Epitope peptide of LGALS3 and complex of the epitope peptide and heat shock protein

Abstract

The present invention relates to the fields of immunology and cancer treatment. Specifically, the present invention relates to epitope peptides of the protein LGALS3 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 LGALS3 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] Liver cancer refers to malignant tumors that occur in the liver, including primary liver cancer and metastatic liver cancer. Among them, primary liver cancer is one of the most common malignant tumors clinically. Among male cancer patients worldwide, liver cancer ranks sixth in terms of incidence and second in terms of mortality; among female cancer patients, liver cancer ranks seventh in terms of incidence and sixth in terms of mortality. In 2008, there were 748,300 newly diagnosed liver cancer cases worldwide, and 695,900 liver cancer patients died. And half of these newly diagnosed 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. 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.

[0003] Pancreatic cancer is one of the digestive tract tumors with extremely high malignancy, and its incidence and mortality are both increasing year by year globally. Currently, the clinical treatment methods for pancreatic cancer mainly include surgical resection, radiotherapy, and chemotherapy. Due to the characteristics of pancreatic cancer such as insidious onset, rapid disease progression, and high malignancy, most patients are in the middle and late stages of cancer once they are diagnosed, losing the opportunity for surgery, resulting in a 5-year survival rate of less than 6%.

[0004] Galectin-3 (LGASL3) belongs to the lectin family, and members of this family have a strong affinity for galactose. This protein has a proline-rich tandem repeat domain at the N-terminus and a C-terminal carbohydrate recognition domain. This protein can self-associate through the N-terminal domain and thus bind to multivalent sugar ligands. This protein is localized in the extracellular matrix, cytoplasm, and nucleus. This protein plays a role in many cellular functions, including apoptosis, innate immunity, cell adhesion, and T cell regulation. At the same time, more and more studies have shown that LGALS3 plays a key role in the occurrence and development of tumors and promotes tumor metastasis. In highly prevalent cancers such as liver cancer, glioblastoma, and pancreatic cancer, the expression of LGALS3 protein is significantly increased, and the expression level of LGALS3 is also directly related to the survival rate of cancer patients.

[0005] Heat shock proteins (HSPs) are a class of proteins that are highly conserved in biological evolution and widely present in prokaryotes and eukaryotes. HSPs can be divided into multiple subfamilies such as HSP110, HSP90, HSP70, HSP60, HSP40, small molecule HSPs, and ubiquitin according to the degree of homology and molecular weight. Heat shock protein (HSP) 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, assist in assembling them onto MHC class I molecules, and present them on the cell membrane. Heat shock protein 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 LGALS3 and their combinations, and specific immune cells induced thereby. The present invention further provides a complex formed by the epitope peptide and a 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: 1 or 2.

[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 1:1:1 to 20:1:1, or 1:1:1 to 1:20:1, or 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 (for example, 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 and a heat shock protein and placing them at 55 °C for 10 min, and then placing them 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 shown in SEQ ID NO: 6.

[0021] In certain embodiments, the complex is prepared by mixing in vitro the polypeptide isolated as described above or the 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.

[0022] In certain embodiments, the complex is immunogenic and capable of inducing a specific immune response against cells expressing LGALS3 (such as tumor cells, such as pancreatic 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 LGALS3 (such as tumor cells, such as pancreatic cancer or liver cancer cells).

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

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

[0028] In certain embodiments, the complex is prepared by mixing in vitro the polypeptide isolated as described above or the polypeptide composition as described above and a heat shock protein. 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 isolated as described above or the polypeptide composition as described above and the heat shock protein and placing them at 55 °C for 10 min, and then placing them at room temperature for 30 min.

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

[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 isolated polypeptide or polypeptide composition as described above with a 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.

[0033] In certain embodiments, the complex is immunogenic and can induce a specific immune response against cells expressing LGALS3 (such as tumor cells, such as pancreatic 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 galectin-3 (LGALS3) or the isolated 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 cell can specifically recognize and kill cells expressing LGALS3 (such as tumor cells, such as pancreatic cancer or liver cancer cells).

[0039] In another aspect, the present invention provides a method for preparing the T cell as described above, the method including stimulating immune cells with the complex or composition as described above. In certain embodiments, the immune cells include 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 an amino acid sequence as shown in SEQ ID NO: 1 or 2.

