Epitope peptides of PDIA3 and complexes of the epitope peptides and heat shock proteins

Abstract

The present invention relates to the fields of immunology and cancer treatment. Specifically, the present invention relates to epitope peptides of the protein PDIA3 and combinations thereof, specific immune cells induced thereby, cancer vaccines comprising them, and their use for preventing or treating cancer. The present invention also relates to complexes formed by the epitope peptides and heat shock proteins, specific immune cells induced thereby, cancer vaccines comprising them, and their use for preventing or treating cancer.

Description

Technical Field

[0001] The present invention relates to the fields of immunology and cancer treatment. Specifically, the present invention relates to epitope peptides of the protein PDIA3 and combinations thereof, specific immune cells induced thereby, tumor vaccines comprising them, and their use for preventing or treating cancer. The present invention also relates to complexes formed by the epitope peptides and heat shock proteins, specific immune cells induced thereby, tumor vaccines comprising them, and their use for preventing or treating cancer. Background Art

[0002] Breast cancer is one of the most common malignant tumors in women. According to statistics, the incidence of breast cancer accounts for 7-10% of all kinds of malignant tumors in the body. In many regions, it has exceeded uterine cancer and become one of the most common tumors seriously affecting women's physical and mental health and even endangering their lives. The incidence of breast cancer is closely related to the patient's genetic genes, living habits, common foods, reproductive status, etc. There are obvious differences in the incidence of breast cancer among different ethnic groups and regions. The high-incidence areas of breast cancer are mainly concentrated in North America, Northern Europe, and Oceania, especially among white women. The medium-incidence areas of breast cancer are concentrated in South America, Southern Europe, and Israel. Asia is a low-incidence area of breast cancer. For example, the lifetime incidence of breast cancer in white American women is 13.1%, that is, on average, 1 in 8-9 people may develop breast cancer, while the incidence of breast cancer in Asian women is 4-7%. According to WHO statistics, there are about 1.3 million new breast cancer cases and about 500,000 deaths globally every year. In big cities such as Beijing, Shanghai, and Tianjin in China, the incidence of breast cancer has ranked first among various cancers in women and shows an obvious upward trend.

[0003] Liver cancer refers to malignant tumors that occur in the liver, including primary liver cancer and metastatic liver cancer. Primary liver cancer is one of the most common malignant tumors clinically. Worldwide, among male cancer patients, liver cancer ranks sixth in terms of proportion and second in terms of mortality; among female cancer patients, liver cancer ranks seventh in terms of proportion and sixth in terms of mortality. In 2008, there were 748,300 new liver cancer cases and 695,900 liver cancer patients died worldwide. And half of these new liver cancer cases and deaths were in China. The regions with the highest incidence of liver cancer are mainly in East Asia, Southeast Asia, Central Africa, and West African countries. The reason for the relatively high incidence of liver cancer in some parts of Asia and sub-Saharan Africa may be the prevalence of HBV in these regions, because 8% of the residents in these regions are chronically infected with HBV, and 60% of liver cancer patients in developing countries have been infected with HBV.

[0004] Protein disulfide isomerase family A, member 3 (PDIA3) is a protein present in the endoplasmic reticulum. It interacts with the lectin chaperones calreticulin and calnexin to regulate the folding of newly synthesized glycoproteins. This protein was once thought to be a phospholipase; however, it has now been shown that this protein actually has protein disulfide isomerase activity. The complex of lectin and this protein mediates protein folding by promoting the formation of disulfide bonds in its glycoprotein substrates. A series of studies have shown that this protein is highly expressed in various cancers, including liver cancer, breast cancer, and skin cancer. Studies have confirmed that PDIA3 plays a key role in the tumorigenesis and development of liver cancer patients through the STAT3 signaling pathway, promoting tumor metastasis. At the same time, the expression level of PDIA3 is also directly related to the survival rate of cancer patients and is a potential target for good tumor treatment intervention.

[0005] Heat shock protein (HSP) is a class of proteins that are highly conserved in biological evolution and widely present in prokaryotes and eukaryotes. HSP can be divided into multiple subfamilies according to the degree of homology and molecular weight, such as HSP110, HSP90, HSP70, HSP60, HSP40, small HSP, and ubiquitin. Heat shock protein gp96 belongs to the members of the HSP90 subfamily, and this protein is the most abundant heat shock protein on the endoplasmic reticulum of cells. Heat shock protein gp96 has the property of polypeptide binding. Inside the endoplasmic reticulum, it can receive polypeptide fragments from the TAP complex and assist in assembling them onto MHC class I molecules, presenting them on the cell membrane. Heat shock proteins gp96 from different tissue sources can carry polypeptide fragments specifically expressed in their source tissues. Summary of the Invention

[0006] The present invention provides epitope peptides of protein PDIA3 and combinations thereof, and specific immune cells induced thereby. The present invention further provides a complex formed by the epitope peptide and heat shock protein, and specific immune cells induced thereby.

[0007] Therefore, in one aspect, the present invention provides an isolated polypeptide having an amino acid sequence as shown in SEQ ID NO: 2 or 3.

[0008] In another aspect, the present invention provides a polypeptide composition comprising any two or three polypeptides selected from the polypeptides shown in any one of SEQ ID NO: 1, 2, and 3.

[0009] In certain embodiments, the polypeptides contained in the polypeptide composition have the same or different ratios.

[0010] In certain embodiments, the polypeptide composition comprises the polypeptide shown in SEQ ID NO: 1, the polypeptide shown in SEQ ID NO: 2, and the polypeptide shown in SEQ ID NO: 3.

[0011] In certain embodiments, the mass ratio of the polypeptide shown in SEQ ID NO: 1, the polypeptide shown in SEQ ID NO: 2, and the polypeptide shown in SEQ ID NO: 3 in the polypeptide composition is from 1:1:1 to 20:1:1, or from 1:1:1 to 1:20:1, or from 1:1:1 to 1:1:20.

[0012] In certain embodiments, the mass ratio of the polypeptide shown in SEQ ID NO: 1, the polypeptide shown in SEQ ID NO: 2, and the polypeptide shown in SEQ ID NO: 3 in the polypeptide composition is from 1:1:1 to 5:1:1, or from 1:1:1 to 1:5:1, or from 1:1:1 to 1:1:5.

[0013] In certain embodiments, the mass ratio of the polypeptide shown in SEQ ID NO: 1, the polypeptide shown in SEQ ID NO: 2, and the polypeptide shown in SEQ ID NO: 3 in the polypeptide composition is 1:1:1, or 5:1:1, or 1:5:1, or 1:1:5.

[0014] In certain embodiments, the ratio of the polypeptide shown in SEQ ID NO: 1, the polypeptide shown in SEQ ID NO: 2, and the polypeptide shown in SEQ ID NO: 3 contained in the polypeptide composition is 1:1:1.

[0015] In another aspect, the present invention provides a complex formed by the isolated polypeptide as described above or the polypeptide composition as described above and a heat shock protein.

[0016] In certain embodiments, the complex is formed by the isolated polypeptide as described above or the polypeptide composition as described above and a heat shock protein by natural adsorption or heat shock.

[0017] In certain embodiments, the complex is prepared by mixing the isolated polypeptide as described above or the polypeptide composition as described above and a heat shock protein in vitro. In certain embodiments, the mixing is carried out at 40 - 80 °C (such as 40 - 70 °C, 40 - 60 °C, or 50 - 60 °C). In certain embodiments, the mixing is carried out at 55 °C.

[0018] In certain embodiments, the complex is prepared by contacting the isolated polypeptide as described above or the polypeptide composition as described above with a heat shock protein and incubating at 55 °C for 10 min, then at room temperature for 30 min.

[0019] In certain embodiments, the heat shock protein is selected from HSP70, HSP90, gp96, HSP110, and mutants or fusion proteins thereof.

[0020] In certain embodiments, the heat shock protein is gp96. In certain embodiments, the heat shock protein has the amino acid sequence as shown in SEQ ID NO: 6.

[0021] In certain embodiments, the complex is prepared by mixing the isolated polypeptide as described above or the polypeptide composition with the heat shock protein in vitro at the following mass ratios: 0.1:1 to 100:1 (such as 0.1:1 to 50:1, 0.1:1 to 10:1, 0.1:1 to 5:1, 0.1:1 to 1:1, 0.5:1 to 100:1, 0.5:1 to 50:1, 0.5:1 to 10:1, 0.5:1 to 5:1, 0.5:1 to 1:1, 1:1 to 100:1, 1:1 to 50:1, 1:1 to 10:1, 1:1 to 5:1), preferably 1:1, 10:1 or 100:1.

[0022] In certain embodiments, the complex is immunogenic and capable of inducing a specific immune response against cells expressing PDIA3 (such as tumor cells, such as breast cancer or liver cancer cells).

