CN103570821A - Mucin-1 antigenic polypeptide and application thereof as tumor vaccine - Google Patents

Abstract

The invention provides a human mucin-1 tumor antigenic polypeptide or continuous 10 amino acid fragments thereof, wherein the polypeptide can be combined with an HLA (Human Leukocyte Antigen) I and can be recognized by a cell CD8<+>T. The invention further provides nucleic acids coding the polypeptide. The invention further provides an antigen presenting cell capable of presenting the polypeptide on the surface of the cell and an immune effector cell capable of recognizing the polypeptide or antigen presenting cell. The invention further provides application of the polypeptide or variants thereof, the nucleic acids, the antigen presenting cell or the immune effector cell in the preparation of vaccines or pharmaceutical compositions for treating or preventing cancer. Tumor vaccines provided by the invention have a good treatment effect on relatively large crowds and are particularly applicable to the Asian, e.g. Chinese.

Description

Mucin-1 antigenic polypeptides and their use as tumor vaccines

Technical Field

The present invention relates to the field of tumor and immune prepayment and therapy. In particular, the invention relates to a human mucin-1 tumor polypeptide derivative and application thereof as a tumor vaccine.

Background

At present, the clinical treatment of cancer is mainly performed by operation and assisted by chemotherapy and radiotherapy, but the specificity of the treatment is insufficient, normal tissues and organs are often damaged, and the problems of high postoperative recurrence and easy metastasis still exist in the surgical treatment. Immunotherapy with tumor vaccines can produce a specific response against tumors, even clearing residual tumor foci, and generating immunological memory. It is now the fourth form of tumor treatment following surgery, chemotherapy and radiotherapy, and has significant complementarity with three major conventional therapies.

As a prophylactic or therapeutic vaccine, tumor vaccines are one form of immunotherapy. The vaccine prepared by the polypeptide is used for treating and preventing tumors by utilizing tumor specific antigens and other immunoregulatory cells. The anti-tumor immune response is initiated by the presentation of tumor antigen to T cells by Antigen Presenting Cells (APC). Dendritic Cells (DCs) are the most powerful professional APCs in humans. The DC loaded with tumor antigen can excite the in-vivo tumor specific T cells, thereby effectively killing the tumor cells and establishing a durable anti-tumor specific immune response.

Human mucin 1(MUC1) is a high molecular weight glycoprotein characterized by high O-glycosylation and containing a continuous repetitive peptide sequence, and is present in a variety of epithelial tissues and has a variety of functions. Mucin is abnormally expressed in cancer tissues and is expressed in a large amount. Human mucin 1(MUC1) is a Tumor Associated Antigen (TAA) molecule present on the surface of 90% of cancer cells. Healthy human cells also contain mucin 1, but in small quantities, do not trigger an immune response. The immune system encounters cancer cells with high mucin 1 concentrations and may trigger an immune response, and activated toxic T lymphocytes may attack and kill the cancer cells.

Cellular immunity, particularly cytotoxic T cells (hereinafter referred to as CTLs), plays an important role in eliminating tumor cells, virus-infected cells, and the like from a living body. CTLs recognize a complex of an antigen peptide (tumor antigen peptide) and an MHC (major histocompatibility complex) class I antigen (which is called an HLA antigen in humans) on tumor cells, and attack and kill the tumor cells.

When a tumor-specific protein (i.e., tumor antigen protein) is synthesized intracellularly, the tumor-specific protein is degraded intracellularly by protease to produce a tumor antigen peptide. The resulting tumor antigen peptide binds to MHC class I antigen (HLA antigen) in the endoplasmic reticulum to form a complex, which is transported to the cell surface and presented as an antigen. When tumor-specific CTLs recognize the complex presented as an antigen, an antitumor effect is exhibited by cytotoxic action or production of lymphokines. Elucidation of a series of these effects makes it possible to improve therapy of tumor-specific CTLs in patients with tumors by using tumor antigen proteins or peptides as so-called cancer immunotherapeutic agents (cancer vaccines). In tumor immunotherapy, the elimination of tumor cells relies on the immunological action of T cells. Antigen recognition by T cells is not a complete antigenic molecule. In Antigen Presenting Cells (APC), tumor-associated antigens are enzymatically decomposed into polypeptides, which are then transported to the lumen of the endoplasmic reticulum in the presence of a peptide chain transporter, bound to newly synthesized HLA molecules (human leukocyte antigens, including HLA I molecules or HLA II molecules) and mobilized to the surface of the APC to form HLA/antigen peptide complexes, and after T cells recognize the HLA/antigen peptide complexes on the surface of the antigen presenting cells via their surface-specific TCRs, they receive a costimulatory signal (interaction of B7 molecules with CD28 molecules). Under dual-signal stimulation, T cells are activated and proliferate, mostly differentiating into effector cells. Wherein the CD₈ ⁺T cells are lethal, breaking down foreign cells and dying. CD (compact disc)₈ ⁺T cells secrete cytokines such as interleukin to make CD₈ ⁺T cells,

And various phagocytic leukocytes are concentrated around the tumor cells to destroy the tumor cells. Some of these T lymphocytes, which become memory cells, will proliferate and differentiate into effector cells more rapidly when they are again stimulated by the same antigen. A few memory cells divide again into memory cells, permanently performing specific immune functions.

In 1991, Rotzschke et al revealed by X-ray crystal diffraction that the antigen-peptide binding region composed of alpha 1 and alpha 2 chains at the top of HLA class I molecules is a groove-like structure and can accommodate short peptides composed of 8-12 amino acid residues. The amino acid composition in the groove is highly conserved in HLA I molecules of different types, and can form stable and strong hydrogen bonds with the N-terminal residue and the C-terminal residue of the epitope peptide, so that the HLA I molecules can identify special epitopes. The amino acids in the grooves of HLA I of different genotypes can be varied, which forms the basis of HLA I polymorphism. Although the binding of antigenic peptides to HLA class I molecules is selective, unlike the mechanism of antibody and antigen binding, the binding motif (binding motif) in the groove can be linked appropriately at the corresponding position as long as the bound polypeptide has 2 to 3 key amino acid residues (anchor sites) with similar chemical properties. The polypeptide binds to the polypeptide and is transported to the cell surface for presentation to CD₈ ⁺T cells. CD (compact disc)₈ ⁺T cells specifically recognize HLA/antigen peptide complexes on the surface of antigen presenting cells via T Cell Receptors (TCRs) on the surfaces thereof, are activated and proliferate, and then differentiate into effector cells. It is this structural basis that each HLA I molecule is capable of binding to a number of different polypeptides. This provides a theoretical basis for epitope prediction of sequences of different types of molecular binding peptides, thereby making possible the analysis and even prediction of CTL epitopes by computer-assisted means.

HLA class II molecules such as HLA-DP, DQ, DR and the like are composed of HThe alpha chain and beta chain encoded by LA II gene are non-covalently linked glucoprotein. After the alpha chain and the beta chain are respectively synthesized in the endoplasmic reticulum, the alpha chain and the beta chain are respectively coiled into an alpha helix and a beta sheet layer to form a groove. There are also anchor sites within the grooves that bind to antigenic peptides to form HLA/polypeptide complexes. Because its ends are open, it can accommodate longer polypeptides (about 12-20 amino acids). When the groove of HLA II is combined with antigen peptide to form MHC/polypeptide complex, and the MHC/polypeptide complex is transported to the cell surface to be presented to CD₄ ⁺T cells. CD (compact disc)₄ ⁺T cells are activated and proliferated after specifically recognizing HLA/antigen peptide complexes on the surface of antigen presenting cells via their surface TCRs, and then differentiate into effector cells. CD (compact disc)₈ ⁺T cells must have CD stimulation₄ ⁺T cell signaling can produce an effective immune response. Activated CD₄ ⁺T cells can effectively promote CD₈ ⁺T cells produce memory cells, thereby allowing the body to produce a long-term anti-tumor CTL response. In addition to activated CDs₄ ⁺T cells can also kill tumor cells directly.

