CN115490764A

CN115490764A - Epitope peptide of LGALS3 and complex of epitope peptide and heat shock protein

Info

Publication number: CN115490764A
Application number: CN202110677901.4A
Authority: CN
Inventors: 张含; 鞠莹; 王力; 杨博; 陈立钊
Original assignee: Foshan Rexiu Biotechnology Co ltd
Current assignee: Foshan Rexiu Biotechnology Co ltd
Priority date: 2021-06-18
Filing date: 2021-06-18
Publication date: 2022-12-20

Abstract

The present invention relates to the fields of immunology and tumor therapy. In particular, 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 tumors. The invention also relates to a complex formed by the epitope peptide and the heat shock protein, a specific immune cell induced by the complex, a tumor vaccine containing the complex and the specific immune cell, and application of the complex and the tumor vaccine in preventing or treating tumors.

Description

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

Technical Field

Background

Liver cancer refers to malignant tumors occurring in the liver, including primary liver cancer and metastatic liver cancer, wherein primary liver cancer is one of the most common clinical malignant tumors. Among male cancer patients worldwide, liver cancer ranks sixth in proportion and mortality ranks second; among female cancer patients, liver cancer ranks seventh in proportion and mortality ranks sixth. In 2008, there were 748,300 new cases of liver cancer worldwide, and 695,900 patients with liver cancer died. Half of these new cases of liver cancer and death are in China. The highest incidence of liver cancer is mainly found in east asia, southeast asia, middle africa and west african countries. The higher incidence of liver cancer in parts of Asia and sub-Saharan areas of Africa may be due to the prevalence of HBV in these areas, with 8% of the residents in these areas chronically infected with HBV and 60% of liver cancer patients in developing countries infected with HBV.

Pancreatic cancer is one of the most malignant tumors of the digestive tract, and its incidence and mortality rate worldwide are increasing year by year. At present, the clinical treatment modes of pancreatic cancer mainly comprise surgical excision and chemoradiotherapy. As the pancreatic cancer is hidden, the disease progresses rapidly, the malignancy is high and the like, once a patient finds that the pancreatic cancer is mostly in the middle and late stage of the cancer, the operation chance is lost, and the 5-year survival rate of the patient is less than 6 percent.

Galectin 3 (Galectin-3, lgasl3) belongs to the lectin family, the members of which have a strong affinity for galactose. The protein has a proline-rich tandem repeat domain at the N-terminal and a C-terminal carbohydrate recognition domain. The protein can self-bind through the N-terminal domain, thereby binding to multivalent sugar ligands. This protein is localized to the extracellular matrix, cytoplasm and nucleus. The protein plays a role in a number of cellular functions, including apoptosis, innate immunity, cell adhesion and T cell regulation. Meanwhile, more and more researches show that LGALS3 plays a key role in the occurrence and development of tumors and promotes tumor metastasis. In high-incidence cancers such as liver cancer, brain glioma, pancreatic cancer and the like, the expression of the LGALS3 protein is obviously improved, and meanwhile, the expression level of the LGALS3 is directly related to the survival rate of cancer patients.

Heat Shock Proteins (HSPs) are a class of proteins that are highly conserved in biological evolution and widely occur in prokaryotes and eukaryotes. HSP can be divided into multiple subfamilies such as HSP110, HSP90, HSP70, HSP60, HSP40, small molecule HSP and ubiquitin according to the homology degree and molecular weight. Heat Shock Protein (HSP) gp96 belongs to a member of the HSP90 subfamily, and is the most abundant Heat shock protein on the endoplasmic reticulum. The heat shock protein gp96 protein has polypeptide binding properties, and can accept polypeptide fragments from TAP complex in endoplasmic reticulum to facilitate the assembly of the protein into MHC class I molecules for presentation on cell membranes. The heat shock protein gp96 derived from different tissues can carry polypeptide fragments specifically expressed in the tissues from which the protein is derived.

Disclosure of Invention

The invention provides epitope peptides of the protein LGALS3 and a combination thereof, and specific immune cells induced by the epitope peptides. The invention further provides a complex formed by the epitope peptide and the heat shock protein, and specific immune cells induced by the complex.

Accordingly, in one aspect, the present invention provides an isolated polypeptide having the amino acid sequence as set forth in SEQ ID NO:1 or 2.

In another aspect, the present invention provides a polypeptide composition comprising any two or three polypeptides selected from the group consisting of: SEQ ID NO: 1. 2 and 3.

In certain embodiments, the polypeptide compositions contain the same or different proportions of polypeptides.

In certain embodiments, the polypeptide composition comprises SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO: 3.

In certain embodiments, the polypeptide composition comprises SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO: the mass ratio of the polypeptide shown in 3 is 1.

In certain embodiments, the polypeptide composition comprises SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, the mass ratio of the polypeptide is 1.

In certain embodiments, the polypeptide composition comprises SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 is 1.

In another aspect, the invention provides a complex formed by an isolated polypeptide as described above or a composition of polypeptides as described above and a heat shock protein.

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

In certain embodiments, the complex is prepared by combining 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 performed at 40-80 ℃ (e.g., 40-70 ℃,40-60 ℃, or 50-60 ℃). In certain embodiments, the mixing is performed at 55 ℃.

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 ℃ for 10min, followed by incubation at room temperature for 30min.

In certain embodiments, the heat shock protein is selected from the group consisting of HSP70, HSP90, gp96, HSP110, and mutants or fusion proteins thereof.

In certain embodiments, the heat shock protein is gp96. In certain embodiments, the heat shock protein has the amino acid sequence as set forth in SEQ ID NO: 6.

In certain embodiments, the complex is prepared by mixing the isolated polypeptide as described above or the polypeptide composition as described above with a heat shock protein in vitro in the following mass ratios: 1 to 100 (e.g. 0.1 to 50.