[0042] In another aspect, the present invention provides a combination of isolated nucleic acid molecules comprising a nucleotide sequence 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 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 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-angiogenesis agents, cytokines, immune checkpoint inhibitors (e.g., anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-LAG-3 antibody or anti-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-4-1BB (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 tumors expressing LGALS3 in a subject and / or for preventing or treating tumors expressing LGALS3 in a subject.

[0054] In certain embodiments, the tumors expressing LGALS3 are selected from colorectal cancer, pancreatic cancer, gastric cancer, lung cancer, endometrial cancer, ovarian cancer, multiple myeloma, melanoma, thyroid cancer, bladder cancer, prostate cancer, breast 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 an additional therapeutic agent, such as simultaneously, separately or sequentially. In certain embodiments, the additional therapeutic agent is an immune stimulant or an anti-tumor agent.

[0059] In certain embodiments, the expression of LGALS3 protein in the tumors expressing LGALS3 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 LGALS3 in a subject and / or for preventing or treating tumors expressing LGALS3 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 LGALS3 are selected from colorectal cancer, pancreatic cancer, gastric cancer, lung cancer, endometrial cancer, ovarian cancer, multiple myeloma, melanoma, thyroid cancer, bladder cancer, prostate cancer, breast cancer, head and neck cancer, or acute myeloid leukemia. In certain embodiments, the subject is a human.

[0062] In certain embodiments, the subject is HLA-A2 positive.

[0063] In certain embodiments, the method further comprises administering to the subject an additional therapeutic agent, such as an immune enhancer or an anti-tumor agent.

[0064] In certain embodiments, the LGALS3 protein expression in the tumors expressing LGALS3 is abnormal (e.g., increased expression compared to healthy subjects).

[0065] 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.

[0066] Term Definitions

[0067] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Also, the virology, biochemistry, and immunology 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.

[0068] As used herein, the term "LGALS3 (Galectin-3)" is also known as galectin-3. Galectins are a family of carbohydrate-binding proteins with diverse biological behaviors. Among them, LGALS3 is not only involved in regulating cellular activities such as cell proliferation, apoptosis, adhesion, and migration, but also participates in physiological and pathological activities such as immune regulation, tumorigenesis, and metastasis. LGALS3 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: CAG33178.1.

[0069] As used herein, the term "gp96" is also known as Grp94 and 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 homodimeric 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.

[0070] As used herein, the term "HSP70" is a heat shock protein with a molecular weight of approximately 70 kD and is an important member of the heat shock protein family, known as the major heat shock protein, including more than 20 proteins with molecular weights of 68, 72, 73, 75, 78 kDa, etc. 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.

[0071] As used herein, the term "HSP90" is a heat shock protein with a molecular weight of approximately 90 kD and is a protein used for co-immunization. It is an important member of the heat shock protein family with a molecular weight of approximately 83 - 90 kDa.

[0072] As used herein, the term "HSP110" is a heat shock protein with a molecular weight of approximately 110 kD and is an important member of the heat shock protein family with a molecular weight of approximately 100 - 110 kDa.

[0073] 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.

[0074] 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 material elements carried by it are 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); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. 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, polyomaviruses (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.

[0075] 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 Drosophila S2 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.

[0076] As used herein, the term "subject" includes, but is not limited to, various animals, especially mammals, such as humans. In certain embodiments, the subject (such as a human) suffers from an autoimmune disease.

[0077] 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.

[0078] 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 the like. 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 (such as aluminum hydroxide), Freund's adjuvants (such as 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 generally understood by those skilled in the art and are capable of stabilizing the desired activity of the active ingredient in the drug (e.g., the inhibitory activity against PSD-95 ubiquitination), and include but are not limited to sodium glutamate, gelatin, SPGA, saccharides (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.

[0079] As used herein, the term "treatment" refers to treating or curing a disease (such as an autoimmune disease), delaying the onset of one or more symptoms of the disease, and / or delaying the progression of the disease.

[0080] As used herein, the term "effective amount" refers to an amount that can effectively achieve the intended purpose. For example, a therapeutically effective amount may be an amount that is effective or sufficient to treat or cure a disease (such as an autoimmune disease), 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.

