HK40010495B - Col14a1-derived tumour antigen polypeptide and use thereof - Google Patents

Description

COL14A 1-derived tumor antigen polypeptide and application thereof

Technical Field

The invention relates to the technical field of cancer diagnosis, prevention and immunotherapy, in particular to a tumor antigen polypeptide caused by COL14A1 gene mutation, a related product thereof and medical application thereof.

Background

Cancer, a disease in which cell proliferation is uncontrolled due to mutations in genes within the cell. The medicine has become a great threat to human health at present and is one of the main causes of human death. The World Health Organization (WHO) has reported in published global cancer report 2014 that worldwide cancer patients and death cases are rapidly increasing in 2012, while nearly half of newly added cancer cases occur in asia, most of which occur in china, and the number of newly added cancer cases in china is higher than the first ^[1] . The data of the annual report of Chinese tumor registration in 2012 show that about 350 ten thousand new cancer cases and about 250 ten thousand new cancer cases die in China every year ^[2] . Therefore, the search for highly effective and specific cancer therapies is of great clinical value.

The traditional methods for treating tumors mainly include surgery, radiotherapy and chemotherapy, but these methods are all adoptedHas great limitations, such as high recurrence rate of tumor metastasis after surgical resection due to proximal invasion or distal metastasis of cancer cells, and severe damage to normal cells of the body, especially hematopoietic system and immune system, caused by radiotherapy and chemotherapy, so that it is difficult to achieve good long-term curative effect on patients with tumor metastasis ^[3] . With the deep research of tumor molecular mechanism and the further development of biotechnology, targeted drug therapy and immunotherapy play an increasingly important role in the comprehensive treatment of tumors. The targeted therapy mainly comprises monoclonal antibodies (sometimes classified as passive immunotherapy) and small molecule targeted drugs, and the immunotherapy mainly comprises cytokine therapy, immune checkpoint monoclonal antibodies, adoptive cell reinfusion, tumor vaccines and the like ^[4,5] . The immunotherapy enhances the anti-tumor immunity of the tumor microenvironment by mobilizing the immune system of the organism so as to control and kill tumor cells, so the immunotherapy has the advantages of high effective rate, strong specificity and good tolerance, and has wide prospect in tumor treatment ^[5,6] 。

The tumor immunotherapy vaccine mainly comprises tumor cell vaccine, dendritic cell vaccine and protein&Polypeptide vaccine, nucleic acid vaccine, genetic engineering vaccine and anti-idiotype tumor vaccine ^[7] . The main mechanism by which these vaccines can kill tumors is by eliciting an immune response in a patient against tumor-specific antigens, including antigen-antibody responses and CTL-specific killing, among others, which CTL-specific killing plays a major role in tumor immune responses. The tumor specific polypeptide is a tumor specific antigen, mainly causes CTL specific killing, and comprises tumor mutant polypeptide and tumor specific high expression polypeptide. The polypeptide of tumor mutation is a specific target of tumor immunotherapy because the polypeptide exists only in tumor tissues of patients, and has the characteristics of good safety, small side effect and the like. The immunotherapy of the target tumor mutation polypeptide takes polypeptide specificity DC-CTL, TIL adoptive reinfusion and other methods as representatives, and has good therapeutic effect ^[8,9] 。

Tumor-specific polypeptides are recognized by CTL or TIL cells and require the antigen presenting function of human leukocyte antigen HLA. Human leukocyte antigens are mainly divided into two subtypes I and II, HLA type I is mainly divided into three subtypes A, B and C, each subtype can be divided intobase:Sub>A plurality of subtypes according to different sequences, HLA-A0201 is one of HLA-A subtypes, accounts for 13% of Chinese population, and hasbase:Sub>A high proportion. The binding capacity of different polypeptides to HLA-A0201 subtype is different. In tumor patients with a particular HLA subtype, the HLA subtype determines that only a portion of the mutant polypeptide has the ability to bind to its HLA and is presented to CTL or TIL cells by its HLA.

The COL14A1 gene encodes the alpha chain of human type XIV collagen, and has 1796 amino acids length and molecular weight of 193,515 dalton. Type XIV collagen binds to the fibril surface and is involved in the regulation of fibril formation, mainly associated with the structural organization of the extracellular matrix and collagen binding.

Reference to the literature：

[1]World Health Organization.Globocan 2012:Estimated cancer incidence,mortality and prevalence worldwide in 2012.

[2]Bernard W.Stewart CPW.World Cancer Report 2014.2014.

[3] Harpsichord, zhang ding di, yangxianghua, zhang jun. New technology of tumor biological treatment-targeted gene therapy, liberty military medicine 2014;26:24-7.

[4]Mellman I,Coukos G,Dranoff G.Cancer immunotherapy comes of age.Nature 2011；480:480-9.

[5]Chen DS,Mellman I.Oncology meets immunology:the cancer-immunity cycle.Immunity 2013；39:1-10.

[6]Currie GA.Eighty years of immunotherapy:a review of immunological methods used for the treatment of human cancer.British journal of cancer 1972；26:141-53.

[7] The research progress of the littletin pepperweed, lihui and royal Xicould in tumor treatment, modern tumor medicine 2013;21:2351-3

[8]Tran E,Turcotte S,Gros A,et al.Cancer immunotherapy based on mutation-specific CD4+T cells in a patient with epithelial cancer.Science.2014.344(6184):641-5.

[9]Cobbold M,De La Pena H,Norris A,et al.MHC class I-associated phosphopeptides are the targets of memory-like immunity in leukemia.Sci Transl Med.2013.5(203):203ra125.

Disclosure of Invention

The invention aims to provide a tumor antigen polypeptide caused by COL14A1 gene mutation, a nucleic acid for encoding the tumor antigen polypeptide, a nucleic acid construct, an expression vector and a host cell containing the nucleic acid, an antigen presenting cell and an immune effector cell of the tumor antigen polypeptide, and medical applications of the tumor antigen polypeptide and the nucleic acid construct, the expression vector and the host cell.