The structural difference between different HLA gene loci or different alleles of the same locus of the same gene can form different HLA molecule groove structures. This results in different HLA allele-encoding molecules being selective for binding to various antigenic peptides. Furthermore, the anchoring sites and residues of antigenic peptides capable of binding to HLA molecules of the same class are often identical or similar. This indicates that a particular HLA molecule can selectively bind to antigenic peptides by virtue of the required consensus motif, in the sense that the binding of both is somewhat selective. In fact, different HLA molecules can selectively bind to peptide fragments with different anchor positions and residues, so that different HLA allele products are likely to present different epitopes of the same antigen molecule, resulting in different responses of different individuals (with different MHC alleles) to the same antigen in different intensities. This is an important mechanism by which HLA participates in and regulates immune responses by its polymorphisms.

Intensive studies have also found that the recognition of antigenic peptides by HLA molecules does not exhibit a strict one-to-one relationship. This variability can be manifested in different ways: antigenic peptides constituting a common basic sequence, the order and structure of which may vary; secondly, the required anchoring residues of the same HLA molecule (e.g. HLA II molecule) are often more than one amino acid, with the result that the number of corresponding peptide chains of a particular common base sequence can be considerably larger, resulting in that one HLA molecule can bind to a plurality of antigenic peptides, activating a plurality of specific T cell clones; third, antigenic peptides that are taken up by different HLA molecules may possess similar common motifs. For example, at least four families, a2, A3, B4, B44, are known in HLA class I molecules, and the members (various allelic products) of these families can selectively recognize antigenic peptides possessing identical or similar anchor residues in common. This means that an antigenic peptide that can be recognized and presented by a certain HLA molecule may also be presented by other molecules in its family. This provides convenience for immunoprophylaxis and immunotherapy using polypeptide vaccines, in vitro primed dendritic cell vaccines or T cell vaccines. Meanwhile, the method provides a basis for identifying and modifying the polypeptide sequence of the tumor vaccine.

Not only can each HLA I molecule bind a certain number of different polypeptides, but T cells also have cross-reactivity phenomena, i.e., a single T cell can recognize protein complexes of two or more different polypeptide antigens with MHC. This in turn provides a basis for identifying and engineering the polypeptide sequences of tumor vaccines at another level.

Research shows that overcoming HLA polymorphism obstacle and finding effective tumor epitope vaccine covering most people is an important prerequisite for the research of tumor specific polypeptide vaccine immunotherapy.

Statistically, only two macros, HLA a2 (including a x 0201, a x 0202, a x 0204, a x 0205, a x 0206, a x 0207, and a x 0208) and HLA A3 (including a x 0301, a x 1101, a x 3101, and a x 6801) had an overall mean distribution frequency in the chinese population of more than 85%. The same large recognized peptide sequences are very similar.

There is also a need in the art for more effective tumor vaccines that cover a large portion of the population.

Disclosure of Invention

The present invention provides antigenic determinants of human mucin I that can be recognized by MHC (major histocompatibility complex) antigens (which are referred to as HLA antigens in humans, including HLA I and HLA II) and thereby stimulate tumor-associated T cells. The antigenic determinant provided by the invention accounts for most of Asians, especially Chinese people, namely the occurrence frequency is higher.

The invention also provides the polypeptide prepared according to the antigenic determinant of the human mucin I and the related tumor polypeptide vaccine. The prepared tumor polypeptide vaccine has good treatment effect in larger people.

Specifically, the present invention provides an isolated polypeptide comprising an amino acid sequence that is identical or substantially identical to the amino acid sequence of (a) or (b) below:

(a) SEQ ID NO: 1;

(b) a fragment of the amino acid sequence of (a).

SEQ ID NO: the amino acid sequence shown in 1 is MTPGTQSPFFLLLLLTVLTVVTGS.

The polypeptides of the invention or variants thereof can bind to HLA I or HLA II molecules and are CD-linked₈ ⁺T cells or CD₄ ⁺T cell recognition. In one aspect of the invention, the polypeptide of the invention or a variant thereof binds to an HLA I molecule and is CD-linked₈ ⁺T cell recognition.

In the present invention, the term "isolated" refers to a form that is not native.

In the present invention, the term "variant" or "variant of a polypeptide" refers to a variant that is equivalent or more active than the polypeptide in protein activity, e.g., antigenically, epitopically or immunologically. In some embodiments, one skilled in the art can obtain such variants by altering a portion of the polypeptide that does not destroy its activity, e.g., by substitution, deletion, insertion, addition of one or more amino acids in the amino acid sequence of the polypeptide. In some embodiments, the variants may be obtained by substituting the polypeptide with related amino acids by identifying residues and portions of the molecule that are conserved between similar polypeptides. One skilled in the art can also analyze three-dimensional structures and amino acid sequences related to structures in similar polypeptides. Based on this information, one skilled in the art can predict amino acid sequence alignments of the three-dimensional structure of antigens, thereby making test variants that contain single amino acid substitutions at each desired amino acid residue. These variants can then be screened using activity assays well known to those skilled in the art.

In the present invention, "comprising" in the expression "a polypeptide comprises an amino acid sequence" includes "having".

The polypeptides provided by the invention comprise a polypeptide having the sequence shown in SEQ ID NO: 1 (immunogenic fragment), i.e. a fragment of a polypeptide having the amino acid sequence shown in SEQ ID NO: 1, which is capable of producing a polypeptide that recognizes the amino acid sequence set forth in SEQ ID NO: 1, or a polypeptide having the amino acid sequence set forth in seq id No. 1. Preferred fragments include, for example, those having the amino acid sequence of SEQ ID NO: 1, or a truncated polypeptide of a portion of the contiguous amino acid sequence of seq id no.

In one aspect of the present invention, there is provided the polypeptide as described above, wherein the fragment in (b) is a polypeptide comprising or having the sequence of SEQ ID NO: 1, or a fragment of consecutive 10 amino acids of the amino acid sequence shown in seq id no.

In still another aspect of the present invention, there is provided the above-mentioned polypeptide having an amino acid sequence of FLLLLLTVLT (SEQ ID NO: 2), LLLLLTVLTV (SEQ ID NO: 3), LLLLTVLTVV (SEQ ID NO: 4), FFLLLLLTVL (SEQ ID NO: 5), GTQSPFFLLL (SEQ ID NO: 6), TQSPFFLLLL (SEQ ID NO: 7).

In one aspect of the invention, the above polypeptide can bind HLA I molecule and is administered by CD₈ ⁺T cell recognition. CD (compact disc)₈ ⁺T cells are also known as cytotoxic T Cells (CTLs). In a further aspect of the invention, wherein said HLA I is HLA a2 or HLA A3 type, preferably HLA a2, more preferably a 0201 or a 0202.

Herein, "substantially identical amino acid sequence" means that one to several (e.g., 2, 3, 4, or 5) amino acids in the amino acid sequence are substituted, deleted, added, or inserted, which have the same or similar or better activity as compared to the amino acid sequence. In the present invention, the activity may refer to, for example, an activity recognized by HLA I and HLA II and further stimulating tumor-associated T cells.