[0023] In another aspect, the present invention provides a composition comprising at least two (such as 2, 3, 4, 5, or more) complexes as described above.

[0024] In certain embodiments, the heat shock proteins in the composition are the same or different.

[0025] In certain embodiments, the composition is immunogenic and capable of inducing a specific immune response against cells expressing PDIA3 (such as tumor cells, such as breast cancer or liver cancer cells).

[0026] In another aspect, the present invention provides a method for preparing a complex as described above, which comprises mixing the isolated polypeptide as described above or the polypeptide composition with a heat shock protein.

[0027] In certain embodiments, the isolated polypeptide as described above or the polypeptide composition forms a complex with the heat shock protein by natural adsorption or heat shock.

[0028] In certain embodiments, the mixing is carried out at 40 - 80 °C (such as 40 - 70 °C, 40 - 60 °C, or 50 - 60 °C). In certain embodiments, the mixing is carried out at 55 °C.

[0029] In certain embodiments, the method includes: contacting the polypeptide separated as described above or the polypeptide composition as described above with a heat shock protein and placing it at 55 °C for 10 min, and then at room temperature for 30 min.

[0030] In certain embodiments, the heat shock protein is selected from HSP70, HSP90, gp96, HSP110, and their mutants or fusion proteins.

[0031] In certain embodiments, the heat shock protein is gp96. In certain embodiments, the heat shock protein has the amino acid sequence shown in SEQ ID NO: 6.

[0032] In certain embodiments, the method includes: mixing the separated polypeptide or polypeptide composition as described above with a heat shock protein in the following mass ratio: 0.1:1 to 100:1 (such as 0.1:1 to 50:1, 0.1:1 to 10:1, 0.1:1 to 5:1, 0.1:1 to 1:1, 0.5:1 to 100:1, 0.5:1 to 50:1, 0.5:1 to 10:1, 0.5:1 to 5:1, 0.5:1 to 1:1, 1:1 to 100:1, 1:1 to 50:1, 1:1 to 10:1, 1:1 to 5:1), preferably 1:1, 10:1 or 100:1.

[0033] In certain embodiments, the complex has immunogenicity and can induce a specific immune response against cells expressing PDIA3 (such as tumor cells, such as breast cancer or liver cancer cells).

[0034] In another aspect, the present invention provides a T cell that expresses a T cell receptor capable of specifically recognizing protein disulfide isomerase family member 3 (PDIA3) or the separated polypeptide or polypeptide composition as described above.

[0035] In certain embodiments, the T cell is obtained by stimulating immune cells with the complex or composition as described above. In certain embodiments, the immune cells include PBMC or T lymphocytes.

[0036] In certain embodiments, the stimulation further includes using an immune - stimulating cytokine (such as IL - 2).

[0037] In certain embodiments, the T cell is a cytotoxic T lymphocyte.

[0038] In certain embodiments, the T cells are capable of specifically recognizing and killing cells that express protein disulfide isomerase family member 3 (e.g., tumor cells, such as breast cancer or liver cancer cells).

[0039] In another aspect, the present invention provides a method for preparing the T cells as described above, which comprises stimulating immune cells with the complex as described above or the composition as described above. In certain embodiments, the immune cells comprise PBMC or T lymphocytes.

[0040] In certain embodiments, the stimulation further comprises using an immunostimulatory cytokine (e.g., IL-2). In certain embodiments, the immunostimulatory cytokine is IL-2.

[0041] In another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the isolated polypeptide as described above. In certain embodiments, the isolated polypeptide has the amino acid sequence shown in SEQ ID NO: 2 or 3.

[0042] In another aspect, the present invention provides a combination of isolated nucleic acid molecules comprising nucleotide sequences encoding the polypeptide composition as described above; the polypeptide composition comprises any two or three polypeptides selected from the polypeptides shown in any one of SEQ ID NO: 1, 2, and 3. In certain embodiments, the polypeptide composition comprises the polypeptide shown in SEQ ID NO: 1, the polypeptide shown in SEQ ID NO: 2, and the polypeptide shown in SEQ ID NO: 3.

[0043] In another aspect, the present invention provides a vector comprising the isolated nucleic acid molecule as described above or the combination of the isolated nucleic acid molecules as described above.

[0044] In certain embodiments, the vector comprises a nucleotide sequence encoding the isolated polypeptide as described above or the polypeptide composition as described above.

[0045] In certain embodiments, the vector is a viral vector, such as a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a baculoviral vector.

[0046] In another aspect, the present invention provides a host cell comprising the isolated nucleic acid molecule as described above or the combination of the isolated nucleic acid molecules as described above or the vector as described above.

[0047] In another aspect, the present invention provides a pharmaceutical composition comprising the isolated polypeptide as described above, the polypeptide composition as described above, the complex as described above, the composition as described above, the T cell as described above, the isolated nucleic acid molecule as described above, the combination of isolated nucleic acid molecules as described above, the vector as described above, or the host cell as described above, and a pharmaceutically acceptable carrier and / or excipient.

[0048] In certain embodiments, the pharmaceutical composition is a tumor vaccine.

[0049] In certain embodiments, the pharmaceutical composition comprises an adjuvant.

[0050] In certain embodiments, the pharmaceutical composition further comprises an additional therapeutic agent, such as an anti-tumor agent or an immune enhancer.

[0051] In certain embodiments, the anti-tumor agent is selected from alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, immune checkpoint inhibitors (e.g., PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, LAG-3 antibody or TIM3 antibody).

[0052] In certain embodiments, the immune enhancer is selected from immune-stimulating antibodies (e.g., anti-CD3 antibody, anti-CD28 antibody, anti-CD40L (CD154) antibody, anti-41BB (CD137) antibody, anti-OX40 antibody, anti-GITR antibody or any combination thereof) or immune-stimulating cytokines (e.g., IL-2).

[0053] In another aspect, the present invention provides the use of the isolated polypeptide as described above, or the polypeptide composition as described above, or the complex as described above, or the composition as described above, or the T cell as described above in the preparation of a medicament for inducing an immune response against a tumor expressing PDIA3 in a subject and / or for preventing or treating a tumor expressing PDIA3 in a subject.

[0054] In certain embodiments, the tumors expressing PDIA3 are selected from breast cancer, liver cancer, glioma, colorectal cancer, pancreatic cancer, gastric cancer, lung cancer, endometrial cancer, ovarian cancer, multiple myeloma, melanoma, thyroid cancer, bladder cancer, prostate cancer, head and neck cancer or acute myeloid leukemia.

[0055] In certain embodiments, the T cells are from the subject.

[0056] In certain embodiments, the subject is a human.

[0057] In certain embodiments, the subject is HLA-A2 positive.

[0058] In certain embodiments, the isolated polypeptide, polypeptide composition, composition or T cell is administered in combination with another therapeutic agent, such as simultaneously, separately or sequentially. In certain embodiments, the other therapeutic agent is an immune stimulant or an anti-tumor agent.

[0059] In certain embodiments, the expression of PDIA3 protein in the tumors expressing PDIA3 is abnormal (e.g., increased expression compared to healthy subjects).

[0060] In another aspect, the present invention provides a method for inducing an immune response against tumors expressing PDIA3 in a subject and / or for preventing or treating tumors expressing PDIA3 in a subject, the method comprising administering to a subject in need thereof an effective amount of the isolated polypeptide as described above, the polypeptide composition as described above, the complex as described above, the composition as described above, the T cell as described above, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the isolated polypeptide or polypeptide composition, or the pharmaceutical composition as described above.

[0061] In certain embodiments, the tumors expressing PDIA3 are selected from breast cancer, liver cancer, glioma, colorectal cancer, pancreatic cancer, gastric cancer, lung cancer, endometrial cancer, ovarian cancer, multiple myeloma, melanoma, thyroid cancer, bladder cancer, prostate cancer, head and neck cancer or acute myeloid leukemia.

[0062] In certain embodiments, the subject is human.

[0063] In certain embodiments, the subject is HLA-A2 positive.

[0064] In certain embodiments, the method further comprises administering to the subject another therapeutic agent, such as an immune enhancer or an anti-tumor agent.

[0065] In certain embodiments, the expression of PDIA3 protein in the tumors expressing PDIA3 is abnormal (e.g., increased expression compared to healthy subjects).

[0066] In certain embodiments, the method comprises: (1) providing immune cells (e.g., PBMC or T cells) from a subject; (2) contacting the complex or composition with the immune cells to obtain the T cell as described above; (3) administering the T cell obtained in step (2) to the subject.