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

In another aspect, the invention provides a composition comprising at least 2 (e.g., 2,3,4, 5, or more) complexes as described above.

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

In certain embodiments, the composition is immunogenic and capable of inducing a specific immune response against a cell expressing LGALS3 (e.g., a tumor cell, such as a pancreatic cancer or liver cancer cell).

In another aspect, the present invention provides a method of preparing a complex as described above comprising mixing an isolated polypeptide as described above or a composition of polypeptides as described above with a heat shock protein.

In certain embodiments, the isolated polypeptide as described above or the polypeptide composition as described above is complexed with a heat shock protein in a natural adsorption or heat shock manner.

In certain embodiments, the method comprises: the isolated polypeptide or the polypeptide composition as described above is contacted with heat shock protein and left at 55 deg.C for 10min, and then left at room temperature for 30min.

In certain embodiments, the method comprises: mixing the isolated polypeptide or the polypeptide composition with heat shock protein in vitro according to the following mass ratio: 1 to 100 (e.g. 0.1 to 50.

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

In another aspect, the invention provides a T cell expressing a T cell receptor capable of specifically recognizing galectin 3 (LGALS 3) or a polypeptide isolated as described above or a polypeptide composition as described above.

In certain embodiments, the T cell is obtained from stimulating an immune cell with a complex as described above or a composition as described above. In certain embodiments, the immune cells comprise PBMCs or T lymphocytes.

In certain embodiments, the stimulation further comprises the use of an immunostimulatory cytokine (e.g., IL-2).

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

In certain embodiments, the T cell is capable of specifically recognizing and killing a cell that expresses LGALS3 (e.g., a tumor cell, such as a pancreatic cancer or liver cancer cell).

In another aspect, the invention provides a method of preparing a T cell as hereinbefore described, the method comprising stimulating an immune cell using a complex as hereinbefore described or a composition as hereinbefore described. In certain embodiments, the immune cells comprise PBMCs or T lymphocytes.

In certain embodiments, the stimulation further comprises the use of an immunostimulatory cytokine (e.g., IL-2). In certain embodiments, the immunostimulatory cytokine is IL-2.

In another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an isolated polypeptide as described above. In certain embodiments, the isolated polypeptide has the amino acid sequence as set forth in SEQ ID NO:1 or 2.

In another aspect, the invention provides a combination of isolated nucleic acid molecules comprising a nucleotide sequence encoding a polypeptide composition as described above; the polypeptide composition comprises any two or three polypeptides selected from the group consisting of: SEQ ID NO: 1. 2 and 3. In certain embodiments, the polypeptide composition comprises SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO: 3.

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

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

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 baculovirus vector.

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

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

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

In certain embodiments, the pharmaceutical composition comprises an adjuvant.

In certain embodiments, the pharmaceutical composition further comprises an additional therapeutic agent, such as an anti-tumor agent or an immunopotentiator.

In certain embodiments, the antineoplastic agent is selected from the group consisting of an alkylating agent, a mitotic inhibitor, an anti-tumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an anti-angiogenic agent, a cytokine, an immune checkpoint inhibitor (e.g., a PD-1 antibody, a PD-L1 antibody, a CTLA-4 antibody, a LAG-3 antibody, or a TIM3 antibody).

In certain embodiments, the immunopotentiator is selected from an immunostimulatory antibody (e.g., an anti-CD 3 antibody, an anti-CD 28 antibody, an anti-CD 40L (CD 154) antibody, an anti-41 BB (CD 137) antibody, an anti-OX 40 antibody, an anti-GITR antibody, or any combination thereof) or an immunostimulatory cytokine (e.g., IL-2).

In another aspect, the invention provides the use of an isolated polypeptide as described above, or a polypeptide composition as described above or a complex as described above, or a composition as described above or a T cell as described above, in the manufacture of a medicament for inducing an immune response against a tumor expressing LGALS3 in a subject, and/or for preventing or treating a tumor expressing LGALS3 in a subject.

In certain embodiments, the tumor expressing LGALS3 is 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 T cell is from the subject.

In certain embodiments, the subject is a human.

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

In certain embodiments, the isolated polypeptide, polypeptide composition, or T cell is administered in combination with an additional therapeutic agent, e.g., simultaneously, separately, or sequentially. In certain embodiments, the additional therapeutic agent is an immunostimulant or an antineoplastic agent.

In certain embodiments, the LGALS3 protein expression is aberrant in the LGALS 3-expressing tumor (e.g., expression is increased as compared to a healthy subject).

In another aspect, the present invention provides a method for inducing an immune response against a tumor expressing LGALS3 in a subject, and/or preventing or treating a tumor expressing LGALS3 in a subject, said method comprising administering to a subject in need thereof an effective amount of an isolated polypeptide as described previously, a polypeptide composition as described previously, a complex as described previously, a composition as described previously, a T cell as described previously, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding said isolated polypeptide or polypeptide composition, or a pharmaceutical composition as described previously.

In certain embodiments, the tumor expressing LGALS3 is 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, and in certain embodiments, the subject is a human.

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

In certain embodiments, the method further comprises administering to the subject an additional therapeutic agent, such as an immunopotentiator or an antineoplastic agent.

In certain embodiments, the method comprises: (1) Providing immune cells (e.g., PBMCs or T cells) from a subject; (2) Contacting the complex or composition with said immune cells to obtain T cells as previously described; (3) Administering the T cells obtained in step (2) to the subject.

Definition of terms

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, virological, biochemical, immunological laboratory procedures used herein are all routine procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

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 cell activities such as proliferation, apoptosis, adhesion and migration of cells, but also involved 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.

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 homodimeric domain). gp96 is well known to those skilled in the art, and its sequence can be found in various public databases, such as NCBI GENBANK database accession numbers: AAH66656.1.