The polypeptides of the invention can be synthesized according to methods used in conventional peptide chemistry. Such known methods include, for example, those described in the following documents: peptide Synthesis, Interscience, New York, 1966.

The polypeptide of the present invention can also be prepared by conventional genetic engineering. For example, the polypeptide may be prepared using nucleotides encoding the polypeptide prepared by conventional DNA synthesis and genetic engineering methods. Namely, the polypeptide is produced by the following method: inserting the polynucleotide into a conventional expression vector; transforming a host cell with the obtained recombinant expression vector; culturing the obtained transformant; and collecting the polypeptide from the culture. This can be done, for example, with reference to the methods described in the following documents: molecular Cloning, T.Maniatis et al, CSH Laboratory (1983).

The present invention also provides an isolated nucleic acid consisting of a base sequence encoding the above-described polypeptide of the present invention. The nucleic acids of the invention may be cDNA, mRNA or DNA/RNA chimeras, preferably DNA. The nucleic acid may be double-stranded or single-stranded. When the nucleic acid is double-stranded, it may be double-stranded DNA, double-stranded RNA, or a DNA: RNA hybrid. When the polynucleotide of the present invention is double-stranded, it may be inserted into an expression vector to produce a recombinant expression vector for expressing the peptide of the present invention. Thus, the nucleic acid of the present invention comprises a recombinant expression vector obtained by inserting the double-stranded polynucleotide of the present invention into an expression vector.

The base sequence encoding the above-mentioned signal peptide of the present invention is not particularly limited as long as it produces any one of the amino acid sequences of the above-mentioned polypeptide of the present invention after translation. The nucleotide sequence encoding the amino acid sequence can be obtained by a PCR method or the like using genomic DNA or RNA containing a mucin sequence as a template. On the other hand, in view of the expression efficiency in host cells, it is generally preferred to select codons which are very often used for the host cells used. Codon usage frequency data in various biological species can be obtained from a database of genetic code usage frequencies. The nucleic acid of the present invention can also be chemically synthesized by an automatic DNA/RNA synthesizer.

In one aspect of the invention, vaccines and pharmaceutical compositions are provided comprising the polypeptides or nucleic acids of the invention described above. The vaccine and the pharmaceutical composition provided in the invention can be used for treating or preventing cancer. The polypeptides of the present invention described above are useful as vaccines and pharmaceutical compositions for treating or preventing cancer. The above-described nucleic acids of the present invention can also be used as vaccines and pharmaceutical compositions for treating or preventing cancer. The nucleic acid of the present invention can be expressed in a conventional manner to obtain the polypeptide of the present invention, and then used for the above-mentioned purpose.

The polypeptide of the present invention can be presented to an HLA antigen of an antigen presenting cell, and thus can treat or prevent a tumor in a patient. The polypeptides of the invention can be presented to HLA of antigen presenting cells, specifically provoking T cells, in particular CD, capable of recognizing the binding complex of HLA antigen and presented polypeptide₈ ⁺T cells, which proliferate to kill tumor cells, can be used to treat or prevent tumors in patients.

Use of the polypeptide of the invention as an active ingredient for inducing T cells, in particular CD₈ ⁺The agent for T cells may be mixed with or administered in combination with a pharmaceutically acceptable carrier, such as a suitable adjuvant, so as to effectively establish cellular immunity. Examples of adjuvants that can be used include those described in Clin. Microbiol. Rev.,7:277-289,1994 (which is incorporated herein as reference)Reference) to the above.

The polypeptides of the invention are capable of being presented and inducing specific cytotoxic T Cells (CTL) particularly efficiently in both HLA a2 and HLA A3 large format. HLA types a2 include a x 0201, a x 0202, a x 0204, a x 0205, a x 0206, a x 0207, and a x 0208. HLA a3 includes a 0301, a 1101, a 3101 and a 6801. The overall mean distribution frequency of these two large population in Chinese is over 85%. Therefore, the polypeptide of the present invention and the vaccine or pharmaceutical composition for preventing and treating tumor based on the polypeptide of the present invention can effectively cover most people. Preferably, the polypeptide of the present invention is capable of being efficiently presented and inducing specific cytotoxic T Cells (CTLs) in HLA class a 2. More preferably, the polypeptide of the invention is capable of being efficiently presented and inducing specific cytotoxic T Cells (CTL) in a 0201, a 0202 types.

A vaccine for inducing a prophylactic or therapeutic immune response, comprising the polypeptide or derivative of the present invention as an active ingredient, may be mixed or administered in combination with a pharmaceutically acceptable carrier, such as a suitable adjuvant, to more effectively establish an immune response.

Examples of adjuvants that can be used include, for example, ingredients of microbial origin or derivatives thereof, cytokines, ingredients of vegetable origin or derivatives thereof, ingredients of marine origin or derivatives thereof, mineral gels such as aluminum hydroxide, lysolecithin, surfactants such as polyhydric alcohols, polyanions, peptides, oily emulsifiers (emulsifier preparations), and the like. Also contemplated are liposome formulations, nanoparticle formulations attached to beads having a diameter of a few microns, formulations with attached lipids, microsphere formulations, microcapsule formulations, and the like.

The methods of administration of the above-described vaccine or pharmaceutical composition of the present invention include intradermal, subcutaneous, intramuscular, intravenous administration, and the like. The dose of the peptide of the present invention in the preparation may be appropriately adjusted depending on the disease to be treated, age and body weight of the patient, and the like. Typically, the peptide of the invention is administered in a dosage of 0.0001 to 1000mg, preferably 0.001 to 1000mg, more preferably 0.1 to 10mg, in a formulation, preferably 1 time every few days or 1 to several months.

The vaccines and pharmaceutical compositions of the present invention described above may also be used to treat patients with tumors by in vitro methods. In other words, the polypeptide or nucleic acid of the invention may be contacted with antigen presenting cells and/or immune effector cells in vitro to generate antigen presenting cells capable of recognizing the antigen or antigen complex of the invention, thereby inducing specific T cells, particularly CD₈ ⁺T cells, which are then returned to the patient for use in the prevention or treatment of cancer. The invention provides T cells, in particular CD, induced by contacting in vitro peripheral blood lymphocytes derived from a patient with a tumor, with a polypeptide or nucleic acid of the invention₈ ⁺T cells, and methods of producing the same.

In one aspect of the invention, a method of producing an antigen presenting cell is provided. In one aspect of the present invention, the method comprises the step of contacting the polypeptide of the present invention described above with a cell having antigen presenting ability. In one aspect of the present invention, the method comprises the step of contacting the nucleic acid of the present invention described above with a cell having antigen presenting ability. The polypeptides and nucleic acids of the present invention as described above can be contacted with cells having antigen presenting ability to produce antigen presenting cells. An antigen presenting cell can be produced by contacting a polypeptide or nucleic acid of the present invention with a cell having antigen presenting ability. The polypeptides and nucleic acids of the invention can be used in vitro for the prevention or treatment of tumors. For example, antigen presenting cells can be generated by contacting a polypeptide or nucleic acid of the invention with cells having antigen presenting ability in vitro. The polypeptides and nucleic acids of the invention may also be used in vivo for the prevention or treatment of tumors.

In the present invention, the cell having antigen presenting ability is a cell expressing an HLA antigen presenting a polypeptide on the surface of the cell. One of the cells having antigen presenting ability is a dendritic cell.