The mutation of COL14A1 gene causes the change of the amino acid sequence, so that the 1040 th site amino acid is mutated from serine to phenylalanine. The mutated COL14A1 gene can be expressed in tumor tissue at high level, thus, the polypeptide can be expressed in tumor tissue at high level. So far, no tumor-specific polypeptide sequence caused by the mutation of the COL14A1 gene is reported at home and abroad, and the polypeptide is not used for immunotherapy research of tumors and affinity research of HLA-A0201; the polypeptide has tumor tissue specificity because it is only expressed in the mutated tumor tissue, thus having very important significance for tumor detection, early prevention and immunotherapy of patients.

In a first aspect, the present invention provides an isolated polypeptide, or immunologically active fragment thereof, selected from the group consisting of:

(a) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 2 (i.e., FMVDGFWSI);

(b) A polypeptide which is formed by substituting, adding and/or deleting one or more amino acid residues in an amino acid sequence shown in SEQ ID NO. 2 and has cytotoxic T lymphocyte inducing ability; and

(c) A variant or derivative of (a) the polypeptide or (b) the polypeptide, which has cytotoxic T lymphocyte inducing capability.

The inventor of the invention finds out frombase:Sub>A breast cancer database that the amino acid at the 1040 th site coded by the COL14A1 gene is mutated from serine (Ser, S) to phenylalanine (Phe, F), and predicts that the mutant polypeptide sequence has high affinity with HLA-A, especially with HLA-A0201 through computer prediction software, wherein the polypeptide consists of 9 amino acids, the molecular weight is 974.23 daltons, and the full-length sequence is: FMVDGFWSI (i.e., SEQ ID NO: 2). The polypeptide is synthesized bybase:Sub>A chemical synthesis method, and T2 affinity test proves that the polypeptide has high affinity with HLA-A, especially HLA-A0201. Proved by in vitro immunogenicity Experiments (ELISPOTs), the polypeptide can induce antigen-specific T cells to secrete IFN-gamma cytokines, and can cause the activation reaction of immune cells; and LDH release experiments have shown that CD8+ T cells can specifically recognize and kill target cells presenting the polypeptide.

The present inventors have also found that (b)base:Sub>A polypeptide formed by substitution, addition or deletion of one or more amino acid residues based on the amino acid sequence of (base:Sub>A)base:Sub>A polypeptide can have the above-mentioned functions of (base:Sub>A)base:Sub>A polypeptide, i.e., having high affinity for HLA-base:Sub>A, particularly HLA-base:Sub>A 0201, and specifically recognized by CD8+ T cells, thereby elicitingbase:Sub>A specific immune response; has cytotoxic T lymphocyte inducing ability.

Preferably, the substitution, addition or deletion of one or more amino acids is the substitution of the amino acid at the 2 nd and/or 9 th position of the amino acid sequence shown in SEQ ID NO. 2;

further preferably, the substitution, addition or deletion of one or more amino acids is that the amino acid at the 2 nd position of the amino acid sequence shown as SEQ ID NO. 2 is substituted by L and/or the amino acid at the 9 th position is substituted by L or V;

still more preferably, the polypeptide of (b) has an amino acid sequence shown as SEQ ID NO. 3 (i.e., FMVDGFWSL), SEQ ID NO. 4 (i.e., FMVDGFWSV), SEQ ID NO. 5 (i.e., FLVDGFWSI), SEQ ID NO. 6 (i.e., FLVDGFWSL), or SEQ ID NO. 7 (i.e., FLVDGFWSV).

Experiments prove that compared with the polypeptide (base:Sub>A), the binding force with HLA-A, especially with HLA-A0201 is enhanced, and the specificity with T cells is unchanged. Therefore, the (b) polypeptide and the (a) polypeptide have the same ability to activate specific T immunity.

The polypeptides of the invention may be synthesized according to methods used in conventional peptide chemistry, including, for example, methods described in: peptide Synthesis, interscience, new York,1966; the polypeptide of the present invention can also be prepared by conventional genetic engineering. For example, the polypeptide may be prepared using conventional DNA synthesis and genetic engineering methods to prepare nucleotides encoding the polypeptide; namely, the polypeptide is produced by the following method: inserting the nucleotide into a common expression vector; transforming a host cell with the obtained recombinant expression vector; culturing the obtained transformant; and collecting the polypeptide from the culture. This can be done, for example, with reference to the methods described in the following documents: molecular Cloning, T.Maniatis et al, CSH Laboratory (1983). The polypeptide obtained by the above method can be confirmed by a reverse phase high performance liquid chromatography-mass spectrometry method.

In a second aspect, the present invention provides an isolated nucleic acid encoding the isolated polypeptide of the first aspect.

In a third aspect, the invention provides a nucleic acid construct comprising a nucleic acid as described in the second aspect operably linked to one or more control sequences capable of directing the production of the polypeptide in an expression host.

In a fourth aspect, the present invention provides an expression vector comprising the nucleic acid construct of the third aspect.

In a fifth aspect, the present invention provides a host cell into which a nucleic acid construct according to the third aspect or an expression vector according to the fourth aspect has been transformed or transfected.

In a sixth aspect, the present invention provides an antigen presenting cell which presents an isolated polypeptide as described in the first aspect on the cell surface.

In a seventh aspect, the present invention provides a method of producing the antigen presenting cell of the sixth aspect, comprising: a step of contacting a polypeptide as described in the first aspect with a cell having antigen presenting ability, or comprising: a step of introducing the nucleic acid according to the second aspect, or the nucleic acid construct according to the third aspect, or the expression vector according to the fourth aspect into a cell having antigen-presenting ability for expression;

preferably, the cell having antigen presenting ability is a dendritic cell.

In an eighth aspect, the invention provides an immune effector cell which recognizes a polypeptide according to the first aspect or recognizes an antigen presenting cell which presents a polypeptide according to the first aspect on the cell surface.

In a ninth aspect, the present invention provides a method of producing an immune effector cell as described in the eighth aspect, comprising: a step of contacting the antigen presenting cell according to the sixth aspect with a cell having an immune effector function;

preferably, the immune effector competent cells are T cells, preferably CD8+ T cells.