[0067] Term Definitions

[0068] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the virological, biochemical, and immunological laboratory procedures used herein are all conventional procedures widely used in the corresponding fields. Meanwhile, to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0069] As used herein, the term "PDIA3" refers to Protein disulfide isomerase family A, member 3, which interacts with the lectin chaperones calreticulin and calnexin to regulate the folding of newly synthesized glycoproteins. Studies have confirmed that it is highly expressed in various cancers including liver cancer, breast cancer, skin cancer, etc., and plays a key role in the tumorigenesis and development of liver cancer patients through the STAT3 signaling pathway, promoting tumor metastasis. PDIA3 is well-known to those skilled in the art, and its sequence can be found in various public databases, such as the NCBI GENBANK database accession number: AAC51518.1.

[0070] As used herein, the term "gp96", also known as Grp94, is a member of the heat shock protein 90 family located on the endoplasmic reticulum membrane of cells. The gp96 protein consists of an N-terminal domain (N-terminal ATP-binding domain), an M domain (charged middle domain), and a C-terminal domain (C-terminal homodimer domain). gp96 is well-known to those skilled in the art, and its sequence can be found in various public databases, such as the NCBI GENBANK database accession number: AAH66656.1.

[0071] As used herein, the term "HSP70" is a heat shock protein with a molecular weight of approximately 70 kD, which is an important member of the heat shock protein family and is called the major heat shock protein, including more than 20 proteins with molecular weights of 68, 72, 73, 75, 78 kDa, etc. The HSP70 family proteins have similar molecular weights, isoelectric points between pH 5.2 - 6.3, and similar tryptic peptide maps, and are often highly induced in stressed cells of almost all organisms, having the function of protecting the body and cells.

[0072] As used herein, the term "HSP90" is a heat shock protein with a molecular weight of approximately 90 kD, which is a protein used for co-immunization. It is an important member of the heat shock protein family, and its molecular weight is approximately 83 - 90 kDa.

[0073] As used herein, the term "HSP110" is a heat shock protein with a molecular weight of approximately 110 kD, which is an important member of the heat shock protein family, and its molecular weight is approximately 100 - 110 kDa.

[0074] As used herein, the term "isolated" or "separated" refers to being obtained by artificial means from its natural state. If a "separated" substance or component occurs in nature, it may be that the natural environment in which it is located has changed, or the substance has been separated from its natural environment, or both situations have occurred. For example, a certain unseparated polynucleotide or polypeptide naturally exists in a living animal body, and the highly purified same polynucleotide or polypeptide separated from this natural state is called isolated. The term "isolated" or "separated" does not exclude the admixture of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0075] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector enables the expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction, or transfection, so that the genetic element carried by it can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); phages such as λ phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector can also contain an origin of replication.

[0076] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0077] As used herein, the term "subject" includes, but is not limited to, various animals, particularly mammals, such as humans. In certain embodiments, the subject (e.g., a human) has a PDIA3-positive tumor.

[0078] As used herein, the term "immune cell" refers to any cell of the immune system that has one or more effector functions. Immune cells typically include cells that play a role in an immune response and generally have a hematopoietic origin. The term "effector function" refers to a specialized function of an immune cell, such as a function or response that enhances or promotes an immune attack on a target cell (e.g., killing of the target cell, or inhibition of its growth or proliferation). For example, the effector function of a T cell, for instance, can be cytolytic activity or an activity that aids or includes the secretion of cytokines. Examples of immune cells include T cells (e.g., α / β T cells and γ / δ T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived macrophages, among others.

[0079] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes but is not limited to: pH regulators, surfactants, ion strength enhancers, osmotic pressure-maintaining agents, absorption-delaying agents, diluents, adjuvants, preservatives, stabilizers, etc. For example, pH regulators include but are not limited to phosphate buffer. Surfactants include but are not limited to cationic, anionic, or nonionic surfactants, such as Tween-80. Ion strength enhancers include but are not limited to sodium chloride. Osmotic pressure-maintaining agents include but are not limited to sugars, NaCl, and their analogs. Absorption-delaying agents include but are not limited to monostearate and gelatin. Diluents include but are not limited to water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol), etc. Adjuvants include but are not limited to aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant), etc. Preservatives include but are not limited to various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art and can stabilize the desired activity of the active ingredient in the drug (e.g., the inhibitory activity against PSD-95 ubiquitination), including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein), or their degradation products (such as lactalbumin hydrolysate), etc.

[0080] As used herein, the term "treatment" refers to treating or curing a disease (such as a tumor), delaying the onset of one or more symptoms of the disease, and / or delaying the progression of the disease.

[0081] As used herein, the term "effective amount" refers to an amount that can effectively achieve the intended purpose. For example, a therapeutically effective amount can be an amount that is effective or sufficient to treat or cure a disease (such as a tumor), delay the onset of one or more symptoms of the disease, and / or delay the progression of the disease. Such an effective amount can be readily determined by those skilled in the art or a doctor, and can be related to the intended purpose, the general health status of the subject, age, gender, weight, the severity of the disease to be treated, complications, the mode of administration, etc. The determination of such an effective amount is entirely within the ability of those skilled in the art.

[0082] Beneficial effects

[0083] The complex of three polypeptides of the protein PDIA3 of the present invention or a combination thereof with a heat shock protein is immunogenic and can induce a specific immune response against cells expressing PDIA3 (such as tumor cells, such as breast cancer or liver cancer cells). Further, the complex can induce the generation of specific immune cells, and the specific immune cells can kill tumor cells expressing PDIA3, thereby being able to prevent or treat tumors expressing PDIA3. Moreover, the three polypeptides of the protein PDIA3 and combinations thereof can be used to prepare a tumor vaccine, and the vaccine can be used to prevent or treat tumors expressing PDIA3.

[0084] The embodiments of the present invention will be described in detail below in conjunction with the drawings and examples. However, those skilled in the art will understand that the following drawings and examples are only used to illustrate the present invention, rather than limiting the scope of the present invention. According to the following detailed description of the drawings and preferred embodiments, various objects and advantageous aspects of the present invention will become apparent to those skilled in the art. Brief description of the drawings

[0085] Figure 1 It is the identification results of SDS-PAGE and Western Blot of gp96 expressed by insects in Example 1 of the present invention.

[0086] Figure 2 It is the killing results of specific CTLs activated by three polypeptides and mixed polypeptides with different ratios complexed with gp96 on human breast cancer cells in Example 2 of the present invention.

[0087] Figure 3The killing results of specific CTLs activated by mixing three polypeptides in equal proportion and complexing with gp96 and the control group (human T cells stimulated by gp96) against MCF-7 cells and T2 cells co-incubated with the three polypeptides in Example 2 of the present invention.

[0088] Figure 4 The effect on the breast cancer tumor volume of specific T cells induced by reinfusing the polypeptide-gp96 complex and the control group (human T cells stimulated by gp96) in Example 3 of the present invention.

[0089] Figure 5 The killing results of specific CTLs activated by complexing three polypeptides and mixed polypeptides in different proportions with gp96 against human liver cancer cells in Example 4 of the present invention.

[0090] Figure 6 The killing results of specific CTLs activated by mixing three polypeptides in equal proportion and complexing with gp96 and the control group (human T cells stimulated by gp96) against human liver cancer cells and T2 cells co-incubated with the three polypeptides in Example 4 of the present invention.

[0091] Figure 7 The effect on the liver cancer tumor volume of specific T cells induced by reinfusing the polypeptide-gp96 complex and the control group (human T cells stimulated by gp96) in Example 5 of the present invention.

[0092] Sequence information

[0093] Table 1: The information description of the sequences involved in this application is shown in the following table.

[0094]

[0095] Detailed implementation manners

[0096] The present invention will now be described with reference to the following examples which are intended to illustrate (but not limit) the present invention.

[0097] Those skilled in the art will appreciate that the examples describe the present invention by way of illustration and are not intended to limit the scope claimed in this application. The experimental methods in the examples are all conventional methods unless otherwise specified. For those conditions not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained commercially. For the quantitative experiments in the examples, three repeated experiments are set, and the results are averaged.

[0098] The sources of the experimental materials involved in the following examples are as follows:

[0099] The nude mice were products of Beijing Vital River Laboratory Animal Technology Co., Ltd., and were referred to as mice for short in the examples. The polypeptides were synthesized by Shanghai Gil Biochemical Co., Ltd.

[0100] HepG2 cells (human liver cancer cells) were purchased from ATCC (American Type Culture Collection), and the product catalog number was HB-8065 TM 。

[0101] MCF-7 cells (human breast cancer cells) were purchased from ATCC, and the product catalog number was HTB-22 TM 。

[0102] Sf9 cells were products of Invitrogen, and the product catalog number was 11496-015.

[0103] Cellfectin II reagent was a product of Life technologies, and the product catalog number was 10362-100.

[0104] Plasmid pFastBac TM 1 was a product of Invitrogen, and the product catalog number was 10359-016.

[0105] The gp96 monoclonal antibody was a product of Santa Cruz, and the product catalog number was sc-56399.