The present invention provides an antigen-presenting cell that expresses on the surface of the cell an HLA antigen presenting the polypeptide of the present invention. One of the cells having antigen presenting ability is a dendritic cell. The antigen-presenting cell of the present invention can be prepared by adding the polypeptide or nucleic acid of the present invention to the above-mentioned cell having antigen-presenting ability.

The antigen-presenting cell of the present invention can be obtained by the following method: isolating cells having antigen presenting ability from a tumor patient, stimulating the cells in vitro with the polypeptide of the present invention, and allowing the antigen presenting cells to present complexes of HLA antigens and the polypeptide. When dendritic cells are used, the antigen-presenting cells of the present invention can be prepared by isolating lymphocytes from peripheral blood of a tumor patient, removing cells that cannot adhere to a culture dish, culturing the adherent cells in the presence of GM-CSF and IL-4 to induce dendritic cells, and culturing and stimulating the dendritic cells with the peptides of the present invention.

The antigen-presenting cell of the present invention is prepared by adding the nucleic acid of the present invention to a cell having antigen-presenting ability. The nucleic acid may be in the form of DNA or RNA. The nucleic acid can express the polypeptide of the present invention in a cell having antigen presenting ability.

The present invention provides an immune effector cell which is capable of specifically recognizing a presenting cell surface-expressing a complex presenting a polypeptide of the present invention and an HLA antigen, and which is activated and proliferated to differentiate into an effector cell. Effector cells include various T cells, e.g., CD₈ ⁺T cells and CD₄ ⁺T cells. In one aspect of the invention, the invention provides an immune effector cell which is a CD₈ ⁺T cells, which are lethal to cancer cells, rupture and die cancer cells, also become memory cells, and when they are again stimulated by the same antigen, they will proliferate and differentiate into effector cells more rapidly.

The immune effector cell of the present invention can be prepared by adding the polypeptide or nucleic acid of the present invention to a cell having antigen-presenting ability, and then contacting the presenting cell presenting a complex of the polypeptide of the present invention and an HLA antigen with the cell having immune effector ability.

The antigen-presenting cells of the present invention provided above can be used as vaccines or pharmaceutical compositions for treating or preventing cancer. The antigen presenting cell of the present invention has an immune-inducing activity and can be used for preparing a reagent for inducing antigen-specific effector cells. Induced CD₈ ⁺T cells are capable of exerting an anti-tumor effect through cytotoxic effects and the production of lymphokines. Thus, the antigen-presenting cell of the present invention may be an active ingredient of a vaccine or pharmaceutical composition for treating or preventing tumors. A vaccine for inducing CTLs, which contains antigen-presenting cells as an active ingredient, may contain saline, Phosphate Buffered Saline (PBS), a culture medium, and the like to stably maintain the antigen-presenting cells. Methods of administration include intravenous administration. Use of antigen presenting cells as active ingredients for inducing CD₈ ⁺The agent for T cells is returned to the patient's body, and thus can effectively induce specific CD in the patient's body responsive to the polypeptide of the present invention₈ ⁺T cells, and as a result, can treat or prevent tumors.

The immune effector cells of the present invention may be used as vaccines or pharmaceutical compositions for treating or preventing cancer. The effector cells of the invention include various T cells, such as CD₈ ⁺T cells. CD of the present invention₈ ⁺T cells are capable of exerting an anti-tumor effect through cytotoxic effects and the production of lymphokines. Therefore, the immune effector cell of the present invention may be an active ingredient of a vaccine or a pharmaceutical composition for treating or preventing tumors. A vaccine for immune effector cells comprising antigen presenting cells as an active ingredient may comprise saline, Phosphate Buffered Saline (PBS), culture medium, and the like to stably maintain the immune effector cells. Methods of administration include intravenous administration. An agent comprising immune effector cells as an active ingredient may be returned to the patient's body, and as a result, tumors may be treated or prevented.

In one aspect of the present invention, there are provided vaccines and pharmaceutical compositions comprising the antigen presenting cells or immune effector cells of the present invention as described above. The vaccine and the pharmaceutical composition provided in the invention can be used for treating or preventing cancer. The antigen presenting cells or immune effector cells of the present invention described above can be used as vaccines and pharmaceutical compositions for treating or preventing cancer.

The vaccines and pharmaceutical compositions of the present invention containing antigen presenting cells or immune effector cells as described above may be used to treat patients with tumors by in vitro methods. The polypeptides or nucleic acids of the invention may be contacted with antigen presenting cells and/or immune effector cells in vitro to generate antigen presenting cells capable of recognizing an antigen or antigen complex of the invention, thereby inducing specific effector cells, such as T cells (particularly CD's)₈ ⁺T cells) and then returning the antigen presenting cells and/or immune effector cells to the patient for use in preventing or treating cancer. In one aspect of the invention, the patient is of HLA type I, preferably HLA type a2 or HLA type A3, more preferably HLA type a2, most preferably a 0201 or a 0202.

Immune effector cells of the invention, e.g., T cells (particularly CD)₈ ⁺T cells) which can be used as an active ingredient of a vaccine or pharmaceutical composition for treating or preventing tumors.

The above-mentioned polypeptide or nucleic acid of the present invention, and the above-mentioned antigen presenting cell or immune effector cell of the present invention can be used for preventing or treating cancer. The cancer includes blood cancer, solid tumor, etc., and more particularly, includes lung cancer, malignant lymphoma (e.g., reticulosarcoma, lymphosarcoma, Hodgkin's disease, etc.), cancer of digestive organs (e.g., gastric cancer, gallbladder cancer, cholangiocarcinoma, pancreatic cancer, liver cancer, colon cancer, rectal cancer, etc.), breast cancer, ovarian cancer, musculoskeletal sarcoma (e.g., osteosarcoma, etc.), bladder cancer, leukemia (e.g., acute leukemia including acute episode of chronic myelogenous leukemia), kidney cancer, prostate cancer, etc., preferably cancer of digestive organs, e.g., gastric cancer, gallbladder cancer, cholangiocarcinoma, pancreatic cancer, liver cancer, colon cancer, rectal cancer, etc. Preferably, the cancer is liver cancer.

The invention also provides the use of a polypeptide of the invention or a variant thereof as described hereinbefore, or a nucleic acid of the invention as described hereinbefore, or an antigen presenting cell of the invention as described hereinbefore, or an immune effector cell of the invention as described hereinbefore, in the preparation of a vaccine or pharmaceutical composition for the treatment or prevention of cancer. The cancer includes blood cancer, solid tumor, etc., and more particularly, includes lung cancer, malignant lymphoma (e.g., reticulosarcoma, lymphosarcoma, Hodgkin's disease, etc.), cancer of digestive organs (e.g., gastric cancer, gallbladder cancer, cholangiocarcinoma, pancreatic cancer, liver cancer, colon cancer, rectal cancer, etc.), breast cancer, ovarian cancer, musculoskeletal sarcoma (e.g., osteosarcoma, etc.), bladder cancer, leukemia (e.g., acute leukemia including acute episode of chronic myelogenous leukemia), kidney cancer, prostate cancer, etc., preferably cancer of digestive organs, e.g., gastric cancer, gallbladder cancer, cholangiocarcinoma, pancreatic cancer, liver cancer, colon cancer, rectal cancer, etc. Preferably, the cancer is liver cancer.