As used herein, the term "HSP70" is a heat shock protein with a molecular weight of about 70kD, is an important member of a group of heat shock protein families, called major heat shock proteins, and includes more than 20 proteins with molecular weights of 68, 72, 73, 75, 78kDa, and the like. The HSP70 family proteins have similar molecular weight, isoelectric points of pH5.2-6.3 and similar trypsin peptide spectrum, are highly induced in stress cells of almost all organisms, and have the function of protecting organisms and cells.

As used herein, the term "HSP90" is a heat shock protein having a molecular weight of about 90kD, a protein used for synergistic immunization. It is an important member of the group in the heat shock protein family and has a molecular weight of about 83-90kDa.

As used herein, the term "HSP110" is a heat shock protein with a molecular weight of about 110kD, an important member of the group of the family of heat shock proteins, and has a molecular weight of about 100-110kDa.

As used herein, the term "isolated" or "isolated" refers to a substance obtained from a natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be altered from its natural environment, or it may be isolated from its natural environment, or both. For example, a polynucleotide or polypeptide that is not isolated naturally occurs in a living animal, and a polynucleotide or polypeptide that is the same in high purity and that is isolated from such a natural state is said to be isolated. The term "isolated" or "isolated" does not exclude the presence of substances mixed artificially or synthetically or other impurities which do not affect the activity of the substance.

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), bacterial Artificial Chromosomes (BACs), or artificial chromosomes of P1 origin (PACs); bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), pox viruses, baculoviruses, papilloma viruses, papova viruses (e.g., SV 40). A vector may 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 may contain a replication initiation site.

As used herein, the term "host cell" refers to a cell which can be used for introducing a vector, and includes, but is 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 fibroblasts, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, HEK 293 cells or human cells.

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., human) has an autoimmune disease.

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 the immune response, which often 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 immune attack on (e.g., killing, or inhibits growth or proliferation of) a target cell. For example, the effector function of a T cell may be, for example, cytolytic activity or activity that aids or includes 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.

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 active ingredient, which is well known in the art (see, e.g., remington's Pharmaceutical sciences. Edited by geno AR,19th ed. Pennsylvania: pH adjusting agents, surfactants, ionic strength enhancers, agents to maintain osmotic pressure, agents to delay absorption, diluents, adjuvants, preservatives, stabilizers and the like. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Agents that maintain osmotic pressure include, but are not limited to, sugars, naCl, and the like. Agents that delay absorption include, but are not limited to, monostearate salts and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols and polyols (e.g., glycerol), and the like. Adjuvants include, but are not limited to, aluminum adjuvants (e.g., aluminum hydroxide), freund's adjuvants (e.g., complete freund's adjuvant), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, for example, thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meaning generally understood by those skilled in the art to be capable of stabilizing the desired activity of the active ingredient in the medicament (e.g., inhibitory activity against PSD-95 ubiquitination), including but not limited to sodium glutamate, gelatin, SPGA, sugars (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin, or casein) or degradation products thereof (e.g., lactalbumin hydrolysate), and the like.

As used herein, the term "treating" refers to treating or curing a disease (e.g., an autoimmune disease), delaying the onset of one or more symptoms of a disease, and/or delaying the progression of a disease.

As used herein, the term "effective amount" refers to an amount effective to achieve the intended purpose. For example, a therapeutically effective amount can be an amount effective or sufficient to treat or cure a disease (e.g., an autoimmune disease), delay the onset of one or more symptoms of a disease, and/or delay the progression of a disease. Such an effective amount can be readily determined by one of skill in the art or a physician, and can be related to the intended purpose, the general health of the subject, the age, sex, weight, severity of the disease to be treated, complications, mode of administration, and the like. Determination of such an effective amount is well within the ability of those skilled in the art.

Advantageous effects

Complexes of the three polypeptides of the protein LGALS of the present invention or combinations thereof with heat shock proteins are immunogenic and are capable of inducing a specific immune response against cells expressing LGALS3 (e.g., 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, so that tumors expressing LGALS3 can be prevented or treated. And, the three polypeptides of the protein LGALS3 and the combination thereof can be used for preparing a tumor vaccine, and the vaccine can be used for preventing or treating tumors expressing LGALS 3.

Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention and do not limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments.

Drawings

FIG. 1 shows the results of SDS-PAGE and Western Blot for insect-expressed gp96 in example 1 of the present invention.

FIG. 2 shows the results of killing pancreatic cancer cells by activated specific CTL after complexing of three polypeptides and mixed polypeptides in different ratios with gp96 in example 2 of the present invention.

FIG. 3 shows the killing results of activated specific CTL and control (gp 96-stimulated human T cells) on PANC-1 cells and T2 cells incubated with the three polypeptides after the three polypeptides are mixed in equal proportion in example 2 of the invention.

FIG. 4 is a graph showing the effect of specific T cell induced by the back-infusion of polypeptide-gp 96 complex and a control group (gp 96-stimulated human T cells) on pancreatic cancer tumor volume in example 3 of the present invention.

FIG. 5 shows the killing effect of the activated specific CTL against human hepatoma cells after the three polypeptides and the mixed polypeptide of different proportions are compounded with gp96 in example 4 of the present invention.

FIG. 6 shows the killing results of the activated specific CTL and the control group (gp 96-stimulated human T cells) on human hepatoma cells and T2 cells incubated with the three polypeptides after the three polypeptides are mixed in equal proportion in example 4 of the present invention.

FIG. 7 is a graph showing the effect of specific T cells induced by the back-infusion of polypeptide-gp 96 complex and the control group (gp 96-stimulated human T cells) on tumor volume in hepatoma in example 5 of the present invention.

Sequence information

Table 1: information on the sequences to which the present application relates is described in the following table.

Detailed Description

The invention will now be described with reference to the following examples which are intended to illustrate the invention, but not to limit it.