[0106] The monoclonal antibody of horseradish peroxidase-labeled goat anti-rat was a product of Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., and the product catalog number was ZB-2307.

[0107] The HiTrap-Q Sepharose ion exchange chromatography column was a product of GE, and the product catalog number was 17-5053-01.

[0108] The Superdex 200 10 / 300GL molecular sieve chromatography column was a product of GE, and the product catalog number was 17517501.

[0109] Escherichia coli DH10Bac competent cells were products of Beijing Yuanpinghao Biotechnology Co., Ltd., and the product catalog number was CL108-01.

[0110] Insect-XPRESSTM Protein-free Insect Cells medium with L-Glutamine was a product of LONZA, and the product catalog number was 12-730Q.

[0111] BSA, PMSF, NaHCO 3 、MnCl 2 、CaCl2 , NaCl 2 , Tris, methyl α-D-mannopyranoside are all products of Sigma-Aldrich, and the product catalog numbers are V900933, P7626, 792519, V900197, 793639, 746398, T1378, M6882 respectively.

[0112] Solution A: The solute is 1 mM PMSF and 30 mM NaHCO 3 ; The solvent is distilled water; The pH value is 7.4.

[0113] Solution B: The solute is 2 mM MnCl 2 , 2 mM CaCl 2 , 500 mM NaCl and 1 mM PMSF; The solvent is 20 mM Tris-HCl buffer solution with pH 7.4.

[0114] Solution C: The solute is 10% (mass / volume ratio) methyl α-D-mannopyranoside, 500 mM NaCl and 1 mM PMSF; The solvent is 20 mM Tris-HCl buffer solution with pH 7.4.

[0115] Washing solution: Dilute Solution B with distilled water to 10 times the volume.

[0116] ConA agarose gel column is a product of GE, and the product catalog number is 17-0440-01. The specification of this column is 1.6×2.5 cm, and the packing medium is Con A-Sepharose 4B.

[0117] Hitrap Q anion exchange column is a product of GE, and the product catalog number is 17-1153-01. The specification of this column is 0.7×2.5 cm.

[0118] HRP-labeled IgG antibody is a product of SEROTEC, and the product catalog number is STAR117P.

[0119] 1×Washing solution is 0.01 mol / L PBS buffer solution with pH 7.4 containing 0.1% (volume percentage) Triton-X100.

[0120] 50 kD and 3 kD ultrafiltration tubes are products of Merck Millipore, and the product catalog numbers are UFC905096, UFC500324 respectively.

[0121] Example 1: Expression of recombinant gp96 protein using insect cells

[0122] I. Recombinant plasmid pFastBac TMConstruction of 1-gp96

[0123] 1. Extract the RNA of HepG2 cells using the Trizol method, and then perform reverse transcription to obtain cDNA.

[0124] 2. According to the nucleotide sequence of the human gp96 gene (GenBank accession number AY040226.1), chemically synthesize primers F1 (SEQ ID NO: 4) and R1 (SEQ ID NO: 5), which carry the recognition sequences of restriction endonucleases EcoRⅠ and XbaⅠ respectively.

[0125] 3. After completing steps 1 and 2, use the cDNA obtained in step 1 as a template and the F1 and R1 synthesized in step 2 as primers for PCR amplification to obtain a PCR amplification product, which contains the nucleotide sequence encoding heat shock protein gp96.

[0126] 4. Double-digest the PCR amplification product with restriction endonucleases EcoRⅠ and XbaⅠ, and recover the digested product.

[0127] 5. Digest the plasmid pFastBacTM1 with restriction endonucleases EcoRⅠ and XbaⅠ, and recover the vector backbone of about 4700 bp.

[0128] 6. Ligate the digested product with the vector backbone to obtain a ligation product.

[0129] 7. Transform the ligation product obtained in step 6 into competent Escherichia coli DH10Bac cells to obtain recombinant Escherichia coli, and then extract the plasmid of this recombinant Escherichia coli to obtain the recombinant plasmid pFastBac1-gp96. The recombinant plasmid pFastBac1-gp96 expresses recombinant heat shock protein gp96 (hereinafter referred to as rgp96), and the amino acid sequence of rgp96 is as shown in SEQ ID NO: 6.

[0130] II. Expression of rgp96

[0131] 1. Co-transfect the recombinant plasmid pFastBac1-gp96 constructed in step one into Sf9 cells (about 4 μg of the recombinant plasmid pFastBac1-gp96 is transfected into every 1×10 6 Sf9 cells; during the co-transfection process, the transfection reagent is Cellfectin II reagent, the culture medium is Insect-XPRESSTM Protein-free Insect Cells medium with L-Glutamine, incubate at 27 °C for 72 h, centrifuge, and the supernatant is the P1 generation virus.

[0132] 2. Suspend the Sf9 cell suspension 1 (containing 1×10 827 °C for 8 - 10 h to obtain cultured cells; then add P1 - generation virus (dose: 0.05 - 0.1 MOI) to the cultured cells, incubate at 27 °C for 72 h, centrifuge at 4000 rpm for 5 min, and the supernatant is the P2 - generation virus.

[0133] 3. Add P2 - generation virus (dose: 0.05 - 0.1 MOI) to Sf9 cell suspension 2 (containing 1.6×10 8 Sf9 cells), culture at 27 °C, 100 - 120 rpm for 72 h, centrifuge at 4000 rpm for 5 min, and the supernatant is the P3 - generation virus.

[0134] Use anti - gp96 monoclonal antibody as the primary antibody and horseradish peroxidase - labeled goat anti - rat monoclonal antibody as the secondary antibody to perform SDS - PAGE and Western blot hybridization on the P3 - generation virus. The specific steps of Western blot hybridization refer to the literature: Zhang Yueming, Duan Yueqiang, Luo Deyan, Yao Huijuan, Wang Xiliang, Li Zhikui. Expression and identification of mouse soluble IL - 5α receptor in the Bac - to - Bac system [J]. Chinese Journal of Biologicals, 2013, 26:5. The results of SDS - PAGE and Western blot hybridization show that rgp96 protein is successfully expressed in Sf9 cells.

[0135] III. Purification of rgp96 protein

[0136] 1. Add P3 - generation virus (dose: 5 MOI) to 300 ml of Sf9 cell suspension 3 (containing 4.5×10 8 Sf9 cells), culture at 27 °C, 100 - 120 rpm for 72 h to obtain a suspension.

[0137] 2. Take the suspension, centrifuge at 7000 rpm for 20 min to obtain supernatant 1.

[0138] 3. Take supernatant 1, filter it through a 0.22 - mm filter membrane to obtain the sample loading solution.

[0139] 4. Load the sample loading solution onto a HiTrap - Q Sepharose ion - exchange chromatography column (flow rate: 1 mL / min), then first rinse with 5 mL of PBS buffer at pH 7.5, 200 mM (flow rate: 1 mL / min); then rinse with 10 mL of PBS buffer at pH 7.5, 300 mM (flow rate: 1 mL / min); finally rinse with 3 mL of PBS buffer at pH 7.5, 600 mM (flow rate: 1 mL / min). Collect the solution after passing through the column and perform ultrafiltration concentration using an ultrafiltration tube with a molecular weight cut - off of 50 KD to obtain about 1 mL of concentrated solution. The concentrated solution contains rgp96.

[0140] 5. Load the concentrated solution obtained in step 4 onto a Superdex 200 10 / 300GL molecular sieve chromatography column (flow rate: 0.25 mL / min), then wash it with PBS buffer at pH 7.5 and 150 mM (flow rate: 0.25 mL / min), collect the breakthrough fraction at 9 - 12 mL, and further ultrafilter and concentrate it using an ultrafiltration tube with a molecular weight cut-off of 50 KD to obtain a solution of rgp96. Determine the protein concentration in the rgp96 solution by the BCA method, and finally aliquot and store it at -80 °C.

[0141] Perform SDS-PAGE electrophoresis analysis and Western blot hybridization on the rgp96 solution obtained in step 5 (using anti-gp96 monoclonal antibody as the primary antibody and horseradish peroxidase-labeled goat anti-rat monoclonal antibody as the secondary antibody). The experimental results are as Figure 1 shown. Among them, lane 1 is the high molecular weight standard protein Marker, lane 2 is the SDS-PAGE electrophoresis result of rgp96, and lane 3 is the Western blot hybridization result of rgp96. Figure 1 The results show that the rgp96 solution shows a single molecular weight band (as indicated by the arrow), and the corresponding molecular weight is consistent with the expectation, that is, through the steps in step three above, rgp96 is effectively expressed and purified.