In a tenth aspect, the present invention provides a targeted immune cell population formed by antigen presenting cells mixed with lymphocytes and co-cultured.

In an eleventh aspect, the present invention provides a conjugate comprising a polypeptide according to the first aspect and an anti-cancer agent.

In a twelfth aspect, the present invention provides an antibody that specifically recognizes the polypeptide of the first aspect.

In a thirteenth aspect, the present invention provides a method of making an antibody, comprising:

immunizing an animal with a polypeptide according to the first aspect;

collecting the serum of the immunized animal; and

purifying the antibody of interest from the serum.

In a fourteenth aspect, the present invention provides a vaccine for the treatment or prevention of cancer in a patient, comprising a polypeptide according to the first aspect, or comprising a nucleic acid according to the second aspect, or comprising a nucleic acid construct according to the third aspect, or comprising an expression vector according to the fourth aspect, or comprising an antigen presenting cell according to the sixth aspect, or comprising an immune effector cell according to the eighth aspect;

preferably, the cancer is a cancer expressing a polypeptide according to the first aspect;

further preferably, the cancer is selected from lung cancer, melanoma, breast cancer, nasopharyngeal cancer, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, skin cancer, prostate cancer, cervical cancer, leukemia and brain tumor.

In a fifteenth aspect, the present invention provides a pharmaceutical composition for treating or preventing cancer in a patient, comprising a polypeptide according to the first aspect, and a pharmaceutically acceptable carrier.

In a sixteenth aspect, the invention provides the use of a polypeptide according to the first aspect in the manufacture of an antibody for the prevention or treatment of a tumour,

optionally, the tumor expresses HLA-A0201 and the polypeptide simultaneously,

optionally, the tumor is lung cancer, melanoma, breast cancer, nasopharyngeal carcinoma, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, skin cancer, prostate cancer, cervical cancer, leukemia and brain tumor.

In a seventeenth aspect, the invention provides the use of a polypeptide according to the first aspect in the manufacture of a medicament for the prevention or treatment of a tumour,

optionally, said tumor expresses both HLA-A0201 and said polypeptide,

optionally, the tumor is lung cancer, melanoma, breast cancer, nasopharyngeal carcinoma, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, skin cancer, prostate cancer, cervical cancer, leukemia and brain tumor.

In an eighteenth aspect, the present invention provides the use of a polypeptide according to the first aspect in the manufacture of a vaccine for the prevention or treatment of a tumour,

optionally, the tumor expresses HLA-A0201 and the polypeptide simultaneously,

optionally, the tumor is lung cancer, melanoma, breast cancer, nasopharyngeal cancer, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, skin cancer, prostate cancer, cervical cancer, leukemia, and brain tumor.

In a nineteenth aspect, the present invention provides use of a detection reagent for a polypeptide according to the first aspect or a nucleic acid according to the second aspect in the preparation of a kit for diagnosing cancer in a patient;

preferably, the cancer is a cancer expressing a polypeptide according to the first aspect;

further preferably, the cancer is selected from lung cancer, melanoma, breast cancer, nasopharyngeal cancer, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, skin cancer, prostate cancer, cervical cancer, leukemia and brain tumor.

In a twentieth aspect, the present invention provides a kit for diagnosing cancer in a patient comprising a detection reagent for a polypeptide according to the first aspect or a nucleic acid according to the second aspect.

In a twenty-first aspect, the present invention provides a method of treatment comprising:

administering to a patient an effective amount of a polypeptide according to the first aspect, an antigen presenting cell according to the sixth aspect, an immune effector cell according to the eighth aspect, a population of targeting immune cells according to the tenth aspect, a conjugate according to the eleventh aspect, an antibody according to the twelfth aspect, a vaccine according to the fourteenth aspect or a pharmaceutical composition according to the fifteenth aspect.

In a twenty-second aspect, the present invention provides a diagnostic method comprising:

detecting whether a patient-derived biological sample carries a polypeptide according to the first aspect;

determining whether the patient has a tumor based on the fact whether the biological sample carries the polypeptide,

optionally, said tumor expresses both HLA-A0201 and said polypeptide,

optionally, the tumor is lung cancer, melanoma, breast cancer, nasopharyngeal cancer, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, skin cancer, prostate cancer, cervical cancer, leukemia, and brain tumor.

Advantageous effects

The tumor antigen polypeptide FMVDGFWSI of the invention is generated by tumor specific mutation of COL14A1 gene, does not exist in normal tissues of human body without the mutation, and only exists in tumor tissues of patients with the mutation; because the antigen exists only in tumor tissues of patients and does not exist in normal tissues, the specificity is high, the specificity of the induced immune response is also high, and the antigen can induce the specific immune response aiming at the tumor; when the vaccine is used as a tumor vaccine, the vaccine is safer than other tumor polypeptide vaccines, has small side effect, rarely causes serious immune response, and is suitable for industrial production due to simple structure and easy artificial synthesis.

Since the modified forms of the above polypeptides have enhanced binding to HLA-A0201 and no change in specificity to T cells, these modified forms have the same ability to activate specific T immunity as the polypeptide FMVDGFWSI. Therefore, the polypeptide FMVDGFWSI or the variant thereof can be used as a target or a vaccine for biological treatment of tumors simultaneously expressing HLA-A0201 and the mutant polypeptide. The polypeptide FMVDGFWSI or its variant can be polypeptide + adjuvant, or polypeptide loaded DC vaccine, or polypeptide specific DC-CTL, DC-CIK vaccine, etc., for preventing and treating tumor; the tumor comprises cancer types such as lung cancer, melanoma, breast cancer, nasopharyngeal carcinoma, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, skin cancer, prostatic cancer, cervical cancer, leukemia, brain tumor and the like expressing the polypeptide sequence.

In addition, the polypeptide of the invention is only present in tumor tissues, and whether the free polypeptide exists in serum can be detected by means of mass spectrometry, so that the polypeptide can be used as a tumor marker for tumor diagnosis.

Drawings

FIG. 1 is a graph showing the results of flow cytometry for detecting the affinity of the polypeptide of the present invention for T2.