It will be appreciated by those skilled in the art that the examples illustrate the invention and are not intended to limit the scope of the invention as claimed. The experimental procedures in the examples are conventional unless otherwise specified. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are commercially available, and are not indicated by manufacturers. In the quantitative experiments in the examples, three repeated experiments are set, and the results are averaged.

The experimental material sources referred to in the following examples are as follows:

the nude mouse is a product of Beijing Weitonglihua laboratory animals, limited liability company; in the examples, the term "mouse" is used for short. The polypeptide was synthesized by Shanghai Jier Biochemical Co., ltd.

HepG2 cells (human hepatoma cells) were purchased from ATCC (American type culture Collection) under the catalog number HB-8065 ^TM 。

PANC-1 (human pancreatic cancer cell) was purchased from the basic medicine cell center of the institute of basic medicine of Chinese academy of medical sciences, and the resource number of the cell strain was 3111C0001CCC000023.

Sf9 cells are Invitrogen, catalog number 11496-015.

Cellfect II reagent is a product of Life technologies, inc. under catalog number 10362-100.

Plasmid pFastBac ^TM Invitrogen corporation, catalog number 10359-016.

The gp96 monoclonal antibody is a product of Santa Cruz company, and the catalog number of the product is sc-56990.

The goat anti-rat monoclonal antibody marked by horseradish peroxidase is a product of Beijing Zhonghua Jinqiao biotechnology limited company, and the catalog number of the product is ZB-2307.

HiTrap-Q Sepharose ion exchange column is product of GE company, and has catalog number of 17-5053-01.

Superdex 200/300 GL molecular sieve chromatographic column is a product of GE company, and the catalog number is 17517501.

The escherichia coli DH10Bac competent cell is a product of Beijing original Hao Biotechnology Limited, and the catalog number of the product is CL108-01.

The extract-XPRESSTM Protein-free insert Cells with L-Glutamine is a product of LONZA corporation, and the product catalog number is 12-730Q.

BSA、PMSF、NaHCO ₃ 、MnCl ₂ 、CaCl ₂ 、NaCl ₂ Tris, methyl alpha-D-mannopyranoside are Sigma-Aldrich company products, and the catalog numbers are V900933, P7626, 792519, V900197, 793639, 746398, T1378 and M6882 respectively.

Solution A: the solute was 1mM PMSF and 30mM NaHCO ₃ (ii) a The solvent is distilled water; the pH was 7.4.

Solution B: solute is 2mM MnCl ₂ 、2mM CaCl ₂ 500mM NaCl and PMSF 1mM; the solvent was 20mM Tris-HCl buffer, pH 7.4.

Solution C: the solute is 10% (mass volume ratio) methyl alpha-D-mannopyranoside, 500mM NaCl and 1mM PMSF; the solvent was Tris-HCl buffer, 20mM, pH 7.4.

Cleaning solution: solution B was diluted to 10 volumes with distilled water.

The ConA Sepharose column is a product of GE corporation, catalog number is 17-0440-01, the specification of the column is 1.6X 2.5cm, and the packing medium is Con A-Sepharose 4B.

The Hitrap Q anion exchange column is available from GE under catalog number 17-1153-01, and the column size is 0.7X 2.5cm.

The HRP-labeled IgG antibody is available from SEROTEC corporation under catalog number STAR117P.

1 × washing solution containing 0.1% (volume percent) Triton-X100, pH 7.4, 0.01mol/L PBS buffer.

50kD and 3kD ultrafiltration tubes are made by Merck Millipore company, and the catalog numbers are UFC905096 and UFC500324 respectively.

Example 1: expression of recombinant gp96 protein by insect cells

1. Recombinant plasmid pFastBac ^TM Construction of 1-gp96

1. The RNA of HepG2 cells is extracted by a Trizol method, and then reverse transcription is carried out to obtain cDNA.

2. Primers F1 (SEQ ID NO: 4) and R1 (SEQ ID NO: 5) carrying the recognition sequences of the restriction enzymes EcoRI and XbaI, respectively, were chemically synthesized based on the nucleotide sequence of the human gp96 gene (GenBank accession No. AY 040226.1).

3. After

steps

1 and 2 are completed, PCR amplification is carried out by taking the cDNA obtained in step 1 as a template and the F1 and R1 synthesized in step 2 as primers to obtain a PCR amplification product which contains a nucleotide sequence coding heat shock protein gp96.

4. The PCR amplification product is double digested with restriction enzymes EcoRI and XbaI, and the digested product is recovered.

5. The plasmid pFastBac was digested with the restriction enzymes EcoRI and Xba I ^TM 1, recovering a vector backbone of about 4700 bp.

6. And connecting the enzyme digestion product with a carrier skeleton to obtain a connection product.

7. And (3) transforming the connecting product obtained in the step (6) into an escherichia coli DH10Bac competent cell to obtain a recombinant escherichia coli, and then extracting the plasmid of the recombinant escherichia coli to obtain a recombinant plasmid pFastBac1-gp96. The recombinant plasmid pFastBac1-gp96 expresses recombinant heat shock protein gp96 (hereinafter referred to as rgp 96), and the amino acid sequence of the rgp96 is shown as SEQ ID NO: 6.

2. Expression of rgp96

1. The recombinant plasmid pFastBac1-gp96 constructed in the step one is co-transfected into Sf9 cells (every 1 × 10) ⁶ About 4. Mu.g of recombinant plasmid pFastBac1-gp96 was transfected into each Sf9 cell; in the co-transfection process, the transfection reagent is Cellffectin II reagent, the culture medium is an insert-XPRESSTM Protein-free insert Cells with L-Glutamine, incubation is carried out for 72h at 27 ℃, centrifugation is carried out, and the supernatant is the P1 generation virus.