[0081] Beneficial effects

[0082] The complex of three polypeptides of the protein LGALS 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 LGALS3 (such as tumor cells, such as pancreatic 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 LGALS3, and thus can prevent or treat tumors expressing LGALS3. Moreover, the three polypeptides of the protein LGALS3 and combinations thereof can be used to prepare a tumor vaccine, and the vaccine can be used to prevent or treat tumors expressing LGALS3.

[0083] 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

[0084] 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.

[0085] Figure 2 It is the killing results of specific CTLs activated by three polypeptides and mixed polypeptides with different ratios after being complexed with gp96 on pancreatic cancer cells in Example 2 of the present invention.

[0086] 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 PANC-1 cells and T2 cells co-incubated with the three polypeptides in Example 2 of the present invention.

[0087] Figure 4 The effect of the specific T cells induced by the polypeptide-gp96 complex reinfusion and the control group (human T cells stimulated by gp96) on the pancreatic cancer tumor volume in Example 3 of the present invention.

[0088] Figure 5 The killing results of specific CTLs activated by mixing three polypeptides and polypeptides mixed in different proportions with gp96 against human liver cancer cells in Example 4 of the present invention.

[0089] 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.

[0090] Figure 7 The effect of the specific T cells induced by the polypeptide-gp96 complex reinfusion and the control group (human T cells stimulated by gp96) on the liver cancer tumor volume in Example 5 of the present invention.

[0091] Sequence information

[0092] Table 1: The information of the sequences involved in this application is described in the following table.

[0093]

[0094] Detailed implementation manners

[0095] The present invention will now be described with reference to the following examples which are intended to illustrate the invention (but not to limit it).

[0096] Those skilled in the art will appreciate that the examples describe the 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 specified, 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.

[0097] The sources of the experimental materials involved in the following examples are as follows:

[0098] The nude mice were products of Beijing Vital River Laboratory Animal Technology Co., Ltd. and were referred to as mice in the examples. The polypeptides were synthesized by Shanghai Gil Biochemical Co., Ltd.

[0099] HepG2 cells (human hepatoma cells) were purchased from ATCC (American Type Culture Collection), and the product catalog number was HB-8065 TM 。

[0100] PANC-1 (human pancreatic cancer cells) were purchased from the Basic Medical Cell Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences, and the cell line resource number was 3111C0001CCC000023.

[0101] Sf9 cells were products of Invitrogen, and the product catalog number was 11496-015.

[0102] Cellfectin II reagent was a product of Life technologies, and the product catalog number was 10362-100.

[0103] Plasmid pFastBac TM 1 was a product of Invitrogen, and the product catalog number was 10359-016.

[0104] The gp96 monoclonal antibody was a product of Santa Cruz, and the product catalog number was sc-56399.

[0105] The horseradish peroxidase-labeled goat anti-rat monoclonal antibody was a product of Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., and the product catalog number was ZB-2307.

[0106] The HiTrap-Q Sepharose ion exchange chromatography column was a product of GE, and the product catalog number was 17-5053-01.

[0107] The Superdex 200 10 / 300GL molecular sieve chromatography column was a product of GE, and the product catalog number was 17517501.

[0108] Escherichia coli DH10Bac competent cells were products of Beijing Yuanpinghao Biotechnology Co., Ltd., and the product catalog number was CL108-01.

[0109] Insect-XPRESSTM Protein-free Insect Cells medium with L-Glutamine was a product of LONZA, and the product catalog number was 12-730Q.

[0110] BSA, PMSF, NaHCO 3 、MnCl2 , CaCl 2 , 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.

[0111] Solution A: The solute is 1 mM PMSF and 30 mM NaHCO 3 ; The solvent is distilled water; The pH value is 7.4.

[0112] Solution B: The solute is 2 mM MnCl 2 , 2 mM CaCl 2 , 500 mM NaCl and 1 mM PMSF; The solvent is a 20 mM Tris-HCl buffer solution with pH 7.4.

[0113] Solution C: The solute is 10% (mass / volume ratio) methyl α-D-mannopyranoside, 500 mM NaCl and 1 mM PMSF; The solvent is a 20 mM Tris-HCl buffer solution with pH 7.4.

[0114] Washing solution: Dilute Solution B with distilled water to 10 times the volume.

[0115] The 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.

[0116] The 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.

[0117] The HRP-labeled IgG antibody is a product of SEROTEC, and the product catalog number is STAR117P.