FIG. 2 is a graph showing the results of specific killing of target cells presenting the polypeptide of the present invention by immune cells.

FIG. 3 shows the tumor growth inhibition effect and mouse survival rate after immunotherapy using the polypeptide of the present invention; wherein, FIG. 3A shows the tumor growth inhibition effect after treatment with adjuvant, adjuvant + wild type polypeptide (FMVDGSWSI, SEQ ID NO: 1), adjuvant + mutant polypeptide FMVDGFWSI or its deformable polypeptide, and FIG. 3B shows the survival rate of mice after treatment with adjuvant, adjuvant + wild type polypeptide (FMVDGSWSI), adjuvant + mutant polypeptide FMVDGFWSI or its deformable polypeptide.

FIG. 4 shows the tumor growth inhibition effect and mouse survival rate after immunotherapy using the polypeptide of the present invention; wherein, fig. 4A shows tumor growth inhibition effect after treatment with DC-loaded wild-type polypeptide (FMVDGSWSI), DC-loaded mutant polypeptide (FMVDGFWSI) or a deformable polypeptide thereof, and fig. 4B shows mouse survival rate after treatment with DC-loaded wild-type polypeptide (FMVDGSWSI), DC-loaded mutant polypeptide FMVDGFWSI or a deformable polypeptide thereof.

FIG. 5 shows the tumor growth inhibition effect and mouse survival rate after immunotherapy using the polypeptide of the present invention; among them, fig. 5A shows a tumor growth inhibition effect after treatment with DC cells infected with lentiviral vectors carrying wild-type polypeptide (FMVDGSWSI), mutant polypeptide (FMVDGFWSI) or a deformable polypeptide thereof, and fig. 5B shows survival rate of mice after treatment with DC cells infected with lentiviral vectors carrying wild-type polypeptide (FMVDGSWSI), mutant polypeptide (FMVDGFWSI) or a deformable polypeptide thereof.

FIG. 6 shows the tumor growth inhibition effect and mouse survival rate after immunotherapy using the polypeptide of the present invention; wherein, fig. 6A shows tumor growth inhibition effect after treatment with DC-loaded wild-type polypeptide (FMVDGSWSI) + CTL, DC-loaded mutant-type polypeptide (FMVDGFWSI) or its transformable polypeptide + CTL, and fig. 6B shows mouse survival rate after treatment with DC-loaded wild-type polypeptide (FMVDGSWSI) + CTL, DC-loaded mutant-type polypeptide (FMVDGFWSI) or its transformable polypeptide + CTL.

Detailed Description

For the purpose of facilitating an understanding of the present invention, the present invention will now be described by way of examples. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

Example 1 prediction of affinity of the polypeptide of the invention

The affinity of the polypeptide was predicted by the following procedure:

according to the selected HLA allele typing, the affinity prediction of the polypeptide is carried out by using self-developed 'mutant polypeptide binding capacity prediction software based on tumor DNA and RNA sequencing' (software copyright number: 2016SR 002835). The predicted results are expressed as IC50 scores, with an IC50 of less than 500nM indicating affinity of the polypeptide and an IC50 of less than 50nM indicating high affinity of the polypeptide.

Wild type polypeptide FMVDGSWSI (SEQ ID NO: 1), mutant polypeptide FMVDGFWSI of the present invention and 5 of its transformable polypeptides (also of the present invention) were obtained by standard solid phase synthesis and purified by reverse phase HPLC. The purity (> 90%) and identity of the polypeptide were determined by HPLC and mass spectrometry, respectively. In this example, the above-described affinities of the polypeptide of the present invention and the wild-type polypeptide to HLA-a0201 were predicted in the above manner, and the prediction scores are shown in table 1 below.

TABLE 1 prediction of the affinity of the polypeptide for HLA-A0201

Mutant polypeptide sequences	IC50(nM)	Wild type polypeptide sequence	IC50(nM)
				FMVDGFWSI	2.18	FMVDGSWSI	3.93
FMVDGFWSL	2.32	-	-
				FMVDGFWSV	2.56	-	-
FLVDGFWSI	2.87	-	-
				FLVDGFWSL	3.01	-	-
FLVDGFWSV	3.14	-	-

As can be seen from Table 1, the IC50 scores of the mutant polypeptides of the present invention were all lower than 50nM as predicted by computer software, indicating that the mutant polypeptides of the present invention have high affinity for HLA-A0201.

Example 2T 2 affinity validation of the Polypeptides of the invention

Obtaining the wild-type polypeptide FMVDGSWSI, the polypeptide FMVDGFWSI of the invention and 5 transformable polypeptides thereof in the manner described in example 1; take 2X 10 ⁵ T2 cells (a lymphocyte, tumor cell line, expressing HLA-A0201; T2 cells are cell lines deficient in the antigen polypeptide Transporter (TAP) essential in the endogenous antigen presentation pathway,t and B lymphocyte hybridoma cells positive for HLA-A2 can be used for researching the affinity of polypeptide and HLA-A2 and the interaction between T cells and MHC-I molecules; ATCC accession No.: CRL-1992 ^TM ) Resuspending the resulting mixture in a 24-well plate using 500. Mu.l of IMDM serum-free medium, adding 10. Mu.g/ml of the above polypeptide and human β 2 microglobulin (final concentration, 3. Mu.g/ml), respectively, in an incubator (37 ℃,5% CO) ₂ ) Cultured overnight in the medium. Each group is provided with 2 repeated holes; t2 cells without added polypeptide were used as background control (i.e. blank) and the group with added CMV polypeptide (NLVPMVATV) was used as positive control.

The cell culture was centrifuged at 200g for 5 minutes to collect the cells. After the collected cells were washed twice with PBS, the cells were directly incubated with FITC monoclonal antibody against HLA-A0201 and maintained at 4 ℃ for 30 minutes. Then using a flow cytometer (BD FACSJazz) ^TM ) And its software detects and analyzes its Mean Fluorescence Intensity (MFI). The Fluorescence Index (FI) was calculated using the following formula:

FI＝[MFI _sample(s) –MFI _Background ]/MFI _Background ；

Wherein, MFI _Background Represents a value without peptide; FI>1.5 shows that the peptide has high affinity for HLA-A0201 molecule, 1.0<FI<1.5 shows that the peptide has moderate affinity for HLA-A0201 molecule, and 0.5<FI<1.0 shows that the peptide is HLA-A0201 molecule with low affinity.