2. Sf9 cell suspension 1 (containing 1X 10) ⁸ Sf9 cells) are cultured for 8 to 10 hours at the temperature of 27 ℃ to obtain cultured cells; then adding P1 generation virus (the dose is 0.05-0.1 MOI) into the cultured cells, incubating for 72h at 27 ℃,centrifuging at 4000rpm for 5min to obtain supernatant as P2 virus.

3. To Sf9 cell suspension 2 (containing 1.6X 10) ⁸ Sf9 cells) are added with P2 generation virus (the dosage is 0.05-0.1 MOI), cultured for 72h at the temperature of 27 ℃ and at the rpm of 100-120, centrifuged for 5min at the rpm of 4000, and the supernatant is the P3 generation virus.

Taking an anti-gp 96 monoclonal antibody as a primary antibody, taking a goat anti-rat monoclonal antibody marked by horseradish peroxidase as a secondary antibody, and carrying out SDS-PAGE and Western blot hybridization on the P3 virus, wherein the Western blot hybridization comprises the following specific steps: zhangyuejing, chuanqiang, rodder inflammation, yaohuan, wanghelian, lissenqiu-mouse soluble IL-5 α receptor expression in Bac-to-Bac system and its identification [ J ]. Journal of chinese biologies, 2013, 26:5.SDS-PAGE and Western Blot hybridization results showed that rgp96 protein was successfully expressed in Sf9 cells.

3. Purification of rgp96 protein

1. To 300ml of Sf9 cell suspension 3 (containing 4.5X 10) ⁸ Sf9 cells) were added with P3 generation virus (5 MOI), cultured at 27 ℃ for 72 hours at 100-120 rpm to obtain a suspension.

2. The suspension was centrifuged at 7000rpm for 20min to obtain supernatant 1.

3. And taking the supernatant fluid 1, and filtering the supernatant fluid through a filter membrane of 0.22mm to obtain a supernatant fluid.

4. Loading the sample solution on a HiTrap-Q Sepharose ion exchange chromatography column (flow rate is 1 mL/min), and then washing with 5mL of PBS buffer solution (flow rate is 1 mL/min) with pH 7.5 and 200 mM; then washed with 10mL of 300mM PBS buffer (flow rate 1 mL/min) at pH 7.5; and finally, washing the column with 3mL of PBS buffer solution with the pH value of 7.5 and the concentration of 600mM (the flow rate is 1 mL/min), collecting the solution after passing through the column, and performing ultrafiltration concentration by using an ultrafiltration tube with the molecular weight cutoff of 50KD to obtain about 1mL of concentrated solution. The concentrate contains rgp96.

5. And (3) loading the concentrated solution obtained in the step (4) on a Superdex 200/300 GL molecular sieve chromatographic column (flow rate is 0.25 mL/min), washing with PBS buffer solution with pH 7.5 and 150mM (flow rate is 0.25 mL/min), collecting the 9-12 mL penetration solution, and further performing ultrafiltration concentration by adopting an ultrafiltration tube with the molecular weight cutoff of 50KD to obtain an rgp96 solution. The protein concentration in the solution of rgp96 was determined by BCA method, finally dispensed and stored at-80 ℃.

The solution of rgp96 obtained in step 5 was subjected to SDS-PAGE electrophoretic analysis and Western blot hybridization (anti-gp 96 monoclonal antibody as the primary antibody, horseradish peroxidase-labeled goat anti-rat monoclonal antibody as the secondary antibody). The results of the experiment are shown in FIG. 1, wherein lane 1 is a high molecular weight standard protein Marker, lane 2 is the result of SDS-PAGE of rgp96, and lane 3 is the result of Western blot hybridization of rgp96. The results in fig. 1 show that the solution of rgp96 shows a single molecular weight band (as indicated by the arrow) and the corresponding molecular weight is consistent with that expected, i.e. rgp96 is efficiently expressed and purified by the steps in step three above.

Example 2: killing effect of specific CTL (cytotoxic T lymphocyte) induced by polypeptide-gp 96 complex on human pancreatic cancer cells

1. Preparation of polypeptide-gp 96 complexes

1-3, preparing each polypeptide into polypeptide solution with the concentration of 20mg/mL by DMSO, taking 1mg of each polypeptide, mixing with 1mg, 0.1mg and 0.01mg of heat shock protein gp96 respectively, dissolving into 4mL of total volume by PBS buffer solution with the pH value of 7.4.01mol/L, then heat shock for 10 minutes at 55 ℃, cooling for 30 minutes at room temperature, and finally washing off the unbound polypeptides by a 50kD ultrafiltration tube to obtain gp 96-polypeptide complexes formed by compounding each polypeptide with gp96 with different mass respectively, wherein the total amount of the gp 96-polypeptide complexes is 9, and the specific formula is shown in the following table 2.

TABLE 2 polypeptide and gp96 complexes

Compound name	Polypeptides 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

1-3 of the 3 polypeptides shown in SEQ ID NO according to 4 different mass ratios (specific ratios are shown in the following table 3), DMSO is used to prepare a polypeptide mixed solution with a concentration of 20mg/mL, for each mixed polypeptide solution, the mixed solution containing 1mg of the polypeptides is taken, each mixed polypeptide solution is respectively mixed with 1mg, 0.1mg and 0.01mg of heat shock protein gp96, the mixed solution is dissolved to a total volume of 4mL by PBS buffer solution with a pH value of 7.4.0.01mol/L, then the mixed solution is subjected to heat shock at 55 ℃ for 10 minutes, cooled at room temperature for 30 minutes, and finally a 50kD ultrafiltration tube is used to wash out unbound polypeptides, so that gp 96-polypeptide complexes formed by respectively compounding 4 mixed polypeptides with gp96 with different masses in different mixing ratios are obtained, and 12 kinds of the mixed polypeptides are specifically shown in the following table 3.

