a structure represented by any one of formula I or formula II,

wherein, X in any structure shown in formula I or formula II is basic amino acid, Z is single epitope T cell antigen peptide, preferably any one of LLLKIFGGV, TLADLLLKI, IPLEELPSV, ILFEYDNPA or its combination.

In a preferred embodiment of the present invention, the basic amino acid is selected from any one of arginine (R), lysine (K), and histidine (H).

In a preferred embodiment of the present invention, the single epitope T cell antigen peptide is any one or a combination of LLLKIFGGV, TLADLLLKI, IPLEELPSV, ILFEYDNPA.

In a preferred embodiment of the invention, said pluripotent T cell epitope peptide is selected from any one of the structures shown in table 1:

TABLE 1

In a preferred embodiment of the present invention, the N-terminus of the pluripotent T cell epitope peptide is modified with palmitoylserine.

In a preferred embodiment of the invention, the palmitoyl serine is modified in a manner of-AAA-flexible linkage to a pluripotent T cell epitope peptide.

It is another object of the present invention to provide a method for inducing the production of DC cells loaded with said pluripotent T cell epitope peptide, comprising the steps of:

1) separating monocytes from peripheral blood and bone marrow, and separating CD14+ cells from the monocytes using CD14 magnetic beads;

2) incubating the CD14+ cells prepared in step 1) with GM-CSF and IL-4 to differentiate into mature DC cells;

3) and (3) impacting the mature DC cell prepared in the step 2) by using the pluripotent T cell epitope peptide to obtain the DC cell.

In a preferred embodiment of the present invention, the mononuclear cells are separated from peripheral blood or bone marrow by a leukocyte separation method.

In a preferred embodiment of the present invention, the mature DC cells in step 2) are subjected to a molecular marker staining method to confirm their differentiation and maturity.

In a preferred technical scheme of the invention, the method comprises the following steps:

1) separating monocytes from peripheral blood and bone marrow by leukapheresis, and separating CD14+ cells from the monocytes using CD14 magnetic beads;

2) suspending the CD14+ cells prepared in the step 1) in RPMI-1640 medium containing 100-200ng/ml IL-4 and 20-60ng/ml GM-CSF for culturing, differentiating the CD14+ cells into DC cells, and staining and molecular marking the DC cells by any one or the combination of CD86, CD40, CD80, HLA-DR and MHC-I to prepare positive DC cells;

3) and (3) impacting the positive DC cells prepared in the step 2) by using the pluripotent T cell epitope peptide to obtain the DC cells.

It is another object of the present invention to provide a method for inducing the production of DC cells loaded with said pluripotent T cell epitope peptide, comprising the steps of:

1) separating mononuclear cells from peripheral blood and bone marrow;

2) incubating the DC cells prepared in step 1) with GM-CSF (20-60ng/ml) and IL-4(100-200 ng/ml);

3) CD11c magnetic beads are adopted to sort out CD11c positive DC cells for later use;

3) impacting the CD11c positive DC cells prepared in the step 3) by using the pluripotent T cell epitope peptide to obtain the CD c positive DC cells.

It is another object of the present invention to provide the DC cells loaded with the pluripotent T cell epitope peptide obtained by the method.

Another objective of the invention is to provide a pharmaceutical composition comprising the single epitope T cell antigen peptide.

Another object of the present invention is to provide a pharmaceutical composition comprising the pluripotent T cell epitope peptide.

It is another object of the present invention to provide a pharmaceutical composition comprising DC cells loaded with said pluripotent T cell epitope peptide.

The invention also aims to provide application of the single epitope T cell antigen peptide or the pharmaceutical composition containing the single epitope T cell antigen peptide in drugs for preventing and/or treating cervical cancer.

The invention also aims to provide application of the pluripotent T cell epitope peptide or the pharmaceutical composition containing the pluripotent T cell epitope peptide in medicines for preventing and/or treating cervical cancer.

The invention also aims to provide application of the DC cell loaded with the pluripotent T cell epitope peptide or the pharmaceutical composition containing the DC cell loaded with the pluripotent T cell epitope peptide in medicines for preventing and/or treating cervical cancer.

In a preferred embodiment of the present invention, the cervical cancer is caused by HPV.

In a preferred embodiment of the invention, the HPV is selected from any one of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68 or complications thereof.

Unless otherwise indicated, when the present invention relates to percentages between liquids, said percentages are volume/volume percentages; the invention relates to the percentage between liquid and solid, said percentage being volume/weight percentage; the invention relates to the percentages between solid and liquid, said percentages being weight/volume percentages; the balance being weight/weight percent.

Compared with the prior art, the invention has the following beneficial technical effects:

1. the invention optimizes and improves the prior dendritic T cell antigen peptide, firstly, arginine and the like are selected as a peptide skeleton, thus improving the stability and immunogenicity of the T cell antigen peptide; polypeptide branches are added, the molecular weight and the biological activity of the antigen peptide are increased, the specific binding capacity and the reaction sensitivity of the epitope peptide are exponentially increased in unit space, the acting quantity and the acting efficiency of CTL are obviously improved, and the CTL cannot enter cell nucleuses after being taken by DC cells, so that the safety is better; thirdly, the single epitope antigen peptide in the HPV L2 protein is selected, the binding specificity with HLA-A2 is higher, and the generation of IFN-gamma can be stimulated more effectively, thereby inducing stronger in vivo antiviral effect.

Drawings

FIG. 1 is a graph showing the change in tumor volume of Hela tumor-bearing mice;

FIG. 2Hela tumor-bearing mouse survival rate.

Detailed Description

The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.