The results of affinity detection of each of the above polypeptides for HLA-A0201 are shown in Table 2 below.

TABLE 2

Sample(s)	Adding polypeptide concentration	Mean fluorescence intensity	FI
				FMVDGSWSI	100μM	772.5	2.38
FMVDGFWSI	100μM	763.9	2.35
				FMVDGFWSL	100μM	748.3	2.28
FMVDGFWSV	100μM	721.2	2.16
				FLVDGFWSI	100μM	701.3	2.07
FLVDGFWSL	100μM	663.9	1.91
				FLVDGFWSV	100μM	642.5	1.81
Blank space	0μM	228.3	0
				CMV	100μM	663.2	1.90

As can be seen from table 2, the affinity verified that FI of the blank group was 0 and FI of the CMV polypeptide as a positive control was 1.90, both of which were normal; while FI of both the wild-type polypeptide and the mutant polypeptide of the present invention is greater than 1.5, further demonstrating that both the wild-type polypeptide and the mutant polypeptide of the present invention are high affinity.

EXAMPLE 3 in vitro stimulation of the Polypeptides of the invention to expand CD8+ T cells

Taking PBMC cells of a volunteer positive for HLA-A0201 subtype, 2X 10 ⁷ The PBMC cells are separated by a wall attaching method for mononuclear cells (3 h), and the CD8+ T cells are separated by a CD8 magnetic bead method. Adherent monocytes are induced to immature DC with GM-CSF (1000U/ml), IL-4 (1000U/ml), and then with IFN-gamma (100U/ml), CD40L (100U/ml), mutant polypeptide FMVDGFWSI of the present invention or any of its 5 transformable polypeptides, to mature DC cells specific for the polypeptide. Mature DC cells loaded with polypeptide are irradiated and co-cultured with CD8+ T cells of volunteers, IL-21 is added, after 3 days, IL-2 and IL-7 are supplemented once in 5 and 7 days later (the final concentrations of IL-21, IL-2 and IL-7 are respectively 30ng/ml, 5ng/ml and 10 ng/ml), co-cultured cells are taken for counting in 10 days, and subsequent ELISPOTs and LDH detection are carried out. The counting results are shown in table 3 below.

TABLE 3 results of cell count after culture

	Total number of cells in well before culture	Total number of cells in well after culture
			FMVDGFWSI	2.5×10^6	1.72×10^7
FMVDGFWSL	2.5×10^6	1.67×10^7
			FMVDGFWSV	2.5×10^6	1.61×10^7
FLVDGFWSI	2.5×10^6	1.56×10^7
			FLVDGFWSL	2.5×10^6	1.46×10^7
FLVDGFWSV	2.5×10^6	1.37×10^7

As can be seen from Table 3, after 10 days of culture, the cells proliferated significantly, and the total cell number was amplified by 5-7 fold, indicating that the addition of the polypeptide of the present invention significantly stimulated the amplification of CD8+ T cells.

Example 4 ELISPOTs method verifies that the polypeptide of the invention activates CD8+ T cell immune response

This example uses an ELISPOTs assay kit (cat # 3420-4AST-10, MABTECH) to verify that the polypeptide of the present invention activates the immune response of CD8+ T cells.

ELISPOTs detection method principle: CD8+ T cells are capable of specifically recognizing complexes of HLA-A0201 and polypeptides, differing in the sequence of the polypeptides, and differing in the population of T cells recognizing complexes of polypeptides with HLA-A0201. Since T2 cells express HLA-A0201, CD8+ T cells are able to specifically recognize polypeptide-loaded T2 cells, and after specifically recognizing complexes of HLA-A0201 and polypeptides, polypeptide-specific CD8+ T cells are able to reactivate and secrete IFN-gamma interferon. And IFN-gamma interferon secreted by CD8+ T cells through activation can be captured by antibodies on ELISPOTs plates, and finally the antibodies recognizing the IFN-gamma can degrade substrates and develop color through enzymes coupled on the antibodies, so that spots are finally generated. The number of spots represents the number of cells activated to secrete IFN-gamma interferon.

The cells cultured in experimental example 3 and T2 cells of mutant polypeptide FMVDGFWSI and wild polypeptide FMVDGSWSI of the invention are added into ELISPOTs plates for culture, and ELISPOTs detection is carried out after 20 hours (see the kit instruction). Finally, the spots generated by the elispot test were counted.

The requirements for immunogenicity of the test polypeptide are as follows: spot number (test polypeptide)/spot number (unrelated polypeptide) >2; that is, the number of spots caused by the test polypeptide is more than twice the number of spots caused by the unrelated polypeptide, indicating that the test polypeptide is immunogenic.

The results of ELISPOTs assay of the polypeptides of the invention are shown in Table 4 below.

TABLE 4 polypeptide stimulation of specific CD8+ T cells to secrete IFN-gamma interferon

From the results in Table 4, it is clear that the polypeptides of the invention and their modified forms are immunogenic and specifically activate the CD8+ T cell immune response.

Example 5 LDH Release assay demonstrating specific killing of target cells presenting a polypeptide of the invention by CD8+ T cells Activity of

The cells cultured in the experimental example 3 and the T2 cells loaded with the mutant polypeptide or the wild-type polypeptide of the invention or unloaded with the polypeptide are co-cultured, the experiment is provided with a maximum release hole, a volume correction hole, a culture medium control hole, a spontaneous release hole, controls of different effective target ratios (number ratio of T cells to T2 cells) and the like, each group is provided with 3 multiple holes, after 4 hours, 50 mul of co-cultured cell supernatant is taken out and added into 50 mul of LDH substrate mixed solution, the cell supernatant is made to catalyze LDH substrate reaction, finally, the 490nm wavelength and 680nm reference wavelength are read, and the killing activity of the target cells to kill T2 is calculated according to the control holes. The results are shown in FIG. 2 and Table 5 below.