TABLE 3 polypeptide and gp96 complexes

2. Preparation of human tumor specific effector cells

1. Anticoagulated fresh whole blood of HLA-A2-positive volunteers was separated using human lymphocyte separation medium (purchased from Cellgro, cat # 25-072-CI) to obtain Peripheral Blood Mononuclear Cells (PBMC), and the cell concentration was adjusted to 1.0X 10 by using RPMI-1640 complete medium (purchased from Gibco, cat # 12633012) containing 10% fetal bovine serum (purchased from Gibco, cat # 10099-141-FBS) ⁶ one/mL, seeded in 24-well plates, 1mL per well.

2. The next day, the polypeptide-gp 96 complex prepared above was added to each group to a final concentration of 10. Mu.g/mL, and the gp96 protein group was added alone as a negative control.

3. IL-2 (from PeproTech, cat: 212-12) was added to a final concentration of 50U/mL on the third day, and half the solution was changed and IL-2 was supplemented to a final concentration of 50U/mL every 2-3 days.

4. The second and third rounds of polypeptide-gp 96 complex/gp 96 stimulation were performed on the seventh and fourteenth days, respectively, and IL-2 was added the following day to a final concentration of 50U/ml.

5. 3 days after the third round of stimulation, effector cells CTL were obtained.

3. Detection of specific killing effect of pancreatic cancer cells

Detection of the target cells used: human pancreatic cancer cell line PANC-1 (HLA-A2 expression positive), T2 cells incubated with three polypeptides together (HLA-A2 expression positive) at a mass ratio of 1.

Use of

Non-radioactive cytotoxicity assay (from Promega, catalog # G1780) cytotoxic activity assay was performed using the following main steps (details see kit instructions):

1. experimental groups: the target cells are T2 cells which are incubated together by PANC-1 cells and three polypeptides according to the mass ratio of 1 ³ The above effector cells were added to the wells at a ratio of effective to target of 5, 1, 10 or 20.

An effector cell spontaneous LDH release group was additionally provided to calibrate the spontaneous LDH release from the effector cells (each group of effector cells was added to a 96-well plate at 50. Mu.L/well and supplemented with 50. Mu.L of RPMI-1640 medium containing 5% fetal bovine serum to a final volume of 100. Mu.L). Target cells spontaneous LDH release group to correct the spontaneous LDH release of the target cells (each group of target cells was added to a 96-well plate at 50 μ 1/well, and 50 μ L RPMI-1640 medium containing 5% fetal bovine serum was supplemented to a final concentration of 100 μ L). The maximum LDH release group of target cells was used as a reference to determine 100% LDH release when calculated (cells were loaded with the target cell spontaneous release group). Volume correction control group, used to correct volume change due to adding lysis solution (adding 5% fetal bovine serum RPMI-1640 medium 100 u L). A control group of media background was used to correct LDH activity produced by serum in the media and background absorption by phenol red (100. Mu.L of RPMI-1640 medium containing 5% fetal bovine serum was added).

2. After cell inoculation, centrifuging for 4min at 250g, and then incubating for 4h in an incubator at 37 ℃; lysis buffer (10 x), 10 μ L/well, was added to the maximal LDH release group of target cells 45min before harvesting the supernatant; the supernatant was then harvested by centrifugation at 250g for 4 min.

3. Transferring 50 mu 1 of supernatant into an ELISA plate, preparing a substrate by using a detection buffer solution, adding 50 mu 1/hole of the prepared substrate into the ELISA plate, covering the plate, reacting for 30min at room temperature in a dark place, adding 50 mu L of stop solution into each hole, and detecting the light absorption value OD at 490nm in an ELISA instrument within 1 h.

4. Calculation of cell killing Rate

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

As shown in fig. 2, under the condition of an effective target ratio of 20. The killing efficiency of activated CTL to tumor cells is as high as 15% -60% after the three polypeptides are mixed according to the mass ratio of 1; and the killing efficiency of the activated CTL to the tumor cells is as high as 40-60% when the polypeptide mixture is mixed with gp96 according to the mass ratio of 1. In conclusion, the CTL activated by mixing the three polypeptides in equal proportion and compounding the mixture with gp96 in equal proportion has the highest killing efficiency on tumor cells. Under the condition that the effective target ratio is 5, 10 or 20.

Example 3: therapeutic effect of specific CTL induced by polypeptide-gp 96 complex on pancreatic cancer

20 female nude mice of 6-8 weeks old are taken, and 10 are back-transfused into each tail vein ⁷ Specific T cells (0.5 mL) induced by the polypeptide-gp 96 complex, wherein the polypeptide-gp 96 complex is a complex formed by mixing three polypeptides in equal proportion and compounding the three polypeptides with gp96 in equal mass proportion (a complex shown as mixed 1-1 in Table 3); 3 days after the reinfusion, all mice were inoculated subcutaneously with 5X 10 injections, respectively ⁶ PANC-1 pancreatic cancer cells; mice were divided into two groups of 10 mice on day 2 post tumor inoculation, and treated as follows:

a first group: the tail vein is used for back transfusion of polypeptide-gp 96 complex activated human T lymphocytes, immunization is carried out three times (0.5 mL each time), and the single back transfusion dosage is 10 ⁷ One/only;

second group: returning gp96 stimulated human T cells via tail vein, immunizing three times (0.5 mL each time), and using single immunization dose as single return dose of 10 ⁷ One/only;

in the above two groups: 3 times of reinfusion was carried out 2,3,4 days after tumor cell inoculation. Starting from the first day of reinfusion, observing the growth condition of the tumor every day, recording the size of the tumor, and calculating the volume of the tumor according to the following formula: v = ab ² V-volume, a-tumor major diameter, b-tumor minor diameter. The change of tumor volume is shown in FIG. 4, and the results show that CTL induced by the three polypeptide-gp 96 complexes can obviously inhibit the growth of pancreatic cancer tumors.