[0118] The 1× washing solution is a 0.01 mol / L PBS buffer solution with pH 7.4 containing 0.1% (volume percentage) Triton-X100.

[0119] The 50 kD and 3 kD ultrafiltration tubes are products of Merck Millipore, and the product catalog numbers are UFC905096, UFC500324 respectively.

[0120] Example 1: Expression of recombinant gp96 protein using insect cells

[0121] I. Recombinant plasmid pFastBacTM Construction of 1-gp96

[0122] 1. Extract the RNA of HepG2 cells using the Trizol method, and then perform reverse transcription to obtain cDNA.

[0123] 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 enzymes EcoRⅠ and XbaⅠ, respectively.

[0124] 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.

[0125] 4. Double-digest the PCR amplification product with restriction enzymes EcoRⅠ and XbaⅠ, and recover the digested product.

[0126] 5. Digest the plasmid pFastBac TM 1 with restriction enzymes EcoRⅠ and XbaⅠ, and recover the vector backbone of approximately 4700 bp.

[0127] 6. Ligate the digested product with the vector backbone to obtain a ligation product.

[0128] 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 shown in SEQ ID NO: 6.

[0129] II. Expression of rgp96

[0130] 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.

[0131] 2. Suspend Sf9 cell suspension 1 (containing 1×10 8 Sf9 cells) in culture at 27°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, and centrifuge at 4000 rpm for 5 min. The supernatant is the P2 generation virus.

[0132] 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 and 100 - 120 rpm for 72 h, centrifuge at 4000 rpm for 5 min. The supernatant is the P3 generation virus.

[0133] Using anti - gp96 monoclonal antibody as the primary antibody and horseradish peroxidase - labeled goat anti - rat monoclonal antibody as the secondary antibody, perform SDS - PAGE and Western blot hybridization on the P3 generation virus. For 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 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.

[0134] III. Purification of rgp96 protein

[0135] 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 and 100 - 120 rpm for 72 h to obtain a suspension.

[0136] 2. Take the suspension, centrifuge at 7000 rpm for 20 min to obtain supernatant 1.

[0137] 3. Take supernatant 1, filter it through a 0.22 mm filter membrane to obtain the sample loading solution.

[0138] 4. Load the sample 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 and 200 mM (flow rate: 1 mL / min); then rinse with 10 mL of PBS buffer at pH 7.5 and 300 mM (flow rate: 1 mL / min); finally rinse with 3 mL of PBS buffer at pH 7.5 and 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 a concentrated solution of about 1 mL. The concentrated solution contains rgp96.

[0139] 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 with PBS buffer at pH 7.5 and 150 mM (flow rate: 0.25 mL / min), and collect the breakthrough fraction at 9 - 12 mL. Further perform ultrafiltration concentration 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 solution of rgp96 using the BCA method, and finally aliquot and store at -80°C.

[0140] Perform SDS-PAGE electrophoresis analysis and Western blot hybridization on the solution of rgp96 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 indicate that the solution of rgp96 shows a single molecular weight band (as indicated by the arrow), and the corresponding molecular weight is consistent with the expected value, that is, through the steps in step three above, rgp96 is effectively expressed and purified.

[0141] Example 2: Killing effect of specific CTLs induced by polypeptide-gp96 complex on human pancreatic cancer cells

[0142] I. Preparation of polypeptide-gp96 complex

[0143] The polypeptides shown in SEQ ID NO:1-3 were chemically synthesized separately. Each polypeptide was formulated into a polypeptide solution with a concentration of 20 mg / mL using DMSO. 1 mg of each polypeptide was taken and mixed with 1 mg, 0.1 mg, and 0.01 mg of heat shock protein gp96 respectively, and dissolved in 0.01 mol / L PBS buffer at pH 7.4 to a total volume of 4 mL. Then, it was heat shocked at 55 °C for 10 minutes, cooled at room temperature for 30 minutes, and finally, the unbound polypeptide was washed away with a 50 kD ultrafiltration tube, obtaining 9 gp96-polypeptide complexes formed by each polypeptide combined with different masses of gp96, as shown in Table 2 below.