TABLE 5T cell specific recognition and killing of target cells presenting the Experimental Polypeptides

As can be seen from the results in fig. 2 and table 5, at an effective target ratio of 1 or 1.

Example 6 construction and packaging of mutant Polypeptides FMVDGFWSI and recombinant lentiviruses of the Deformable Polypeptides thereof

Synthesizing a DNA sequence 'TTCATGGGTGGATCTTGGAGCATA' (SEQ ID NO: 8) corresponding to a wild-type polypeptide FMVDGSWSI, synthesizing a DNA sequence 'TTCATGGGTGGATGTCGGAGCATA' (SEQ ID NO: 9) corresponding to a mutant-type polypeptide FMVDGFWSI, synthesizing a DNA sequence 'TTCATGGGTGGATTCTGGAGGCCTG' (SEQ ID NO: 10) corresponding to a deformable-type polypeptide FMVDWSL, synthesizing a DNA sequence 'TTCATGGGTGGATTCTGGAGCGTC' (SEQ ID NO: 11) corresponding to a deformable-type polypeptide FMVDGFWSV, synthesizing a DNA sequence 'TTCCTGGGTGGAGATCTGGAGCTGCGTC' (SEQ ID NO: 11) corresponding to a deformable-type polypeptide FLVDGFWSI, synthesizing a DNA sequence 'TTCCTGGGAGTGGAGTTGGAGCCTG' (SEQ ID NO: 13) corresponding to a deformable-type polypeptide FLVDGFWSI, synthesizing a DNA sequence 'TTGGTGGAGTGGTTGGAGTCTGGGAGCTTGGAGCTTC' (SEQ ID NO: 12) corresponding to a deformable-type polypeptide FLVDGFGTGGAGTC) (SEQ ID NO: 14); and respectively constructing a lentiviral vector pHBLV-Puro of a wild type polypeptide FMVDGSWSI, a mutant type polypeptide FMVDGFWSI and a deformable polypeptide thereof, which are respectively named as pHBLV-Puro-FMVDGSWSI, pHBLV-Puro-FMVDGFWSI, pHBLV-Puro-FMVDGFWSL, pHBLV-Puro-FMVDGFWSV, pHBLV-Puro-FLVDGFWSI, pHBLV-Puro-FLVDWSL and pHBLV-Puro-FLVDGFWSV. The 7 lentivirus plasmids were co-transfected with pSPAX2 and pMD2G helper plasmids into 293T cells, respectively, and packaged into lentiviruses of the wild-type polypeptide FMVDGSWSI, the mutant polypeptide FMVDGFWSI, and their transformable polypeptides.

Example 7 establishment of human Lung cancer cell lines expressing mutant polypeptide FMVDGFWSI

The human non-small cell lung adenocarcinoma cell line NCI-H2087 was purchased from ATCC and was HLA-A0201 positive for its HLA subtype. Cells were cultured in DMEM medium containing 10% fetal bovine serum, 100U/mL penicillin and 100U/mL streptomycin at 37 ℃ with 5% CO ₂ Culturing in an incubator. The FMVDGFWSI lentivirus packaged in example 6 is transfected into an H2087 cell line, puromycin antibiotics are adopted to continuously screen a survival H2087 cell line, and finally, the H2087 cell line expressing FMVDGFWSI polypeptide is established and named as H2087-FMVDGFWSI cell line.

Example 8 NOD SCID mouse human immune reconstitution

Collecting 600-900ml of anticoagulated peripheral blood of healthy volunteers, separating Peripheral Blood Mononuclear Cells (PBMC) by Ficoll, and collecting the cells for later use. 300 NOD SCID mice with immunodiffusion excluded, 2X 10 PBMC per intraperitoneal injection ⁷ 0.5ml, NOD SCID mice were human reconstituted immunologically. Mice after 4 weeks were selected for inoculation with the human lung cancer cell line model.

Example 9 H2087 establishment of subcutaneous transplantation tumor model for FMVDGFWSI

The human non-small cell lung adenocarcinoma cell line H2087-FMVDGFWSI established in example 7 was cultured in DMEM medium containing 10% fetal bovine serum, 100U/mL penicillin and 100U/mL streptomycin and was subjected to 5% CO at 37% ₂ Culturing in an incubator. H2087-FMVDGFWSI tumor cells were collected, centrifuged at 1500rpm for 5min, and the tumor cells were washed 3 times with sterile saline. Diluting properly, adding 10 μ l of 0.4% phloroglucinol blue into 40 μ l of cell suspension, and counting by microscopic examination to obtain a solution with concentration of 1 × 10 ⁸ One/ml tumor cell suspension, NOD/SCID mice or NOD/SCID mice after immune reconstitution were selected, and each mouse was inoculated with 100. Mu.l of tumor cell suspension subcutaneously. After inoculation is finished, observing whether the inoculated part is infected or not day by day, whether the tumor naturally regresses or not after growing, measuring the long diameter a and the short diameter b of the tumor by using a vernier caliper every 2 to 3 days, calculating the size of the tumor = a b/2, and weighing and recording the weight and the tumor of the mouse every day; after 7 days, mice were palpable subcutaneously to approximately rice grain size tumors, at which time NOD/SCID mice, a H2087-FMVDGFWSI subcutaneous tumor model, were treated with the DC-CTL vaccine. H2087-FMVDGFWSI subcutaneous tumor model NOD/SCID mice with 4 weeks of immune reconstitution were treated with polypeptide + complete Freund's adjuvant, or polypeptide + DC vaccine, or lentivirus-infected DC cell vaccine, and DC-CTL vaccine, respectively, and the tumor volume and the survival rate of the mice were recorded every 2 days.