[0142] Example 2: Killing effect of specific CTLs induced by polypeptide-gp96 complex on human breast cancer cells

[0143] I. Preparation of polypeptide-gp96 complex

[0144] Chemically synthesize the polypeptides shown in SEQ ID NO: 1 - 3 respectively. Prepare a polypeptide solution with a concentration of 20 mg / mL for each polypeptide using DMSO. Take 1 mg of each polypeptide and mix it with 1 mg, 0.1 mg, and 0.01 mg of heat shock protein gp96 respectively, dissolve it in 0.01 mol / L PBS buffer at pH 7.4 to a total volume of 4 mL, then heat shock at 55 °C for 10 minutes, cool at room temperature for 30 minutes, and finally wash away the unbound polypeptide with a 50 kD ultrafiltration tube to obtain 9 gp96-polypeptide complexes formed by each polypeptide combined with different masses of gp96, as shown in Table 2 below.

[0145] Table 2. Polypeptide and gp96 complex

[0146] Complex Name Polypeptide Used Mass Ratio of Polypeptide to gp96 Peptide 1-1 SEQ ID NO:1 1:1 Peptide 1-0.1 SEQ ID NO:1 10:1 Peptide 1-0.01 SEQ ID NO:1 100:1 Peptide 2-1 SEQ ID NO:2 1:1 Peptide 2-0.1 SEQ ID NO:2 10:1 Peptide 2-0.01 SEQ ID NO:2 100:1 Peptide 3-1 SEQ ID NO:3 1:1 Peptide 3-0.1 SEQ ID NO:3 10:1 Peptide 3-0.01 SEQ ID NO:3 100:1

[0147] Mix the three polypeptides shown in SEQ ID NO: 1 - 3 in four different mass ratios (the specific ratios are shown in Table 3 below), and prepare a polypeptide mixed solution with a concentration of 20 mg / mL using DMSO. For each mixed polypeptide solution, take a mixed solution containing 1 mg of polypeptide, and mix each mixed polypeptide solution with 1 mg, 0.1 mg, and 0.01 mg of heat shock protein gp96 respectively. Dissolve it in 0.01 mol / L PBS buffer at pH 7.4 to a total volume of 4 mL, then heat shock at 55 °C for 10 minutes, cool at room temperature for 30 minutes, and finally wash away the unbound polypeptide with a 50 kD ultrafiltration tube to obtain 12 gp96 - polypeptide complexes formed by the combination of four different mixed ratios of mixed polypeptides and gp96 with different masses, as shown in Table 3 below.

[0148] Table 3. Polypeptide - gp96 Complex

[0149]

[0150] II. Preparation of Human Tumor - Specific Effector Cells

[0151] 1. Use human lymphocyte separation medium (purchased from Cellgro, catalog number: 25 - 072 - CI) to separate anticoagulated fresh whole blood from HLA - A2 - positive volunteers to obtain peripheral blood mononuclear cells (PBMC), and adjust the cell concentration to 1.0×10 6 cells / mL with RPMI - 1640 complete medium (purchased from Gibco, catalog number: 12633012) containing 10% fetal bovine serum (purchased from Gibco, catalog number: 10099 - 141 - FBS), and inoculate into a 24 - well plate, 1 mL per well.

[0152] 2. Add the polypeptide - gp96 complex prepared above to each group the next day to a final concentration of 10 μg / mL, and the group with only gp96 protein added serves as a negative control.

[0153] 3. Add IL - 2 (purchased from PeproTech, catalog number: 212 - 12) to each well on the third day to a final concentration of 50 U / mL, and replace half of the medium and supplement IL - 2 to a final concentration of 50 U / ml every 2 - 3 days.

[0154] 4. Perform the second - round and third - round polypeptide - gp96 complex / gp96 stimulation on the seventh day and fourteenth day respectively, and add IL - 2 to a final concentration of 50 U / ml the next day.

[0155] 5. Three days after the third - round stimulation, obtain effector cells CTL.

[0156] III. Detection of Specific Killing Effect on Breast Cancer Cells

[0157] Target cells used for detection: human breast cancer cell line MCF-7 (positive for HLA-A2 expression), T2 cells (positive for HLA-A2 expression) co-incubated with three polypeptides at a mass ratio of 1:1:1, and the incubated T2 cells present the polypeptides.

[0158] Use Non-radioactive cytotoxicity assay (purchased from Promega, catalog number: G1780) was used for cytotoxic activity detection. The main steps are as follows (see the kit instruction manual for details):

[0159] 1. Experimental group: MCF-7 cells and T2 cells co-incubated with three polypeptides at a mass ratio of 1:1:1 were used as target cells. The number of target cells inoculated was 5×10 3 / well, and the above effector cells were added at an effector-to-target ratio of 5:1, 10:1, or 20:1. The effector cells were inoculated at 50 μL / well in a 96-well culture plate, and the final volume was 100 μL.

[0160] In addition, an effector cell spontaneous LDH release group was set up to calibrate the LDH spontaneously released by effector cells (each group of effector cells was added at 50 μL / well to a 96-well plate, and 50 μL of RPMI-1640 medium containing 5% fetal bovine serum was supplemented to a final volume of 100 μL). A target cell spontaneous LDH release group was set up to correct the LDH spontaneously released by target cells (each group of target cells was added at 50 μL / well to a 96-well plate, and 50 μL of RPMI-1640 medium containing 5% fetal bovine serum was supplemented to a final concentration of 100 μL). A target cell maximum LDH release group was set up as a reference for determining 100% LDH release during calculation (the cell loading was the same as that of the target cell spontaneous release group). A volume correction control group was set up to correct the volume change caused by the addition of lysis buffer (100 μL of RPMI-1640 medium containing 5% fetal bovine serum was added). A medium background control group was set up to correct the LDH activity generated by the serum in the medium and the background absorption caused by phenol red (100 μL of RPMI-1640 medium containing 5% fetal bovine serum was added).

[0161] 2. After cell inoculation, centrifuge at 250 g for 4 min, then incubate in a 37°C incubator for 4 h; 45 min before harvesting the supernatant, add lysis buffer (10×), 10 μL / well, to the target cell maximum LDH release group; then centrifuge at 250 g for 4 min and harvest the supernatant.

[0162] 3. Transfer 50 μL of the supernatant to an enzyme-linked immunosorbent assay (ELISA) plate. Prepare the substrate with the detection buffer, add 50 μL of the prepared substrate per well to the ELISA plate, cover the plate, and react in the dark at room temperature for 30 min. Add 50 μL of the stop solution to each well, and measure the absorbance OD at 490 nm with an ELISA reader within 1 h.

[0163] 4. Calculate the cell killing rate

[0164] Killing rate (%) = [(OD value of the experimental group - OD value of the effector cell spontaneous release group - OD value of the target cell spontaneous release group) / (OD value of the target cell maximum release group - OD value of the target cell spontaneous release group)] × 100%

[0165] The experimental results are as Figure 2 shown. Under the condition of an effector - target ratio of 20:1, CTLs activated by 21 complexes of polypeptide and gp96 all have tumor killing activity, while the control without polypeptide (only containing gp96) has a killing effect on tumor cells of less than 7%. Among them, CTLs activated by the mixture of three polypeptides in a mass ratio of 1:1:1 and complexed with gp96 have a tumor killing efficiency as high as 15% - 60%; moreover, when the polypeptide mixture and gp96 are mixed in a mass ratio of 1:1, CTLs activated by them have a tumor killing efficiency as high as 44% - 60%. In summary, the CTLs activated by the equal - proportion mixture of the three polypeptides themselves and the equal - proportion complex of their mixture and gp96 have the highest tumor killing efficiency. Among them, under the conditions of an effector - target ratio of 5:1, 10:1 or 20:1, the three polypeptides are mixed in equal proportion by themselves, and the CTLs activated after the polypeptide mixture and gp96 are complexed in an equal - mass ratio (such as the complex shown as mix 1 - 1 in Table 3) have tumor killing results as Figure 3 shown, and the results show that CTLs activated by the three polypeptide - gp96 complexes have obvious killing effects on human breast cancer cells, while CTLs activated by gp96 as the control group have basically no killing activity.

[0166] Example 3: Therapeutic effect of specific CTLs induced by polypeptide - gp96 complexes on breast cancer

[0167] Take 20 female nude mice aged 6 - 8 weeks, and retro - inject 10 7 specific T cells (0.5 mL) induced by polypeptide - gp96 complexes into the tail vein of each mouse. The polypeptide - gp96 complex is a complex formed by mixing three polypeptides in equal proportion and complexing with gp96 in an equal - mass ratio (such as the complex shown as mix 1 - 1 in Table 3); 3 days after retro - injection, 5×10 6 MCF - 7 cells are subcutaneously inoculated into all mice; on the second day after tumor inoculation, the mice are divided into two groups, with 10 mice in each group, and the following treatments are carried out respectively:

[0168] The first group: Retro - inject human T lymphocytes activated by polypeptide - gp96 complexes into the tail vein, immunize three times (0.5 mL each time), and the single - retro - injection dose is 10 7 cells / mouse;

[0169] Group 2: Tail vein injection of gp96-stimulated human T cells, immunized three times (0.5 mL each time), and the single immunization dose and single injection dose were 10 7 cells / mouse;

[0170] In the above two groups: Three injections were given on the 2nd, 3rd, and 4th days after inoculation with tumor cells. Starting from the first injection, the tumor growth was observed every day, the tumor size was recorded, and the tumor volume was calculated according to the following formula: V = ab 2 / 2 (V—volume, a—long diameter of the tumor, b—short diameter of the tumor). The changes in tumor volume are shown in Figure 4 , and the results showed that CTLs induced by the three polypeptide-gp96 complexes could significantly inhibit the growth of breast cancer.