Drawings

FIG. 1a cell line MUC1 protein expression western immunoblot assay;

FIG. 1b cell line HLA-A2 protein expression western immunoblot assay;

FIG. 2a proliferation of T cells primed by dendritic cells presenting a polypeptide of the invention;

FIG. 2b T cell morphology of control group;

FIG. 2c DCs-stimulated T cell morphology loaded with antigenic peptides;

FIG. 3T cell killing effect on tumor cells;

FIG. 4T cell killing effect on tumor cells;

FIG. 5T cell killing effect on tumor cells;

FIG. 6T cell killing effect on tumor cells;

FIG. 7T cell killing effect on tumor cells;

figure 8T cells secrete the cytokine IFN γ;

FIG. 9 mature DC cell surface markers;

FIG. 10a T effect of cell killing tumor cells;

FIG. 10b T effect of cell killing tumor cells;

FIG. 11 cell line MUC1 protein expression western immunoblot assay;

FIG. 12a tumor treatment effect of antigenic peptides in mice;

FIG. 12b tumor prevention effect of antigenic peptides in mice.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to be limiting in any way.

EXAMPLE 1 Synthesis of antigenic peptides

Polypeptide MTPGTQSPFFLLLLLTVLTVVTGS (SEQ ID NO: 1), hereinafter referred to simply as antigenic peptide, was artificially synthesized. The synthesized polypeptide was purified to 97%. The lyophilized antigenic peptide was dissolved in dimethyl sulfoxide (25 mM) and stored at-80 ℃.

Polypeptides FLLLLLTVLT (SEQ ID NO: 2), LLLLLTVLTV (SEQ ID NO: 3), LLLLTVLTVV (SEQ ID NO: 4), FFLLLLLTVL (SEQ ID NO: 5), GTQSPFFLLL (SEQ ID NO: 6), TQSPFFLLLL (SEQ ID NO: 7), abbreviated as antigenic peptide 1, antigenic peptide 2, antigenic peptide 3, antigenic peptide 4, antigenic peptide 5 and antigenic peptide 6, respectively, were synthesized in the same manner.

Example 2 preparation of Dendritic Cells (DCs)

Mononuclear Cells (PBMCs) in peripheral blood include lymphocytes and monocytes, and have a specific gravity of about 1.075 in volume and shape. The Ficoll-Hypaque separating medium with specific gravity of 1.077 is used for density gradient centrifugation to ensure that cells with certain specific gravity are distributed according to corresponding density gradient, and various blood cells can be separated.

A white blood cell concentrated solution of a healthy volunteer is obtained from a hong Kong cross blood center, is diluted by a proper amount by PBS, is added on the surface of a Ficoll-Hypaque separating medium, is centrifuged for 30min at a density gradient of 800g at 18-20 ℃, the content of a centrifuge tube is divided into three layers after centrifugation, the upper layer is plasma (containing platelets), the middle layer is the separating medium, the bottom layer is red blood cells and granulocytes, and a milky turbid PBMC layer can be seen at the interface of the upper layer and the middle layer. Taking out the PBMC layer, suspending in PBS, centrifuging for 10min at 400g, repeating the above steps, suspending the cell sediment in RPMI1640 containing 10% fetal calf serum, culturing at 37 ℃ for 2 hours, taking out non-adherent cells for separating T cells, adding an RPMI1640 culture medium containing 1000U/ml hGM-CSF and 5OOU/ml hIL-4 into the adherent cells, culturing at 37 ℃ for 1 week, inducing the differentiation of monocytes into DCs, wherein the culture medium and cell factors are changed every two days, and harvesting non-adsorbed and loosely adsorbed cells, namely DCs.

EXAMPLE 3 preparation of T cells (Nylon cotton method)

T cells have short but little villi on their surface, B cells have many but long villi, B cells tend to adhere to nylon cotton fibers at 37 ℃ due to the difference in smoothness of the cell surface, and T cells do not have this ability. By using this property, T cells and B cells can be separated.

Preparing nylon cotton columns: after 1g of nylon cotton/column was sterilized, RPMI1640 medium was added thereto, and the mixture was left at 37 ℃ for 2 hours to allow RPMI1640 to flow out. Resuspending the above non-adherent cells in an appropriate volume of RPMI1640 to give a cell concentration of 0.5-1.0X10⁸Adding the mixture into a prepared nylon cotton column, keeping the temperature of the mixture at 2 ml/column for 1 hour at 37 ℃, adding RPMI1640 and 10 ml/column into the nylon cotton column, eluting cells which are not adsorbed on the nylon cotton, and collecting eluent, namely T cell suspension. Centrifuging at 400g for 5min, and suspending the cell sediment in a cell freezing medium for freezing and storing for later use.

Example 4T cell proliferation assay (3H-TdR incorporation method)

The premise of cell proliferation is the replication of cytoplasm and nucleus, and generally, one cell cycle is divided into 4 stages, i.e., G1, S, G2, and M stages. Wherein, the S phase is a DNA synthesis phase, and the main function is DNA synthesis. 3H-TdR (methyl-3H) thymine nucleotide is the precursor of DNA synthesis, and after being added into cell culture solution, it will be taken up by cells and can be used as raw material for DNA synthesis. The more DNA synthesized by the cells, the more 3H-TdR is incorporated, and the degree of cell proliferation can be reflected by detecting the incorporated 3H-TdR.

Adding an antigen with a proper concentration such as each antigen peptide of the experiment into a culture solution of DCs, incubating for 4 hours at 37 ℃, harvesting the DCs, washing the DCs once by PBS, adding the DCs and T cells from the same volunteer into a 96-hole flat-bottom culture plate according to a certain cell proportion, culturing for 5 days at 37 ℃, adding 3H-TdR at 1 uCi/hole, preserving the temperature for 18 hours at 37 ℃, collecting the cells on glass fiber filter paper by using a multi-head cell collector, washing for three times by PBS, 5% trichloroacetic acid and absolute ethyl alcohol in sequence, drying the filter paper, putting the filter paper into a scintillation solution, and determining the cpm value on a beta liquid scintillation counter.

Example 5 Cytotoxic T Lymphocyte (CTLs) assay (LDH method)

Lactate Dehydrogenase (LDH), one of the cytosolic enzymes of living cells, is normally impermeable to the cell membrane, but when target cells are damaged by attack from effector cells, the permeability of the cell membrane changes and LDH is released into the medium. LDH can change oxidation coenzyme I (NAD) into reduction coenzyme I (NADH) in the process of catalyzing lactic acid to generate pyruvic acid, the reduction coenzyme I (NADH) is reduced by the reduction of iodonitroclorac tetrazoxazole (INT) through the reduction of the hydrogen-transferring phenazine dimethyl sulfate (PMS), INT receives H2+ and is reduced into a purple-red formazan ribs and ribs compound, the compound has a high absorption peak at 490nm, and the read A490nm is used as an index, so that the degree of killing of target cells by effector cells can be known.

Adding appropriate concentration of antigen such as each antigen peptide of the experiment into the culture solution of DCs, incubating for 4 hours at 37 ℃, harvesting the DCs, washing with PBS once, adding DCs and T cells from the same volunteer into a 24-well culture plate according to a certain proportion, co-culturing for 7-10 days at 37 ℃, wherein 20U/ml hIL-2 is added into the culture solution, and collecting the T cells as effector cells by Ficoll-Hypaque density gradient centrifugation. 5mM EDTA digestion of tumor cell lines (such as SMMC-7721 or K562) cells as target cells.