Example 10 polypeptide vaccine treatment regimens

H2087-FMVDGFWSI subcutaneous tumor model NOD/SCID mice immunized for 4 weeks of reconstitution were randomized into 8 groups: adjuvant + wild type polypeptide group, adjuvant + FMVDGFWSI polypeptide group andadjuvant + its 5 variable polypeptide groups, 6 each per group. The first immunization dose of the polypeptide is 100 mu g/mouse. The polypeptides were resuspended in PBS, mixed with 150. Mu.l/mouse Freund's complete adjuvant, adjusted to 300. Mu.l/mouse with PBS, and injected subcutaneously into the back at two sites. Booster immunizations (complete freund's adjuvant used 1 st time, incomplete freund's adjuvant used thereafter) were performed 4 times at the same dose after 2 weeks. The general characteristics of the tumor-bearing mice, including mental state, activity, reaction, diet, body weight, tumor growth and the like, were observed daily. Tumor longest (a) and shortest (b) diameters were measured every 2 days with a vernier caliper. Tumor volume was calculated as 1/2x length x width ² . The results are shown in FIG. 3. The results show that FMVDGFWSI or its modified form plus Freund's adjuvant can effectively inhibit tumor growth and prolong the survival of mice compared with the adjuvant-only group and the wild-type polypeptide group. The life cycle calculation formula is as follows: survival over time = 100% mice alive over time/(mice alive over time + mice dead over time).

Example 11 preparation of a DC polypeptide vaccine and treatment regimens using the same

Collecting 100-150ml of anticoagulated peripheral blood of healthy volunteers, separating peripheral blood mononuclear cells by Ficoll, collecting PBMC cells, and performing filtration according to the ratio of 2-3 multiplied by 10 ⁶ Resuspending in RPMI 1640 culture medium at 37 deg.C for 2h to obtain adherent cells as DC, and inducing adherent cells as mature DC cells by using 1000U/ml GM-CSF,1000U/ml IL-4, 100U/ml IFN-gamma, and 100U/ml CD 40L; after harvesting mature DCs, wild type polypeptide, mutant polypeptide FMVDGFWSI and 5 transformable polypeptides (10. Mu.g/ml) were added, incubated for 4h, and washed 3 times with physiological saline. The polypeptide-loaded DCs were adjusted to (4.0. + -. 0.5). Times.10 with physiological saline ⁷ Pieces/ml for subsequent experiments.

Tumor-bearing mice were randomly divided into 7 groups: DC-load wild type polypeptide group, DC-load mutant type polypeptide FMVDGFWSI and DC-load 5 kinds of deformable polypeptides, each group has 6 polypeptides. Cell suspensions were prepared that DC-loaded with wild-type polypeptide, DC-loaded with FMVDGFWSI polypeptide and DC-loaded with its 5 transformable polypeptides. Intradermal injection of tumor-bearing mice near the inner thigh of groin0.1ml was injected on each side, 1 time per week. The dosage is (4.0 +/-0.5) multiplied by 10 ⁶ Each cell/time, 2 total injections. After the injection, the vital signs of the mice were observed, and the size of the tumor was measured every 2 days with a vernier caliper. Tumor volume is calculated as tumor volume =1/2x length x width ² . Meanwhile, the weight change and survival of the mice were recorded. The results are shown in fig. 4, and the results in fig. 4 show that FMVDGFWSI or its deformable polypeptide loaded DC vaccine can significantly prolong the survival of mice and slow down the growth of mouse tumors relative to the wild type polypeptide loaded DC vaccine group.

Example 12 preparation of a vaccine for DC infection with recombinant lentivirus of polypeptide genes and treatment protocol Using the vaccine

Collecting 100-150ml of anticoagulated peripheral blood of healthy volunteers, separating peripheral blood mononuclear cells by Ficoll, collecting PBMC cells, incubating for 2h at 37 ℃, washing off non-adherent cells, and culturing DC cells by recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) and recombinant human leukocyte-4 (rhIL-4). Culturing to the fifth day, half the amount of medium was changed and cell density was adjusted to 1 × 10 ⁶ Per ml; recombinant lentiviral fluids (constructed as described in example 6) containing appropriate amounts of wild-type polypeptide, mutant polypeptide FMVDGFWSI or 5 of its transformable polypeptides were added. After 24h, the virus culture medium was removed, the medium containing 50ng/ml rhIL-4, 100ng/ml rh GM-CSF,100U/ml IFN-. Gamma.and 100U/ml CD40L was added, incubated at 37 ℃ and 5% CO ₂ The cells were cultured in an incubator for 16h and DC cells adjusted to (4.0. + -. 0.5) × 10 ⁷ Pieces/ml for subsequent experiments.

Tumor-bearing mice were randomly divided into 7 groups: the wild type polypeptide-DC group, FMVDGFWSI polypeptide-DC group and 5 deformable polypeptide-DC groups thereof, each group comprising 6 polypeptides. Cell suspensions were prepared that were DC-loaded with wild-type polypeptide, DC-loaded with FMVDGFWSI polypeptide and DC-loaded with its 5 transformable polypeptides. The immuno-reconstituted tumor-bearing mice were injected intradermally, 0.1ml per side, 1 time per week, near the inner thigh of the groin. The dosage is (4.0 +/-0.5) multiplied by 10 ⁶ One cell/time, 2 total injections. After the injection, the vital signs of the mice were observed, and the size of the tumor was measured every 2 days with a vernier caliper. Tumor bodyThe product is calculated as tumor volume =1/2 × length × width ² . Meanwhile, the weight change and survival of the mice were recorded. The results are shown in fig. 5, and fig. 5 shows that the DC vaccine infected by the recombinant lentivirus of mutant polypeptide FMVDGFWSI or its 5 kinds of deformable polypeptide genes has a significant tumor-inhibiting effect and prolongs the survival of mice, but the wild-type polypeptide is not responsive to the tumor, compared with the wild-type polypeptide group.

EXAMPLE 13 preparation of polypeptide-specific CTL vaccines and in vivo treatment regimens using the same

Collecting 100-150ml of anticoagulated peripheral blood of healthy volunteers, separating peripheral blood mononuclear cells by Ficoll, collecting PBMC cells, and performing filtration according to the ratio of 2-3 multiplied by 10 ⁶ The cells/ml were resuspended in RPMI 1640 medium, incubated at 37 ℃ for 2h, and the nonadherent cells were aspirated as Peripheral Blood Lymphocytes (PBL).