[0171] Example 4: Killing effect of specific CTLs induced by polypeptide-gp96 complexes on human hepatoma cells

[0172] I. Preparation of polypeptide-gp96 complexes was the same as in Example 2

[0173] II. Preparation of human tumor-specific effector cells was as described in Example 2

[0174] III. Detection of the killing effect on hepatoma cells

[0175] Target cells used for detection: Human hepatoma cells HepG2 (positive for HLA-A2 expression), and T2 cells (positive for HLA-A2 expression) co-incubated with the three polypeptides in a mass ratio of 1:1:1. The incubated T2 cells present the polypeptides

[0176] The detection method was the same as in Example 2. In the experimental group, effector cells and target cells were added at a ratio of 5:1, 10:1, or 20:1 according to the corresponding ratio. At the same time, an effector cell spontaneous release group, a target cell spontaneous release group, a target cell maximum release group, a background control group, and a volume correction control group were established. After incubation at 37 °C for 4 h, cell lysate was added, and the supernatant was harvested for LDH detection.

[0177] The experimental results are shown in Figure 5As shown, under the condition of an effector-to-target ratio of 20:1, CTLs activated by 21 complexes of polypeptide and gp96 all had tumor-killing activity, while the control without polypeptide (only containing gp96) had less than 7% killing effect on tumor cells. Among them, CTLs activated by mixing three polypeptides in a mass ratio of 1:1:1 and complexing with gp96 had a tumor cell killing efficiency as high as 15% - 60%; moreover, when the polypeptide mixture and gp96 were mixed in a mass ratio of 1:1, the killing efficiency of CTLs activated on tumor cells was as high as 43% - 60%. In summary, the CTLs activated by mixing the three polypeptides in equal proportion themselves and complexing their mixture with gp96 in equal proportion had the highest killing efficiency on tumor cells. Among them, under the conditions of an effector-to-target ratio of 5:1, 10:1 or 20:1, the three polypeptides were mixed in equal proportion themselves, and the killing results of CTLs activated after complexing the polypeptide mixture with gp96 in an equal mass ratio (such as the complex shown as mix 1-1 in Table 3) on tumor cells were as Figure 6 shown, and the results showed that CTLs activated by the three polypeptide-gp96 complexes had obvious killing effects on liver cancer cells, while CTLs activated by gp96 had basically no killing activity as the control group.

[0178] Example 5: Therapeutic effect of specific CTLs induced by polypeptide-gp96 complexes on liver cancer model

[0179] Twenty 6-8-week-old female nude mice were taken, and 10 7 specific T cells (0.5 mL) induced by polypeptide-gp96 complexes were transfused into the tail vein of each mouse. The polypeptide-gp96 complex was a complex formed by mixing three polypeptides in equal proportion and complexing with gp96 in an equal mass ratio (such as the complex shown as mix 1-1 in Table 3); 3 days after the transfusion, 5×10 6 HepG2 liver cancer cells were subcutaneously inoculated into all mice; on the second day after the tumor inoculation, the mice were divided into two groups, with 10 mice in each group, and the following treatments were carried out respectively:

[0180] The first group: Human T lymphocytes activated by polypeptide-gp96 complexes were transfused into the tail vein, immunized three times (0.5 mL each time), and the single transfusion dose was 10 7 / mouse;

[0181] The second group: Human-derived T cells stimulated by gp96 were transfused into the tail vein, immunized three times (0.5 mL each time), and the single immunization dose was the single transfusion dose of 10 7 cells / mouse;

[0182] In the above two groups: The transfusions were carried out 3 times respectively on the 2nd, 3rd, and 4th days after inoculating the tumor cells. Starting from the first day of transfusion, the tumor growth was observed every day, the tumor size was recorded, and the tumor volume was calculated according to the following formula: V = ab 2 / 2 (V—volume, a—major axis of the tumor, b—minor axis of the tumor). The changes in tumor volume are shown in Figure 7 , and the results show that CTLs induced by the three polypeptide-gp96 complexes can significantly inhibit the growth of liver cancer tumors.