Example 4: killing effect of specific CTL induced by polypeptide-gp 96 complex on human liver cancer cells

1. Preparation of polypeptide-gp 96 Complex As in example 2

2. Preparation of human tumor-specific effector cells see example 2

3. Detection of specific killing effect of liver cancer cells

Detection of the target cells used: human hepatoma cells HEPG2 (HLA-A2 expression positive), T2 cells (HLA-A2 expression positive) which are co-incubated according to the mass ratio of 1.

The detection method is the same as that in example 2, the experimental group is added with effector cells and target cells according to the proportion of 5, 10 or 20. And incubating for 4h at 37 ℃, adding cell lysate, and harvesting supernatant for LDH detection.

As shown in fig. 5, under the condition of effective target ratio of 20. The killing efficiency of activated CTL on tumor cells is as high as 15% -60% after the three polypeptides are mixed according to the mass ratio of 1; and the killing efficiency of the activated CTL to the tumor cells is as high as 40-60% when the polypeptide mixture is mixed with gp96 according to the mass ratio of 1. In conclusion, the CTL activated by the three polypeptides which are mixed in equal proportion and the mixture of the three polypeptides and gp96 which are compounded in equal proportion has the highest killing efficiency on tumor cells. Wherein, under the condition that the effective target ratio is 5, 10 or 20, the results of killing tumor cells by activated CTLs after three polypeptides are mixed according to equal proportion and the mixture is compounded with gp96 according to equal mass proportion (as shown by the compound mixed with 1-1 in Table 3) are shown in figure 6, and the results show that the CTLs activated by the three polypeptides-gp 96 compound have obvious killing effect on liver cancer cells, while the CTLs activated by gp96 have no killing activity basically as a control group.

Example 5: therapeutic action of specific CTL induced by polypeptide-gp 96 complex on liver cancer model

20 female nude mice of 6-8 weeks old are taken, and 10 are returned to each tail vein ⁷ Specific T cells (0.5 mL) induced by the polypeptide-gp 96 compound, wherein the polypeptide-gp 96 compound is a compound formed by mixing three polypeptides in equal proportion and compounding the three polypeptides with gp96 in equal mass proportion; 3 days after the reinfusion, all mice were inoculated subcutaneously with 5X 10 cells each ⁶ An HEPG2 liver cancer cell; mice were divided into two groups of 10 mice on day 2 post tumor inoculation, and treated as follows:

a first group: the tail vein is used for back transfusion of polypeptide-gp 96 complex activated human T lymphocytes, immunization is carried out three times (0.5 mL each time), and the single back transfusion dosage is 10 ⁷ A/only;

second group: returning gp96 stimulated human T cells via tail vein, immunizing three times (0.5 mL each time), and using single immunization dose as single return dose of 10 ⁷ One/one;

in the above two groups: 3 times of reinfusion was carried out 2,3,4 days after tumor cell inoculation. Starting from the first day of reinfusion, observing the growth condition of the tumor every day, recording the size of the tumor, and calculating the volume of the tumor according to the following formula: v = ab ² 2 (V-volume, a-tumor major diameter, b-)Tumor minor axis). The change of tumor volume is shown in figure 7, and the result shows that CTL induced by the three polypeptide-gp 96 complexes can obviously inhibit the growth of the liver cancer tumor.

While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and changes in detail can be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. A full appreciation of the invention is gained by taking the entire specification as a whole in the light of the appended claims and any equivalents thereof.

SEQUENCE LISTING

<110> Foshan thermal shock Biotechnology Ltd

<120> epitope peptide of LGALS3 and complex of said epitope peptide and heat shock protein

<130> IDC210142

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 9

<212> PRT

<213> artificial

<220>

<223> amino acid sequence of fragment 1 of human LGALS3 polypeptide

<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 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 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> gp96 amplification primer F1 sequence

<400> 4

ggaattcatg gacgatgaag ttgat 25

<210> 5

<211> 29

<212> DNA

<213> artificial

<220>

<223> gp96 amplimer R1 sequence

<400> 5

gctctagact attagaattc atctttttc 29

<210> 6

<211> 803

<212> PRT

<213> artificial

<220>

<223> human source 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 for coding human source 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. An isolated polypeptide having the amino acid sequence set forth in SEQ ID NO:1 or 2.

2. A polypeptide composition comprising any two or three polypeptides selected from the group consisting of: SEQ ID NO: 1. 2 and 3;

preferably, the polypeptide compositions contain the same or different proportions of polypeptides;

preferably, the polypeptide composition comprises SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO: 3.

3. A complex formed by the isolated polypeptide of claim 1 or the polypeptide composition of claim 2 and a heat shock protein;

preferably, the complex is formed by the isolated polypeptide of claim 1 or the polypeptide composition of claim 2 and a heat shock protein in a natural adsorption or heat shock manner;

preferably, the complex is prepared by mixing the isolated polypeptide of claim 1 or the polypeptide composition of claim 2 and a heat shock protein in vitro; preferably, the mixing is performed at 40-80 ℃ (e.g., 40-70 ℃,40-60 ℃, or 50-60 ℃); preferably, the mixing is carried out at 55 ℃;

preferably, the complex is prepared by contacting the isolated polypeptide of claim 1 or the polypeptide composition of claim 2 with a heat shock protein and holding at 55 ℃ for 10min, followed by holding at room temperature for 30min;

preferably, the heat shock protein is selected from the group consisting of HSP70, HSP90, gp96, HSP110, and mutants or fusion proteins thereof;

preferably, the heat shock protein is gp96; preferably, the heat shock protein has the amino acid sequence as shown in SEQ ID NO: 6;

preferably, the complex is prepared by mixing the isolated polypeptide of claim 1 or the polypeptide composition of claim 2 with a heat shock protein in vitro at a mass ratio of 0.1 to 100 (e.g. 0.1 to 50;

preferably, the complex is immunogenic and capable of inducing a specific immune response against a cell expressing LGALS3 (e.g., a tumor cell, such as a pancreatic cancer or liver cancer cell).