[0144] Table 2. Polypeptide-gp96 complexes

[0145] 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

[0146] The 3 polypeptides shown in SEQ ID NO:1-3 were mixed according to 4 different mass ratios (the specific ratios are shown in Table 3 below), and formulated into a polypeptide mixed solution with a concentration of 20 mg / mL using DMSO. For each mixed polypeptide solution, a mixed solution containing 1 mg of polypeptide was taken. Each mixed polypeptide solution was mixed with 1 mg, 0.1 mg, and 0.01 mg of heat shock protein gp96 respectively, and dissolved in 0.01 mol / L PBS buffer at pH 7.4 to a total volume of 4 mL. Then, it was heat shocked at 55 °C for 10 minutes, cooled at room temperature for 30 minutes, and finally, the unbound polypeptide was washed away with a 50 kD ultrafiltration tube, obtaining 12 gp96-polypeptide complexes formed by 4 different mixed ratios of mixed polypeptides combined with different masses of gp96, as shown in Table 3 below.

[0147] Table 3. Polypeptide-gp96 complexes

[0148]

[0149] II. Preparation of human tumor-specific effector cells

[0150] 1. Use human lymphocyte separation medium (purchased from Cellgro, product 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, product number: 12633012) containing 10% fetal bovine serum (purchased from Gibco, product number: 10099-141-FBS), and inoculate into a 24-well plate, 1 mL per well.

[0151] 2. On the next day, the polypeptide-gp96 complex prepared above was added to each group to a final concentration of 10 μg / mL, and the group with only gp96 protein added served as the negative control.

[0152] 3. On the third day, IL-2 (purchased from PeproTech, catalog number: 212-12) was added to each well to a final concentration of 50 U / mL. The medium was changed by half every 2-3 days and IL-2 was supplemented to a final concentration of 50 U / mL.

[0153] 4. On the seventh day and the fourteenth day, the second and third rounds of stimulation with the polypeptide-gp96 complex / gp96 were performed, and IL-2 was added to a final concentration of 50 U / mL on the next day.

[0154] 5. Three days after the third round of stimulation, effector cells CTL were obtained.

[0155] III. Detection of specific killing effect on pancreatic cancer cells

[0156] Target cells used for detection: human pancreatic cancer cell line PANC-1 (positive for HLA-A2 expression), T2 cells (positive for HLA-A2 expression) co-incubated with three polypeptides in a mass ratio of 1:1:1, and the incubated T2 cells presenting the polypeptides.

[0157] Use Non-radioactive cytotoxicity assay (purchased from Promega, catalog number: G1780) was used to detect cytotoxic activity. The main steps are as follows (see the kit instruction manual for details):

[0158] 1. Experimental group: Using PANC-1 cells and T2 cells co-incubated with three polypeptides in a mass ratio of 1:1:1 as target cells, the number of target cells inoculated was 5×10 3 / well, and the above effector cells were added according to the effector-to-target ratio of 5:1, 10:1 or 20:1. The effector cells were inoculated into a 96-well culture plate at 50 μL / well, and the final volume was 100 μL.

[0159] In addition, a group of effector cell spontaneous LDH release was set up to calibrate the LDH spontaneously released by effector cells (each group of effector cells was added to a 96-well plate at 50 μL / well, and supplemented with 50 μL of RPMI-1640 medium containing 5% fetal bovine serum to a final volume of 100 μL). A group of target cell spontaneous LDH release was set up to correct the LDH spontaneously released by target cells (each group of target cells was added to a 96-well plate at 50 μ1 / well, and supplemented with 50 μL of RPMI-1640 medium containing 5% fetal bovine serum to a final concentration of 100 μL). A group of maximum LDH release of target cells was used 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 the lysis solution (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 produced 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).

[0160] 2. After cell inoculation, centrifuge at 250 g for 4 min, and then incubate in a 37 °C incubator for 4 h; 45 min before harvesting the supernatant, add the lysis solution (10×), 10 μL / well, to the group of maximum LDH release of target cells; then centrifuge at 250 g for 4 min and harvest the supernatant.

[0161] 3. Transfer 50 μ1 of the supernatant to an enzyme-linked immunosorbent assay (ELISA) plate, prepare the substrate with the detection buffer, add 50 μ1 / well of the prepared substrate 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 detect the absorbance OD at 490 nm with an ELISA reader within 1 h.