[0183] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings that have been published, and these changes are within the protection scope of the present invention. The entire scope of the present invention is given by the appended claims and any equivalents thereof. SEQUENCE LISTING <110> Foshan Resit Biotechnology Co., Ltd. <120> Epitope peptides of PDIA3 and complexes of the epitope peptides and heat shock proteins <130> IDC210145 <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of human-derived PDIA3 polypeptide fragment 1 <400> 1 Ala Leu Phe Pro Gly Val Ala Leu Leu 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of human-derived PDIA3 polypeptide fragment 2 <400> 2 Glu Leu Ser Asp Phe Ile Ser Tyr Leu 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of human PDIA3 polypeptide fragment 3 <400> 3 Phe Ile Ser Asp Lys Asp Ala Ser Ile 1 5 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> gp96 amplification primer F1 sequence <400> 4 ggaattcatg gacgatgaag ttgat 25 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> gp96 amplification primer R1 sequence <400> 5 gctctagact attagaattc atctttttc 29 <210> 6 <211> 803 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of human heat shock protein gp96 <400> 6 Met Arg Ala Leu Trp Val Leu Gly Leu Cys Cys Val Leu Leu Thr Phe 1 5 10 15 Gly Ser Val Arg Ala Asp Asp Glu Val Asp Val Asp Gly Thr Val Glu 20 25 30 Glu Asp Leu Gly Lys Ser Arg Glu Gly Ser Arg Thr Asp Asp Glu Val 35 40 45 Val Gln Arg Glu Glu Glu Ala Ile Gln Leu Asp Gly Leu Asn Ala Ser 50 55 60 Gln Ile Arg Glu Leu Arg Glu Lys Ser Glu Lys Phe Ala Phe Gln Ala 65 70 75 80 Glu Val Asn Arg Met Met Lys Leu Ile Ile Asn Ser Leu Tyr Lys Asn 85 90 95 Lys Glu Ile Phe Leu Arg Glu Leu Ile Ser Asn Ala Ser Asp Ala Leu 100 105 110 Asp Lys Ile Arg Leu Ile Ser Leu Thr Asp Glu Asn Ala Leu Ser Gly 115 120 125 Asn Glu Glu Leu Thr Val Lys Ile Lys Cys Asp Lys Glu Lys Asn Leu 130 135 140 Leu His Val Thr Asp Thr Gly Val Gly Met Thr Arg Glu Glu Leu Val 145 150 155 160 Lys Asn Leu Gly Thr Ile Ala Lys Ser Gly Thr Ser Glu Phe Leu Asn 165 170 175 Lys Met Thr Glu Ala Gln Glu Asp Gly Gln Ser Thr Ser Glu Leu Ile 180 185 190 Gly Gln Phe Gly Val Gly Phe Tyr Ser Ala Phe Leu Val Ala Asp Lys 195 200 205 Val Ile Val Thr Ser Lys His Asn Asn Asp Thr Gln His Ile Trp Glu 210 215 220 Ser Asp Ser Asn Glu Phe Ser Val Ile Ala Asp Pro Arg Gly Asn Thr 225 230 235 240 Leu Gly Arg Gly Thr Thr Ile Thr Leu Val Leu Lys Glu Glu Ala Ser 245 250 255 Asp Tyr Leu Glu Leu Asp Thr Ile Lys Asn Leu Val Lys Lys Tyr Ser 260 265 270 Gln Phe Ile Asn Phe Pro Ile Tyr Val Trp Ser Ser Lys Thr Glu Thr 275 280 285 Val Glu Glu Pro Met Glu Glu Glu Glu Ala Ala Lys Glu Glu Lys Glu 290 295 300 Glu Ser Asp Asp Glu Ala Ala Val Glu Glu Glu Glu Glu Glu Lys Lys 305 310 315 320 Pro Lys Thr Lys Lys Val Glu Lys Thr Val Trp Asp Trp Glu Leu Met 325 330 335 Asn Asp Ile Lys Pro Ile Trp Gln Arg Pro Ser Lys Glu Val Glu Glu 340 345 350 Asp Glu Tyr Lys Ala Phe Tyr Lys Ser Phe Ser Lys Glu Ser Asp Asp 355 360 365 Pro Met Ala Tyr Ile His Phe Thr Ala Glu Gly Glu Val Thr Phe Lys 370 375 380 Ser Ile Leu Phe Val Pro Thr Ser Ala Pro Arg Gly Leu Phe Asp Glu 385 390 395 400 Tyr Gly Ser Lys Lys Ser Asp Tyr Ile Lys Leu Tyr Val Arg Arg Val 405 410 415 Phe Ile Thr Asp Asp Phe His Asp Met Met Pro Lys Tyr Leu Asn Phe 420 425 430 Val Lys Gly Val Val Asp Ser Asp Asp Leu Pro Leu Asn Val Ser Arg 435 440 445 Glu Thr Leu Gln Gln His Lys Leu Leu Lys Val Ile Arg Lys Lys Leu 450 455 460 Val Arg Lys Thr Leu Asp Met Ile Lys Lys Ile Ala Asp Asp Lys Tyr 465 470 475 480 Asn Asp Thr Phe Trp Lys Glu Phe Gly Thr Asn Ile Lys Leu Gly Val 485 490 495 Ile Glu Asp His Ser Asn Arg Thr Arg Leu Ala Lys Leu Leu Arg Phe 500 505 510 Gln Ser Ser His His Pro Thr Asp Ile Thr Ser Leu Asp Gln Tyr Val 515 520 525 Glu Arg Met Lys Glu Lys Gln Asp Lys Ile Tyr Phe Met Ala Gly Ser 530 535 540 Ser Arg Lys Glu Ala Glu Ser Ser Pro Phe Val Glu Arg Leu Leu Lys 545 550 555 560 Lys Gly Tyr Glu Val Ile Tyr Leu Thr Glu Pro Val Asp Glu Tyr Cys 565 570 575 Ile Gln Ala Leu Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala 580 585 590 Lys Glu Gly Val Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg 595 600 605 Glu Ala Val Glu Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp 610 615 620 Lys Ala Leu Lys Asp Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu 625 630 635 640 Thr Glu Ser Pro Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly 645 650 655 Asn Met Glu Arg Ile Met Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp 660 665 670 Ile Ser Thr Asn Tyr Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Asn 675 680 685 Pro Arg His Pro Leu Ile Arg Asp Met Leu Arg Arg Ile Lys Glu Asp 690 695 700 Glu Asp Asp Lys Thr Val Leu Asp Leu Ala Val Val Leu Phe Glu Thr 705 710 715 720 Ala Thr Leu Arg Ser Gly Tyr Leu Leu Pro Asp Thr Lys Ala Tyr Gly 725 730 735 Asp Arg Ile Glu Arg Met Leu Arg Leu Ser Leu Asn Ile Asp Pro Asp 740 745 750 Ala Lys Val Glu Glu Glu Pro Glu Glu Glu Pro Glu Glu Thr Ala Glu 755 760 765 Asp Thr Thr Glu Asp Thr Glu Gln Asp Glu Asp Glu Glu Met Asp Val 770 775 780 Gly Thr Asp Glu Glu Glu Glu Thr Ala Lys Glu Ser Thr Ala Glu Lys 785 790 795 800 Asp Glu Leu <210> 7 <211> 2412 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence encoding human heat shock protein gp96 <400> 7 atgagggccc tgtgggtgct gggcctctgc tgcgtcctgc tgaccttcgg gtcggtcaga 60 gctgacgatg aagttgatgt ggatggtaca gtagaagagg atctgggtaa aagtagagaa 120 ggatcaagga cggatgatga agtagtacag agagaggaag aagctattca gttggatgga 180 ttaaatgcat cacaaataag agaacttaga gagaagtcgg aaaagtttgc cttccaagcc 240 gaagttaaca gaatgatgaa acttatcatc aattcattgt ataaaaataa agagattttc 300 ctgagagaac tgatttcaaa tgcttctgat gctttagata agataaggct aatatcactg 360 actgatgaaa atgctctttc tggaaatgag gaactaacag tcaaaattaa gtgtgataag 420 gagaagaacc tgctgcatgt cacagacacc ggtgtaggaa tgaccagaga agagttggtt 480 aaaaaccttg gtaccatagc caaatctggg acaagcgagt ttttaaacaa aatgactgaa 540 gcacaggaag atggccagtc aacttctgaa ttgattggcc agtttggtgt cggtttctat 600 tccgccttcc ttgtagcaga taaggttatt gtcacttcaa aacacaacaa cgatacccag 660 cacatctggg agtctgactc caatgaattt tctgtaattg ctgacccaag aggaaacact 720 ctaggacggg gaacgacaat tacccttgtc ttaaaagaag aagcatctga ttaccttgaa 780 ttggatacaa ttaaaaatct cgtcaaaaaa tattcacagt tcataaactt tcctatttat 840 gtatggagca gcaagactga aactgttgag gagcccatgg aggaagaaga agcagccaaa 900 gaagagaaag aagaatctga tgatgaagct gcagtagagg aagaagaaga agaaaagaaa 960 ccaaagacta aaaaagttga aaaaactgtc tgggactggg aacttatgaa tgatatcaaa 1020 ccaatatggc agagaccatc aaaagaagta gaagaagatg aatacaaagc tttctacaaa 1080 tcattttcaa aggaaagtga tgaccccatg gcttatattc actttactgc tgaaggggaa 1140 gttaccttca aatcaatttt atttgtaccc acatctgctc cacgtggtct gtttgacgaa 1200 tatggatcta aaaagagcga ttacattaag ctctatgtgc gccgtgtatt catcacagac 1260 gacttccatg atatgatgcc taaatacctc aattttgtca agggtgtggt ggactcagat 1320 gatctcccct tgaatgtttc ccgcgagact cttcagcaac ataaactgct taaggtgatt 1380 aggaagaagc ttgttcgtaa aacgctggac atgatcaaga agattgctga tgataaatac 1440 aatgatactt tttggaaaga atttggtacc aacatcaagc ttggtgtgat tgaagaccac 1500 tcgaatcgaa cacgtcttgc taaacttctt aggttccagt cttctcatca tccaactgac 1560 attactagcc tagaccagta tgtggaaaga atgaaggaaa aacaagacaa aatctacttc 1620 atggctgggt ccagcagaaa agaggctgaa tcttctccat ttgttgagcg acttctgaaa 1680 aagggctatg aagttattta cctcacagaa cctgtggatg aatactgtat tcaggccctt 1740 cccgaatttg atgggaagag gttccagaat gttgccaagg aaggagtgaa gttcgatgaa 1800 agtgagaaaa ctaaggagag tcgtgaagca gttgagaaag aatttgagcc tctgctgaat 1860 tggatgaaag ataaagccct taaggacaag attgaaaagg ctgtggtgtc tcagcgcctg 1920 acagaatctc cgtgtgcttt ggtggccagc cagtacggat ggtctggcaa catggagaga 1980 atcatgaaag cacaagcgta ccaaacgggc aaggacatct ctacaaatta ctatgcgagt 2040 cagaagaaaa catttgaaat taatcccaga cacccgctga tcagagacat gcttcgacga 2100 attaaggaag atgaagatga taaaacagtt ttggatcttg ctgtggtttt gtttgaaaca 2160 gcaacgcttc ggtcagggta tcttttacca gacactaaag catatggaga tagaatagaa 2220 agaatgcttc gcctcagttt gaacattgac cctgatgcaa aggtggaaga agagcctgaa 2280 gaagaacctg aagagacagc agaagacaca acagaagaca cagagcaaga cgaagatgaa 2340 gaaatggatg tgggaacaga tgaagaagaa gaaacagcaa aggaatctac agctgaaaaa 2400 gatgaattgt aa 2412

Claims

1. A separated polypeptide, the separated polypeptide having an amino acid sequence as shown in any one of SEQ ID NO: 2 or 3.

2. A polypeptide composition, the polypeptide composition comprising any two or three polypeptides selected from the following: the polypeptides shown in any one of SEQ ID NO: 1, 2, and 3.

3. The polypeptide composition of claim 2, wherein, the ratios of the polypeptides contained in the polypeptide composition are the same or different.

4. The polypeptide composition of claim 2, wherein, the polypeptide composition comprises the polypeptide shown in SEQ ID NO: 1, the polypeptide shown in SEQ ID NO: 2, and the polypeptide shown in SEQ ID NO:

3.

5. The polypeptide composition of claim 4, wherein, the mass ratios of the polypeptide shown in SEQ ID NO: 1, the polypeptide shown in SEQ ID NO: 2, and the polypeptide shown in SEQ ID NO: 3 in the polypeptide composition are 1:1:1, or 5:1:1, or 1:5:1, or 1:1:

5.

6. A complex formed by the separated polypeptide according to claim 1 or the polypeptide composition according to any one of claims 2 - 5 and a heat shock protein.