Respectively suspending effector cells and target cells in a certain volume of analysis culture medium (RPMI 1640 containing 1% BSA), adding the effector cells (100ul) and the target cells (100ul) into a 96-well round-bottom culture plate according to different proportions, carrying out heat preservation at 37 ℃ for 5 hours, taking out 100 ul/well supernatant, adding the supernatant into a 96-well enzyme label plate, adding 100 ul/well LDH reaction solution, carrying out reaction in a dark place at room temperature for 30min, adding 1MHCL stop solution and 50 ul/well, and measuring A490nm and A630nm as reference wavelengths. Calculating the cytotoxicity: cytotoxicity (%) ﹦ [ (killing test well-target cell spontaneous release well-effector cell spontaneous release well)/(maximum release well-target cell spontaneous release well) ] × 100%.

Injecting effector cells and target cells in a killing experiment hole; the target cell spontaneous release hole is the target cell + analysis culture medium; the effector cell spontaneous release hole is an effector cell + analysis culture medium; the maximum release pore was the target cell +2% TritioX 100.

Example 6 determination of IFN γ by ELISA-spot (ELISPOT) method

The cell factor ELISPOT method is used for measuring the number of cell factors secreting cell in single cell suspension, and has the advantages of high detection speed, high sensitivity and easy operation. The principle is that firstly, an antibody with high affinity and anti-cytokine is coated on an ELISPOT plate, when a cell to be detected is added into the ELISPOT plate, the secreted cytokine is captured by the coated antibody, so that after a cell suspension to be detected is removed, another labeled antibody with anti-cytokine is added, and then a corresponding chromogenic reagent is added, so that spots representing the secretion quantity and position of the cytokine can be generated on the ELISPOT plate.

Adding appropriate concentration of antigen such as each antigen peptide of the experiment to the culture solution of DCs, incubating for 4 hours at 37 ℃, harvesting the DCs, washing once with PBS, adding DCs and T cells from the same volunteer into a 24-well culture plate according to a certain proportion, co-culturing for 7-10 days at 37 ℃, wherein 20U/ml hIL-2 is added to the culture solution, using Ficoll-Hypaque density gradient centrifugation and collecting T cells as effector cells, digesting SMMC-7721 with 5mM EDTA, treating with gamma ray radiation as target cells, respectively resuspending the effector cells and target cells in a certain volume of culture medium, adding the effector cells (100ul) and target cells (100ul) into a 96-well ELISPOT reaction plate coated with IFN gamma antibody in advance according to a certain proportion, incubating for 18 hours at 37 ℃, removing cell suspension, washing 10 times with PBST, adding biotin-labeled IFN gamma antibody, incubation at 37 ℃ for 2 hours, PBST washing 10 times, addition of enzyme-labeled anti-biotin antibody, 37' C incubation for 2 hours, PBST washing 10 times, addition of substrate solution, reaction at room temperature in the dark for 15-30min, counting using ELISPOT automatic reader.

Example 7 flow cytometry assay (FACS)

Preparing single cell suspension: the cells to be tested were harvested and washed with cold PBS, resuspended in 50 ul/tube PBS, the corresponding antibody was added, placed on ice for 1 hour, washed 2 times with cold PBS, FITC-labeled secondary antibody was added, placed on ice for 30min, washed 2 times with cold PBS, and resuspended in 2% paraformaldehyde for flow cytometry.

Example 8Western identification of tumor cell lines with MUC1 protein and HLA-A2 protein

The proteins contained in the tumor cells are identified by SDS-PAGE and Western immunoblotting methods commonly used in the art, i.e., a method in which the proteins are separated after single-direction electrophoresis and transferred to a nitrocellulose filter, and then the presence of the corresponding antigen is detected by using a radioactive or enzyme-labeled specific antibody.

The tumor cell lines SMMC-7721 and SMMC-7721-. SMMC-7721 is a commercially available hepatoma tumor cell line provided by Shanghai cell biology research institute (see Lu J., XuR.B. and Doung R.C. (1985) The transcriptional and expression transcriptional amino transferase by transcriptional in The human liver cell line (SMMC-7721) in vitro Shi Yan Sheng Wu Xue Bao18: 231:. sup.238). SMMC-7721-0201 is a cell line transfected and stably expressing the human HLA-0201 gene in SMMC-7721.

Tumor cells SMMC-7721 and SMMC-7721-plus 0201 were first digested with 0.25% trypsin and then blown out in 1640 complete medium (containing 10% FBS and 1% penicillin-streptomycin) and collected in 15ml centrifuge tubes. Centrifuge at 1000rpm for 5min and discard the supernatant. Resuspend the cells with 80. mu.l PBS (PBS volume can be increased properly for larger cell), add 5 XSDS-PAGE sample Buffer, mix well and boil in boiling water bath for 5 min.

After sample treatment, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblotting were carried out in accordance with a conventional method. In Western immunoblotting experiment, the primary antibody is anti-Muc 1 monoclonal antibody and anti-HLA-A2 monoclonal antibody (BB 7.2 cell supernatant), and the secondary antibody is goat anti-mouse IgG-HRP.

FIGS. 1a and 1b show the results of SDS-PAGE and Western immunoblotting experiments.

Wherein the sample of the SMMC-7721-0201 cell line is shown in lane 1 of FIG. 1a, and the sample of the SMMC-7721 cell line is shown in lane 1.

Wherein the first lane of FIG. 1b is a sample of SMMC-7721-0201 cell line, and the second lane is a sample of SMMC-7721 cell line.

As shown in FIG. 1a, both the tumor cell lines SMMC-7721 and SMMC-7721-0201 expressed MUC1 protein.

As shown in FIG. 1b, the tumor cell line SMMC-7721 was HLA-A2 negative; SMMC-7721-0201 is positive for HLA-A2.

Additional experiments also tested the expression of MHC I in SMMC-7721 and SMMC-7721-containing 0201, demonstrating that cell lines SMMC-7721 and SMMC-7721-containing 0201 are MHC I positive.

Example 9 stimulation of T cell proliferation by DCs loaded with respective antigenic peptides

DCs were stimulated with the antigenic peptide prepared in example 1, namely MTPGTQSPFFLLLLLTVLTVVTGS (SEQ ID NO: 1), and then the obtained DCs loaded with the antigenic peptide were used to stimulate autologous T cells, and whether or not the DCs loaded with the antigenic peptide could stimulate proliferation of autologous T cells was determined.

DCs were loaded with 10ug/ml of antigenic peptide under conditions of 37 ℃ incubation for 4 hours, then co-cultured with autologous T cells at different ratios (DCs: T =1:10 and DCs: T =1: 3), 3H-TdR was added after 5 days, and 3H-TdR uptake was measured 18 hours later to determine the proliferation status of T cells. As controls, T cells were co-cultured with DCs not loaded with antigenic peptides, or T cells were cultured alone. As shown in fig. 2a, the proliferation levels of T cells in each group were significantly different, with T =1:10 for DCs, 10 times higher for DCs loaded with antigenic peptide than for T cells not loaded with DCs, and 15 times higher for T cells cultured alone; when DCs T was increased to 1:3, the level of T cell proliferation was also significantly increased. The results of these experiments show that: DCs loaded with antigenic peptides are able to effectively stimulate the proliferation of autologous T cells, whereas DCs not loaded with antigenic peptides are unable to stimulate the proliferation of autologous T cells.

In addition, as shown in fig. 2b and 2c, morphologically, T cells stimulated by DCs loaded with antigen peptides were significantly different from those of the control group, and exhibited significant activation states, such as increased cell volume and number.