[0162] 4. Calculate the cell killing rate

[0163] 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%

[0164] The experimental results are as follows Figure 2As 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. The CTLs activated after mixing three polypeptides in a mass ratio of 1:1:1 and complexing them 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 CTLs activated had a tumor cell killing efficiency as high as 40% - 60%. In summary, the CTLs activated by mixing the three polypeptides in equal proportions themselves and complexing their mixture with gp96 in equal proportions had the highest tumor cell killing efficiency. 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 proportions themselves, and their mixture was complexed with gp96 in an equal mass ratio (the complex shown as Mix 1-1 in Table 3), and the killing results of the CTLs activated on tumor cells were as Figure 3 shown. The results showed that the CTLs activated by the three polypeptide-gp96 complexes had obvious killing effects on pancreatic cancer cells, while the CTLs activated by gp96 had basically no killing activity as the control group.

[0165] Example 3: Therapeutic effect of polypeptide-gp96 complex-induced specific CTLs on pancreatic cancer

[0166] Twenty 6 - 8-week-old female nude mice were taken, and 10 7 specific T cells (0.5 mL) induced by the polypeptide-gp96 complex were transfused into the tail vein of each mouse. The polypeptide-gp96 complex was a complex formed by mixing three polypeptides in equal proportions and complexing them with gp96 in an equal mass ratio (the complex shown as Mix 1-1 in Table 3); 3 days after the transfusion, 5×10 6 PANC-1 pancreatic 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:

[0167] The first group: Human T lymphocytes activated by the polypeptide-gp96 complex were transfused into the tail vein, immunized three times (0.5 mL each time), and the single transfusion dose was 10 7 cells / mouse;

[0168] 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;

[0169] In the above two groups: They were transfused three times 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 4 , and the results show that CTLs induced by the three polypeptide-gp96 complexes can significantly inhibit the growth of pancreatic cancer tumors.

[0170] Example 4: Killing effect of specific CTLs induced by polypeptide-gp96 complexes on human liver cancer cells

[0171] I. Preparation of polypeptide-gp96 complexes is the same as in Example 2

[0172] II. Preparation of human tumor-specific effector cells is as described in Example 2

[0173] III. Detection of the killing effect on liver cancer cells

[0174] Target cells used for detection: human liver cancer cells HEPG2 (positive for HLA-A2 expression), T2 cells (positive for HLA-A2 expression) co-incubated with the three polypeptides in a mass ratio of 1:1:1, and the incubated T2 cells present the polypeptides.

[0175] The detection method is 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.

[0176] The experimental results are as Figure 5 shown. Under the condition of an effector-to-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 less than 7% killing effect on tumor cells. CTLs activated by the three polypeptides mixed in a mass ratio of 1:1:1 and complexed with gp96 have a tumor-killing efficiency as high as 15% - 60%; moreover, CTLs activated by the polypeptide mixture and gp96 mixed in a mass ratio of 1:1 have a tumor-killing efficiency as high as 40% - 60%. In summary, CTLs activated by the three polypeptides mixed in equal proportions themselves and their mixture complexed with gp96 in equal proportions have the highest tumor-killing efficiency. Among them, under the condition of an effector-to-target ratio of 5:1, 10:1, or 20:1, CTLs activated by the three polypeptides mixed in equal proportions themselves and their mixture complexed with gp96 in equal mass proportions (such as the complexes shown as Mix 1-1 in Table 3) have the killing results of tumor cells as Figure 6 shown, and the results show that CTLs activated by the three polypeptide-gp96 complexes have an obvious killing effect on liver cancer cells, while CTLs activated by gp96 as the control group have basically no killing activity.

[0177] Example 5: Therapeutic effect of specific CTLs induced by polypeptide-gp96 complex on liver cancer model

[0178] Twenty 6-8-week-old female nude mice were taken, and 10 7 specific T cells (0.5 mL) induced by polypeptide-gp96 complex 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 equal mass ratio; 3 days after the transfusion, 5×10 6 HEPG2 liver cancer cells were subcutaneously inoculated into all mice; on the 2nd day after tumor inoculation, the mice were divided into two groups, with 10 mice in each group, and the following treatments were carried out respectively:

[0179] The first group: Human T lymphocytes activated by polypeptide-gp96 complex were transfused into the tail vein, immunized three times (0.5 mL each time), and the single transfusion dose was 10 7 cells / mouse;

[0180] 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;

[0181] In the above two groups: Transfusions were carried out 3 times on the 2nd, 3rd, and 4th days after tumor cell inoculation. 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—long diameter of the tumor, b—short diameter of the tumor). The change in tumor volume is 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.