4. A composition comprising at least 2 (e.g., 2,3,4, 5, or more) of the complexes of claim 3;

preferably, the heat shock proteins in the composition are the same or different;

preferably, the composition is immunogenic and is capable of inducing a specific immune response against cells expressing LGALS3 (e.g., tumor cells, such as pancreatic cancer or liver cancer cells).

5. A method of preparing the complex of claim 3, comprising mixing the isolated polypeptide of claim 1 or the polypeptide composition of claim 2 with a heat shock protein;

preferably, the isolated polypeptide of claim 1 or the polypeptide composition of claim 2 is complexed with a heat shock protein by natural adsorption or heat shock;

preferably, the mixing is carried out at 40-80 ℃ (e.g., 40-70 ℃,40-60 ℃, or 50-60 ℃); preferably, the mixing is carried out at 55 ℃;

preferably, the method comprises: contacting the isolated polypeptide of claim 1 or the polypeptide composition of claim 2 with a heat shock protein at 55 ℃ for 10min and then at room temperature for 30min;

preferably, the heat shock protein is gp96; preferably, the heat shock protein has the amino acid sequence as set forth in SEQ ID NO: 6;

preferably, the method comprises: the isolated polypeptide of claim 1 or the polypeptide composition of claim 2 is mixed with a heat shock protein in the following ratio of 0.1 to 100 (e.g. 0.1 to 50;

preferably, the complex is immunogenic and is capable of inducing a specific immune response against a cell expressing LGALS3 (e.g., a tumor cell, such as a pancreatic cancer or liver cancer cell).

6. A T cell expressing a T cell receptor capable of specifically recognizing galectin 3 (LGALS 3) or the isolated polypeptide of claim 1 or the polypeptide composition of claim 2;

preferably, the T cell is obtained by stimulating an immune cell with the complex of claim 3 or the composition of claim 4; preferably, the immune cells comprise PBMCs or T lymphocytes;

preferably, the stimulation further comprises the use of an immunostimulatory cytokine (e.g., IL-2);

preferably, the T cell is a cytotoxic T lymphocyte;

preferably, the T cell is capable of specifically recognizing and killing a cell expressing LGALS3 (e.g., a tumor cell, such as a pancreatic cancer or liver cancer cell).

7. A method of making the T cell of claim 6, the method comprising stimulating an immune cell with the complex of claim 3 or the composition of claim 4; preferably, the immune cells comprise PBMCs or T lymphocytes;

preferably, the stimulation further comprises the use of an immunostimulatory cytokine (e.g., IL-2).

8. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the isolated polypeptide of claim 1; preferably, the isolated polypeptide has the amino acid sequence as set forth in SEQ ID NO:1 or 2.

9. An isolated combination of nucleic acid molecules comprising a nucleotide sequence encoding the polypeptide composition of claim 2; the polypeptide composition comprises any two or three polypeptides selected from the group consisting of: the amino acid sequence of SEQ ID NO: 1. 2 and 3; more preferably, the polypeptide composition comprises SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO: 3.

10. A vector comprising the isolated nucleic acid molecule of claim 8 or a combination of the isolated nucleic acid molecules of claim 9;

alternatively, the vector comprises a nucleotide sequence encoding the isolated polypeptide of claim 1 or the polypeptide composition of claim 2;

preferably, the vector is a viral vector, such as a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector or a baculovirus vector.

11. A host cell comprising the isolated nucleic acid molecule of claim 8 or the combination of isolated nucleic acid molecules of claim 9 or the vector of claim 10.

12. A pharmaceutical composition comprising the isolated polypeptide of claim 1, the polypeptide composition of claim 2, the complex of claim 3, the composition of claim 4, the T cell of claim 6, the isolated nucleic acid molecule of claim 8, the isolated nucleic acid molecule combination of claim 9, the vector of claim 10, or the host cell of claim 11, and a pharmaceutically acceptable carrier and/or excipient;

preferably, the pharmaceutical composition is a tumor vaccine;

preferably, the pharmaceutical composition comprises an adjuvant;

preferably, the pharmaceutical composition further comprises an additional therapeutic agent, such as an anti-tumor agent or an immunopotentiator;

preferably, the antineoplastic agent is selected from the group consisting of alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, immune checkpoint inhibitors (e.g., PD-1 antibodies, PD-L1 antibodies, CTLA-4 antibodies, LAG-3 antibodies, or TIM3 antibodies);

preferably, the immunopotentiator is selected from the group consisting of an immunostimulatory antibody (e.g., an anti-CD 3 antibody, an anti-CD 28 antibody, an anti-CD 40L (CD 154) antibody, an anti-41 BB (CD 137) antibody, an anti-OX 40 antibody, an anti-GITR antibody, or any combination thereof).

13. Use of an isolated polypeptide of claim 1, or a polypeptide composition of claim 2 or a complex of claim 3, or a composition of claim 4 or a T cell of claim 6, in the manufacture of a medicament for inducing an immune response against a tumor expressing LGALS3 in a subject, and/or preventing or treating a tumor expressing LGALS3 in a subject;

preferably, the LGALS3 expressing tumor is 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;

preferably, the T cell is from the subject;

preferably, the subject is a human;

preferably, the subject is HLA-A2 positive;

preferably, the isolated polypeptide, polypeptide composition, composition or T cell is administered in combination, e.g., simultaneously, separately or sequentially, with an additional therapeutic agent; preferably, the additional therapeutic agent is an immunostimulant or an antineoplastic agent;

preferably, the LGALS3 protein expression is aberrant in the LGALS 3-expressing tumor (e.g., increased expression compared to a healthy subject).