And (3) sorting the collected PBL by magnetic beads to obtain CD8+ T, co-breeding and sensitizing the CD8+ T and DC loaded with wild type polypeptide, FMVDGFWSI loaded with mutant type polypeptide and five transformable polypeptides thereof, wherein the cell ratio is DC: CD8+ T = 1. Adding IL-2 at 500IU/ml and IL-7 at 50ng/ml to the culture medium, 5% CO at 37 ℃ ₂ Incubating together in an incubator, and counting cells after 1 week of culture; additional DCs loaded with FMVDGFWSI polypeptide and 5 of its transformable polypeptides, DCs loaded with wild-type polypeptide and 500IU/ml IL-2 were added for a second stimulation cycle at week 2. The same treatment was performed at week 3. Thus, three rounds of co-stimulation were performed, with appropriate addition of medium during the culture. The number of lymphocytes was counted on days 0, 7, 14 and 21 of culture, respectively, and the cell Proliferation Index (PI) was calculated. PI = number of cells after expansion/number of seeded cells. Cells, i.e., cytotoxic T Lymphocytes (CTL), were harvested 7 days after 3 rd stimulation (i.e., day 21 of culture). Resuspending the cells in physiological saline, the volume is 0.2ml, and the cells are returned through tail vein, and the number of the returned cells of each tumor model mouse is about 1x10 ⁸ And (4) one cell. After the injection, the mice were observed with care for vital signs, and the length and width of the tumor were measured with a vernier caliper every 2 days.

The results are shown in FIG. 6; the results in fig. 6 show that the mutant polypeptide FMVDGFWSI or the DC-CTL vaccine activated by 5 of its transformable polypeptides has significant tumor-suppression effect and prolonged survival of mice compared to the wild-type polypeptide group.

The applicant states that the product of the present invention and the application and use effects thereof are illustrated by the above embodiments, but the present invention is not limited thereto, and those skilled in the art should understand that any modification to the present invention, equivalent replacement of the product of the present invention, addition of auxiliary components, selection of specific modes, etc. fall within the protection scope and disclosure scope of the present invention.

SEQUENCE LISTING

<110> Shenzhen Hua Dagenea institute

<120> COL14A1 derived tumor antigen polypeptide and application thereof

<130> PCT160063PPC

<160> 14

<170> PatentIn version 3.3

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Claims (21)

1. An isolated polypeptide or immunologically active fragment thereof, wherein the polypeptide has an amino acid sequence as shown in SEQ ID NO. 2, 3, 4, 5, 6 or 7.

2. An isolated nucleic acid encoding the polypeptide of claim 1.

3. A nucleic acid construct comprising the nucleic acid of claim 2 operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.

4. An expression vector comprising the nucleic acid construct of claim 3.

5. A host cell into which the nucleic acid construct of claim 3 or the expression vector of claim 4 has been transformed or transfected.

6. An antigen presenting cell which presents the polypeptide of claim 1 on the cell surface.

7. A method of producing the antigen presenting cell of claim 6, comprising: a step of contacting the polypeptide of claim 1 with a cell having antigen presenting ability, or comprising: a step of introducing the nucleic acid according to claim 2, or the nucleic acid construct according to claim 3, or the expression vector according to claim 4 into a cell having antigen-presenting ability for expression.

8. The method according to claim 7, wherein the antigen-presenting cell is a dendritic cell.

9. A conjugate comprising the polypeptide of claim 1 and an anti-cancer agent.

10. A vaccine for treating or preventing cancer in a patient comprising the polypeptide of claim 1, or comprising the nucleic acid of claim 2, or comprising the nucleic acid construct of claim 3, or comprising the expression vector of claim 4, or comprising the antigen presenting cell of claim 6;

the cancer is a cancer expressing the polypeptide of claim 1.

11. The vaccine of claim 10, wherein the cancer is selected from the group consisting of lung cancer, melanoma, breast cancer, nasopharyngeal cancer, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, skin cancer, prostate cancer, cervical cancer, leukemia, and brain tumor.

12. A pharmaceutical composition for treating or preventing cancer in a patient comprising the polypeptide of claim 1, and a pharmaceutically acceptable carrier.

13. Use of the polypeptide of claim 1 in the preparation of an antibody for the prevention or treatment of a tumor;

the tumor expresses HLA-A0201 and the polypeptide of claim 1 simultaneously.

14. The use according to claim 13, wherein the tumor is lung cancer, melanoma, breast cancer, nasopharyngeal cancer, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, skin cancer, prostate cancer, cervical cancer, leukemia or brain tumor.

15. Use of the polypeptide of claim 1 in the manufacture of a medicament for the prevention or treatment of a tumor;

the tumor expresses HLA-A0201 and the polypeptide of claim 1 simultaneously.

16. The use according to claim 15, wherein the tumor is lung cancer, melanoma, breast cancer, nasopharyngeal cancer, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, skin cancer, prostate cancer, cervical cancer, leukemia or brain tumor.

17. Use of a polypeptide according to claim 1 for the preparation of a vaccine for the prevention or treatment of a tumour;

the tumor expresses HLA-A0201 and the polypeptide of claim 1 simultaneously.

18. The use according to claim 17, wherein the tumor is lung cancer, melanoma, breast cancer, nasopharyngeal cancer, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, skin cancer, prostate cancer, cervical cancer, leukemia or brain tumor.

19. Use of a polypeptide according to any one of claim 1 or a nucleic acid according to claim 2 in the preparation of a kit for diagnosing cancer in a patient;

the cancer is a cancer expressing the polypeptide of claim 1.

20. The use according to claim 19, wherein the cancer is selected from the group consisting of lung cancer, melanoma, breast cancer, nasopharyngeal carcinoma, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, skin cancer, prostate cancer, cervical cancer, leukemia and brain tumors.

21. A kit for diagnosing cancer in a patient comprising the polypeptide of claim 1 or the nucleic acid of claim 2.