[0182] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that: according to all the teachings that have been published, various modifications and changes can be made to the details, and these changes are all 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 Reshue Biotechnology Co., Ltd. <120> Epitope peptides of LGALS3 and complexes of the epitope peptides and heat shock proteins <130> IDC210142 <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 9 <212> PRT <213> artificial <220> <223> Amino acid sequence of human-derived LGALS3 polypeptide fragment 1 <400> 1 Met Leu Ile Thr Ile Leu Gly Thr Val 1 5 <210> 2 <211> 10 <212> PRT <213> artificial <220> <223> Amino acid sequence of human-derived LGALS3 polypeptide fragment 2 <400> 2 Val Leu Val Glu Pro Asp His Phe Lys Val 1 5 10 <210> 3 <211> 9 <212> PRT <213> artificial <220> <223> Amino acid sequence of human-derived LGALS3 polypeptide fragment 3 <400> 3 His Leu Leu Gln Tyr Asn His Arg Val 1 5 <210> 4 <211> 25 <212> DNA <213> artificial <220> <223> Sequence of gp96 amplification primer F1 <400> 4 ggaattcatg gacgatgaag ttgat 25 <210> 5 <211> 29 <212> DNA <213> artificial <220> <223> Sequence of gp96 amplification primer R1 <400> 5 gctctagact attagaattc atctttttc 29 <210> 6 <211> 803 <212> PRT <213> artificial <220> <223> Human heat shock protein gp96 amino acid sequence <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 <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 tcattttcaa aggaaagtga tgaccccatg gcttatattc actttactgc tgaaggggaa 1140 gttaccttca aatcaatttt atttgtaccc acatctgctc cacgtggtct gtttgacgaa 1200 gttaccttca aatcaatttt atttgtaccc acatctgctc cacgtggtct gtttgacgaa 1200 tatggatcta aaaagagcga ttacattaag ctctatgtgc gccgtgtatt catcacagac 1260 tatggatcta aaaagagcga ttacattaag ctctatgtgc gccgtgtatt catcacagac 1260 gacttccatg atatgatgcc taaatacctc aattttgtca agggtgtggt ggactcagat 1320 gacttccatg atatgatgcc taaatacctc aattttgtca agggtgtggt ggactcagat 1320 gatctcccct tgaatgtttc ccgcgagact cttcagcaac ataaactgct taaggtgatt 1380 gatctcccct tgaatgtttc ccgcgagact cttcagcaac ataaactgct taaggtgatt 1380 aggaagaagc ttgttcgtaa aacgctggac atgatcaaga agattgctga tgataaatac 1440 aggaagaagc ttgttcgtaa aacgctggac atgatcaaga agattgctga tgataaatac 1440 aatgatactt tttggaaaga atttggtacc aacatcaagc ttggtgtgat tgaagaccac 1500 aatgatactt tttggaaaga atttggtacc aacatcaagc ttggtgtgat tgaagaccac 1500 tcgaatcgaa cacgtcttgc taaacttctt aggttccagt cttctcatca tccaactgac 1560 tcgaatcgaa cacgtcttgc taaacttctt aggttccagt cttctcatca tccaactgac 1560 attactagcc tagaccagta tgtggaaaga atgaaggaaa aacaagacaa aatctacttc 1620 attactagcc tagaccagta tgtggaaaga atgaaggaaa aacaagacaa aatctacttc 1620 atggctgggt ccagcagaaa agaggctgaa tcttctccat ttgttgagcg acttctgaaa 1680 atggctgggt ccagcagaaa agaggctgaa tcttctccat ttgttgagcg acttctgaaa 1680 aagggctatg aagttattta cctcacagaa cctgtggatg aatactgtat tcaggccctt 1740 aagggctatg aagttattta cctcacagaa cctgtggatg aatactgtat tcaggccctt 1740 cccgaatttg atgggaagag gttccagaat gttgccaagg aaggagtgaa gttcgatgaa 1800 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 complex formed by a polypeptide composition and heat shock protein gp96; Wherein, The polypeptide composition comprises any two or three polypeptides selected from the following: 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.

2. The complex according to claim 1, wherein the complex is formed by natural adsorption or heat shock of the polypeptide composition and heat shock protein gp96.