7. The complex of claim 6, wherein, the complex is formed by the separated polypeptide according to claim 1 or the polypeptide composition according to any one of claims 2 - 5 and a heat shock protein in a natural adsorption or heat shock manner.

8. The complex of claim 6, wherein, the complex is prepared by mixing the separated polypeptide according to claim 1 or the polypeptide composition according to any one of claims 2 - 5 and a heat shock protein in vitro.

9. The complex of claim 8, wherein, the mixing is carried out at 40 - 80 °C.

10. The complex of claim 8, wherein, the mixing is carried out at 55 °C.

11. The complex of claim 6, wherein, the complex is prepared by contacting the separated polypeptide according to claim 1 or the polypeptide composition according to any one of claims 2 - 5 and a heat shock protein, placing them at 55 °C for 10 min, and then placing them at room temperature for 30 min.

12. The complex of claim 6, wherein, the complex is prepared by mixing the separated polypeptide according to claim 1 or the polypeptide composition according to any one of claims 2 - 5 and a heat shock protein in vitro at a mass ratio of 0.1:1 to 100:

1.

13. The complex of claim 6, wherein, the complex is prepared by mixing the separated polypeptide according to claim 1 or the polypeptide composition according to any one of claims 2 - 5 and a heat shock protein in vitro at a mass ratio of 1:1 to 100:

1.

14. The complex of claim 6, wherein, the complex is prepared by mixing the separated polypeptide according to claim 1 or the polypeptide composition according to any one of claims 2 - 5 and a heat shock protein in vitro at a mass ratio of 1:1 to 10:

1.

15. The complex of claim 6, wherein, The complex is prepared by mixing the isolated polypeptide according to claim 1 or the polypeptide composition according to any one of claims 2-5 with a heat shock protein in vitro at a mass ratio of 1:1, 10:1 or 100:

1.

16. The complex of claim 6, wherein, the heat shock protein is selected from HSP70, HSP90, gp96, HSP110.

17. The complex of claim 6, wherein, the heat shock protein is gp96.

18. The complex of claim 6, wherein, the heat shock protein has the amino acid sequence shown in SEQ ID NO:

6.

19. The complex of claim 6, wherein, the complex has immunogenicity and can induce a specific immune response against cells expressing PDIA3.

20. The complex of claim 19, wherein, the cells expressing PDIA3 are tumor cells.

21. A composition comprising at least two complexes according to any one of claims 6-20.

22. The composition of claim 21, wherein, the heat shock proteins in the composition are the same or different.

23. The composition of claim 21, wherein, the composition has immunogenicity and can induce a specific immune response against cells expressing PDIA3.

24. The composition of claim 23, wherein, the cells expressing PDIA3 are tumor cells.

25. A method for preparing the complex according to any one of claims 6-20, which comprises mixing the isolated polypeptide according to claim 1 or the polypeptide composition according to any one of claims 2-5 with a heat shock protein.

26. The method of claim 25, wherein, the isolated polypeptide according to claim 1 or the polypeptide composition according to any one of claims 2-5 and the heat shock protein form a complex by natural adsorption or heat shock.

27. The method of claim 25, wherein, the mixing is carried out at 40-80 °C.

28. The method of claim 25, wherein, the mixing is carried out at 55 °C.

29. The method of claim 25, which comprises: contacting the isolated polypeptide according to claim 1 or the polypeptide composition according to any one of claims 2-5 and the heat shock protein and placing them at 55 °C for 10 min, and then placing them at room temperature for 30 min.

30. The method of claim 25, which comprises: mixing the isolated polypeptide according to claim 1 or the polypeptide composition according to any one of claims 2-5 with the heat shock protein at a mass ratio of 0.1:1 to 100:

1.

31. The method of claim 25, which comprises: mixing the isolated polypeptide according to claim 1 or the polypeptide composition according to any one of claims 2-5 with the heat shock protein at a mass ratio of 1:1 to 100:

1.

32. The method of claim 25, which comprises: mixing the isolated polypeptide according to claim 1 or the polypeptide composition according to any one of claims 2-5 with the heat shock protein at a mass ratio of 1:1 to 10:

1.

33. The method of claim 25, which comprises: The isolated polypeptide according to claim 1 or the polypeptide composition according to any one of claims 2 - 5 is mixed with a heat shock protein in a mass ratio of 1:1, 10:1 or 100:

1.

34. The method of claim 25, wherein, the heat shock protein is selected from HSP70, HSP90, gp96, HSP110.

35. The method of claim 25, wherein, the heat shock protein is gp96.

36. The method of claim 25, wherein, the heat shock protein has the amino acid sequence shown in SEQ ID NO:

6.

37. The method of claim 25, wherein, the complex has immunogenicity and is capable of inducing a specific immune response against cells expressing PDIA3.

38. The method of claim 37, wherein, the cells expressing PDIA3 are tumor cells.

39. A method for preparing T cells, the method comprising obtaining by stimulating immune cells with the complex according to any one of claims 6 - 20 or the composition according to any one of claims 21 - 24; the T cells express a T cell receptor capable of specifically recognizing protein disulfide isomerase family member 3 (PDIA3) or the isolated polypeptide according to claim 1 or the polypeptide composition according to any one of claims 2 - 5.

40. The method of claim 39, wherein, the immune cells comprise PBMC or T lymphocytes.

41. The method of claim 39, wherein, the stimulation further comprises using an immunostimulatory cytokine.

42. The method of claim 41, wherein, the immunostimulatory cytokine is IL - 2.

43. An isolated nucleic acid molecule, which comprises a nucleotide sequence encoding the isolated polypeptide according to claim 1.

44. A combination of isolated nucleic acid molecules, which comprises a nucleotide sequence encoding the polypeptide composition according to any one of claims 2 - 5.

45. A vector, which comprises the isolated nucleic acid molecule according to claim 43 or the combination of isolated nucleic acid molecules according to claim 44; or, the vector comprises a nucleotide sequence encoding the isolated polypeptide of claim 1 or the polypeptide composition according to any one of claims 2 - 5.

46. The vector of claim 45, wherein, the vector is a viral vector.

47. A host cell, which comprises the isolated nucleic acid molecule according to claim 43 or the combination of isolated nucleic acid molecules according to claim 44 or the vector according to claim 45 or 46.

48. A pharmaceutical composition, which comprises the isolated polypeptide according to claim 1, the polypeptide composition according to any one of claims 2 - 5, the complex according to any one of claims 6 - 20, the composition according to any one of claims 21 - 24, the isolated nucleic acid molecule according to claim 43, the combination of isolated nucleic acid molecules according to claim 44, the vector according to claim 45 or 46, or the host cell according to claim 47, and a pharmaceutically acceptable carrier and / or excipient.

49. The pharmaceutical composition of claim 48, wherein, the pharmaceutical composition is a tumor vaccine.

50. The pharmaceutical composition of claim 48, wherein, The pharmaceutical composition comprises an adjuvant.

51. The pharmaceutical composition of claim 48, wherein, the pharmaceutical composition further comprises an anti-tumor agent or an immune enhancer.

52. The pharmaceutical composition of claim 51, wherein, the anti-tumor agent is selected from alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, radiosensitizers, anti-angiogenesis agents, cytokines, immune checkpoint inhibitors.

53. The pharmaceutical composition of claim 52, wherein, the immune checkpoint inhibitor is a PD-1 antibody, a PD-L1 antibody, a CTLA-4 antibody, a LAG-3 antibody or a TIM3 antibody.

54. The pharmaceutical composition of claim 51, wherein, the immune enhancer is selected from immune stimulatory antibodies.

55. The pharmaceutical composition of claim 54, wherein, the immune stimulatory antibody is an anti-CD3 antibody, an anti-CD28 antibody, an anti-CD40L (CD154) antibody, an anti-41BB (CD137) antibody, an anti-OX40 antibody, an anti-GITR antibody or any combination thereof.

56. Use of the isolated polypeptide of claim 1, or the polypeptide composition of any one of claims 2-5, or the complex of any one of claims 6-20, or the composition of any one of claims 21-24 in the preparation of a medicament for inducing an immune response against a tumor expressing PDIA3 in a subject and / or for preventing or treating a tumor expressing PDIA3 in a subject; wherein, the tumor expressing PDIA3 is selected from liver cancer, breast cancer, glioma, colorectal cancer, pancreatic cancer, gastric cancer, lung cancer, endometrial cancer, ovarian cancer, multiple myeloma, melanoma, thyroid cancer, bladder cancer, prostate cancer, head and neck cancer or acute myeloid leukemia.

57. The use of claim 56, wherein, the subject is a human.

58. The use of claim 56, wherein, the subject is HLA-A2 positive.

59. The use of claim 56, wherein, the isolated polypeptide, polypeptide composition, or composition is administered in combination with an immune stimulant or an anti-tumor agent.

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