By performing the same tests on the antigenic peptide 1, the antigenic peptide 2, the antigenic peptide 3, the antigenic peptide 4, the antigenic peptide 5 and the antigenic peptide 6, the DCs loaded with the antigenic peptides can effectively stimulate the proliferation of autologous T cells.

Example 10 DCs loaded with respective antigen peptides were able to activate tumor cell-specific CTLs

CTLs play an extremely important role in the immune response of the body against tumors. DCs were stimulated with the antigen peptide prepared in example 1, and then autologous T cells were stimulated with the antigen peptide-loaded DCs, and whether tumor cell-specific CTLs were activated by the antigen peptide-loaded DCs was determined.

DCs were loaded with different concentrations of antigenic peptides, incubated at 37 ℃ for 4 hours, then co-cultured with T cells at a ratio of DCs: T =1:5, and after 7-10 days, the lethality of T cells against the MUC1 protein-expressing hepatoma cells SMMC-7721-0201 was determined by the LDH method. In the experiment, T cells were co-cultured with DCs loaded with the antigen peptide, and as a control, T cells were co-cultured with DCs not loaded with the antigen peptide, or T cells were cultured alone. As shown in fig. 3, T cells stimulated by DCs loaded with antigenic peptides were able to effectively kill tumor cells expressing MUC1 protein, whereas T cells co-cultured with DCs not loaded with antigenic peptides and T cells cultured alone were not able to kill tumor cells. As shown in FIG. 4, when the concentration of the antigen peptide loaded with DCs was increased, the killing ability of the tumor cells by the T cells stimulated by the DCs was also increased.

In order to determine that the lethality of effector cells to tumor cells was due to the activation of tumor cell-specific CTLs rather than the presence of non-specific killer NK cells, in the cytotoxicity assay, using a commercially available NK cell-sensitive cell line K562(atcc. cat. No.: CCL-243) as a target cell, T cells stimulated with DCs loaded with antigenic peptides were able to effectively kill the hepatoma cell SMMC-7721 expressing MUC1 protein, as shown in fig. 5, while SMMC-7721 was the source cell of the antigenic peptide. However, T cells stimulated with DCs loaded with antigenic peptides were unable to kill K562 cells. It is shown that specific CTLs rather than non-specific NK cells kill liver cancer cells.

To further determine the specificity of killing activity against hepatoma cells, human hepatoma cell lines SMMC-7721-0201 and SMMC-7721 were used as target cells in the cytotoxicity assay, and in addition, to determine whether the killing activity was MHC I restricted, the MHC I on the surface of the target cells was blocked with an anti-MHC I antibody as described in the results of example 8, where SMMC-7721-0201 and SMMC-7721 were both positive for MHC I and antigenic peptide. As shown in the experimental results of FIG. 6, the killing activity of CTLs on SMMC-7721-0201 is significantly higher than that of SMMC-7721, and the killing power of CTLs on SMMC-7721-0201 is also lost after the MHC I on the surface is blocked, and on the contrary, the killing power of CTLs on SMMC-7721 is not significantly changed after the MHC I on the surface is blocked. This indicates that the killing power of CTLs against SMMC-7721 is MHC I restricted, thus indicating that the killing activity against SMMC-7721 is mediated by CTLs.

Since the cell SMMC-7721 as the antigen source and the T cell as the effector cell are not from the same individual, the killing activity of the T cell against SMMC-7721 may be an allogenic response. To eliminate the possibility of such allotype reactions, DCs loaded with antigen peptides and DCs not loaded with antigen peptides were used as target cells in cytotoxicity assays. As shown in fig. 7, since DCs and T cells are derived from the same individual, T cells showed no killing activity against unloaded DCs, and in contrast, T cells showed significant killing activity against DCs loaded with antigen peptides, because DCs loaded with antigen peptides processed and presented tumor polypeptides bound by antigen peptides, expressing tumor antigens on the cell surface, indicating that the killing activity of CTLs against SMMC-7721 is an anti-tumor immune response specific to tumor antigens rather than a heterogeneous response.

The above experimental results show that: tumor antigens bound by the antigen peptides are successfully processed by DCs and presented to T cells, thereby effectively activating tumor cell-specific CTLs.

The same tests were carried out for the antigenic peptide 1, the antigenic peptide 2, the antigenic peptide 3, the antigenic peptide 4, the antigenic peptide 5 and the antigenic peptide 6, which were effective in activating tumor cell-specific CTLs.

Example 11 DCs loaded with antigenic peptides are able to stimulate T cells to secrete the cytokine IFN γ

In the anti-tumor immune response, cell-mediated anti-tumor response plays a key role, and among them, CTLs reaction and secretion of IFN γ are the main features of the cellular response.

Whether DCs loaded with the antigen peptide can stimulate autologous T cells to secrete the cytokine IFN gamma or not was determined. DCs were loaded with 40ug/ml of antigenic peptide as described in example 6, incubated at 37 ℃ for 4 hours, then co-cultured with T cells at a ratio of DCs: T =1:5, and after 7-10 days, the secretion of IFN γ by T cells was determined. As controls, T cells were co-cultured with DCs not loaded with antigenic peptides, or T cells were cultured alone. Stimulated autologous T cells were harvested by gradient centrifugation and IFN γ secretion was measured by ELISPOT. Autologous T cells were co-cultured with DCs not loaded with antigen peptides and autologous T cells themselves served as controls. As shown in fig. 8, T cells stimulated by DCs loaded with antigen peptides were able to secrete high levels of IFN γ when encountering target cell SMMC-7721, whereas T cells stimulated by DCs not loaded with antigen peptides and T cells cultured alone did not secrete significant levels of IFN γ when encountering target cell SMMC-7721. The experimental result is consistent with the above CTLs experimental result, which shows that DCs loaded with antigen peptides can activate tumor cell specific T cells, and the T cells can secrete high-level cell factors IFN gamma.

Example 12 antigenic peptides stimulate maturation of DCs

As described above, DCs loaded with antigenic peptides were able to activate tumor cell-specific CTLs, indicating that DCs have processed to present the tumor antigens to which the antigenic peptides bind and elicit a specific anti-tumor immune response. Since the phenotypic change of DCs, i.e., the change from immature DCs to mature DCs, is a prerequisite for the action of DCs, it can be concluded that DCs loaded with antigenic peptides must undergo phenotypic change. To test this inference, it was determined whether the antigenic peptides could stimulate maturation of DCs.

DCs were further cultured for 48 hours after incubation with 40ug/ml of the antigen peptide at 37 ℃ for 4 hours, and then the expression profiles of HLA-DR, CD80, CD86, CD83 and CD40 on the DCs surface were determined by flow cytometry. As shown in FIG. 9, after stimulation with antigenic peptides, the expression levels of HLA-DR, co-stimulatory molecules CD80 and CD86, and cell maturation marker molecules CD83 and CD40 on the surface of DCs were significantly increased. This indicates that the antigenic peptide can effectively stimulate the maturation of DCs and is an effective activating molecule for DCs.

The experimental results show that each antigen peptide separated and purified from the tumor cells is combined with the tumor antigen, and the maturation of DCs can be effectively stimulated; moreover, DCs loaded with antigen peptides can effectively process and present tumor antigens and activate tumor cell-specific anti-tumor cell immune responses, including stimulation of T cell proliferation, activation of tumor cell-specific CTLs, stimulation of T cell secretion of cytokine IFN gamma, and the like.