3. The complex according to claim 1, wherein the complex is prepared by mixing the polypeptide composition and heat shock protein gp96 in vitro.

4. The complex according to claim 3, wherein the mixing is carried out at 40 - 80 °C.

5. The complex according to claim 3, wherein the complex is prepared by contacting the polypeptide composition and heat shock protein gp96 and placing them at 55 °C for 10 min, and then placing them at room temperature for 30 min.

6. The complex according to claim 1, wherein the heat shock protein gp96 has the amino acid sequence shown in SEQ ID NO:

6.

7. The complex according to claim 1, wherein the complex is prepared by mixing the polypeptide composition and heat shock protein gp96 in vitro at a mass ratio of 0.1:1 to 100:

1.

8. The complex according to claim 7, Wherein, The mass ratio is 0.1:1 to 50:

1.

9. A composition comprising at least two complexes according to any one of claims 1 - 8.

10. A method for preparing the complex according to any one of claims 1 - 8, which comprises mixing a polypeptide composition and heat shock protein gp96.

11. The method according to claim 10, wherein the polypeptide composition and heat shock protein gp96 form a complex by natural adsorption or heat shock.

12. The method according to claim 10, wherein the mixing is carried out at 40 - 80 °C.

13. The method according to claim 10, the method Comprises: Contacting the polypeptide composition and heat shock protein gp96 and placing them at 55 °C for 10 min, and then placing them at room temperature for 30 min.

14. The method according to claim 10, wherein the heat shock protein gp96 has the amino acid sequence shown in SEQ ID NO:

6.

15. The method according to claim 10, the method Comprises: Mixing the polypeptide composition and heat shock protein gp96 at a mass ratio of 0.1:1 to 100:

1.

16. The method according to claim 15, Wherein, The mass ratio is 0.1:1 to 50:

1.

17. A method for preparing T cells, the method comprises stimulating immune cells with the complex according to any one of claims 1 - 8 or the composition according to claim 9; Wherein, The T cells express a T cell receptor that can specifically recognize a polypeptide composition containing any two or three polypeptides selected from the following: 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.

18. The method according to claim 17, wherein the immune cells comprise PBMC or T lymphocytes.

19. The method according to claim 17, wherein the stimulation further comprises using an immunostimulatory cytokine.

20. A combination of isolated nucleic acid molecules, which comprises a nucleotide sequence encoding the complex according to any one of claims 1-8.

21. A vector, which comprises the combination of isolated nucleic acid molecules according to claim 20.

22. The vector according to claim 21, wherein the vector is a viral vector.

23. The vector according to claim 21, wherein the vector is a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector or a baculoviral vector.

24. A host cell, which comprises the combination of isolated nucleic acid molecules according to claim 20 or the vector according to any one of claims 21-23.

25. A pharmaceutical composition, which comprises the complex according to any one of claims 1-8, the composition according to claim 9, the combination of isolated nucleic acid molecules according to claim 20, the vector according to any one of claims 21-23 or the host cell according to claim 24, and a pharmaceutically acceptable excipient.

26. The pharmaceutical composition according to claim 25, wherein the pharmaceutical composition is a tumor vaccine.

27. The pharmaceutical composition according to claim 25, wherein the pharmaceutical composition comprises an adjuvant.

28. The pharmaceutical composition according to claim 25, wherein the pharmaceutical composition further comprises an additional therapeutic agent.

29. The pharmaceutical composition according to claim 28, wherein the additional therapeutic agent is selected from alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, radiosensitizers, antiangiogenic agents, cytokines, immune checkpoint inhibitors.

30. The pharmaceutical composition according to claim 29, wherein, the immune checkpoint inhibitor is selected from a PD-1 antibody, a PD-L1 antibody, a CTLA-4 antibody, a LAG-3 antibody or a TIM3 antibody.

31. Use of the complex according to any one of claims 1-8, or the composition according to claim 9, in the preparation of a medicament for inducing an immune response against pancreatic cancer and / or liver cancer in a subject and / or for preventing or treating pancreatic cancer and / or liver cancer in a subject.

32. The use according to claim 31, wherein the subject is a human.

33. The use according to claim 31, wherein the subject is HLA-A2 positive.

34. The use according to claim 31, wherein the composition is administered in combination with an additional therapeutic agent.

35. The use according to claim 34, wherein the additional therapeutic agent is an immunostimulant or an antitumor agent.

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