Example 13 DCs loaded with antigenic peptides were able to activate tumor cell-specific CTLs and efficiently attack tumor cells

This experiment tested the HLA phenotypic specificity of each antigenic peptide of the present invention.

Experiments cytotoxic T lymphocyte killing assays were performed using a kit purchased from PerkinElmer (AD 0116):

after digesting the target cells (tumor cells SMMC-7721 and SMMC-7721-020, wherein SMMC-7721 and SMMC-7721-0201 are both positive to MUC1 protein; SMMC-7721 is negative to HLA-A2; SMMC-7721-0201 is positive to HLA-A2) with 0.25% trypsin, washing the cells once with 1640 complete medium (containing 10% FBS and 1% penicillin-streptomycin); then suspending the cells by 1640 complete culture medium, adjusting the cell count to 1X 10⁶cells/ml. Adding 1 μ l BATDA to 1ml of cells at 37 deg.C and 5% CO₂The incubator is placed for 15 min. Then 200g, centrifugation for 5min, discarding the supernatant, washing 5 times with PBS containing 2% FBS, each 200g centrifugation for 5 min. Finally, resuspend the cells in 1640 full culture medium and adjust the number of cells to 5X 10⁴cells/ml。

DCs were stimulated by adding appropriate concentrations of antigens including the antigen peptide and the antigen peptide 2 to the culture broth of DCs, respectively, as described in example 9, and then the resulting DCs loaded with the antigen peptide were used to stimulate autologous T cells.

The stimulated CD8+ T cells were collected, centrifuged at 1000rpm, the supernatant discarded, and the CD8+ T cells were resuspended in 1640 complete medium,adjusting the cell count to 1X 10⁶cells/ml。

Mu.l of CD8+ T cells and 100. mu.l of tumor cells (BATDA-loaded) were added to a 96-well plate. A blank control group (culture medium control), a tumor cell spontaneous release group (100 mu.l of tumor cells +100 mu.l of culture medium) and a tumor cell maximum release group (10 mu.l of lysate +90 mu.l of culture medium +100 mu.l of tumor cells) are simultaneously set, and T cells are set as unstimulated groups. The experimental group required 3 replicate wells to be established.

After incubation of 96-well plates for 2h at 37 ℃ in a 5% CO2 incubator, 200g were centrifuged for 5min, 20 □ l of supernatant was pipetted into an Elisa plate (kit-of-parts) and 200 □ l of Europium solution was added and shaken horizontally for 15min at room temperature. Detection was carried out using a detection instrument (Victor 2V multilabel counter) from Perkinelmer, with an excitation filter (emission filter) of 615 nm.

Calculation of experimental results:

the results are shown in FIGS. 10a and 10 b. Wherein, the result of the tumor cell attack after loading DCs with the antigen peptide is shown in FIG. 10a, and the result of the tumor cell attack after loading DCs with the antigen peptide 2 is shown in FIG. 10 b.

The experimental result shows that the DCs stimulated T cells loaded with antigens (including the antigen peptide and the antigen peptide 2) can kill the SMMC-7721 and SMMC-7721-0201 which are positive to the MUC1 protein. Wherein the capacity of killing SMMC-7721-0201 cells (HLA-A2 +, MUC1 +) is higher than that of SMMC-7721 cells (HLA-A2-, MUC1 +). The antigenic peptide of the invention is proved to be more effective in attacking HLA-A2 positive cells.

Example 14 animal experiments: antigen peptide-loaded DCs for inhibiting tumors in mice

1. Construction of B16 (for animal experiments) cell line stably expressing MUC1 protein

B16 is a commercially available mouse melanoma cell line (ATCC cat. No. crl-6322). A plasmid containing the MUC1 gene was purchased from origin (Cat. No. RC221390).

B16 cells were digested with 0.25% trypsin and blown out in 1640 complete medium (containing 10% FBS and 1% penicillin-streptomycin). Then, an appropriate amount of cells were plated in a 24-well plate (three wells were co-inoculated), and after 36 hours, a plasmid containing the MUC1 gene was transfected into B16 cells (0.5. mu.g/well of the plasmid) by the lipofection method. The following day, transfected cells were digested with 0.25% trypsin, aliquoted into all 24 wells, and culture continued. On the third day, the culture medium was changed, and 1640 complete medium containing 1.2mg/ml G418 was added to continue the culture.

Observing the death condition of the cells every day, and changing the liquid every other day; four days later, the surviving cells became stable, and the cells were subjected to limiting dilution and seeded into 96-well plates at a cell amount of 0.5 cells/well (10-20 96-well plates). The growth of cells in 96-well plates was observed, and monoclonal cells were searched and labeled. The monoclonal cells that had grown over the well were expanded and collected for identification of MUC1 positive cells by Western methods as described previously. And performing amplification culture on the cells which are identified to be positive, and freezing and storing.

In FIG. 11, lane 1 shows an untransfected B16 cell strain, lane 2 shows a MUC1-4 monoclonal cell, and lane 3 shows a MUC1-6 monoclonal cell.

From the results in FIG. 11, the monoclonal cells of both MUC1-4 and MUC1-6 had distinct bands and were detected as positive. Therefore, the monoclonal cells of the two strains of MUC1-4 and MUC1-6 are determined to be a B16 cell line which stably expresses MUC1 protein.

2. Animal experiments

Experimental mice (C57 BL/6 mice, 6-8 weeks old) were divided into two groups: a treatment group and an immune protection group. Wherein the treatment group was the administration of tumor cells to mice followed by the administration of the polypeptides of the present invention (antigenic peptide and antigenic peptide 2, respectively). Wherein the immunoprotection group is to administer the polypeptide of the present invention (antigenic peptide and antigenic peptide 2, respectively) to mice first, followed by administration to tumor cells.

Treatment groups: a total of 32 mice were tested and divided into 4 groups of 8 mice each, and labeled A, B, C, D. Of these, 8 mice in group A were injected with B16 cells, and the remaining 3 groups were injected with B16-MUC1-4 cells. The inoculation site is subcutaneous injection of the right axilla of the forelimb, 1 × 10 for each mouse⁴Cells were dosed at 200. mu.l. One week later, polypeptides were injected, 16 mice in groups a and B were injected with PBS, and two groups C and D were injected with two polypeptides, respectively: antigenic peptide and antigenic peptide 2, 40. mu.g/100. mu.l per mouse. The same polypeptides were injected in duplicate weekly groups over the following two weeks at the same dose, and mice were observed daily for tumor growth and data were recorded.

FIG. 12a is a graph of the tumor treatment effect of antigenic peptides in mice.

Wherein,

b16-g: group a, injection of B16 cells;

b16-mg: group B, injected with B16-MUC1-4 cells;

b16-mg 53: group C, B16-MUC1-4 cells and antigenic peptide 2 were injected;

b16-mg 64: group D, B16-MUC1-4 cells and antigenic peptides were injected;

since the results for the two groups B16-g and B16-mg were identical, the lines overlap.

FIG. 12b shows the tumor-preventive effect of antigenic peptide 2 in mice.

Wherein

B16-g: group E, injection of B16 cells;

b16-mg: group F, injection of B16-MUC1-4 cells;

b16-mg 53: group G, B16-MUC1-4 cells and antigenic peptide 2;

b16-mg 64: group H, B16-MUC1-4 cells and antigenic peptides were injected.

From the experimental results, mice injected with the polypeptide group were able to prolong the lifespan by more than two weeks, as compared with the control group. The survival rate of mice given the group of antigenic peptides was high by comparison between the antigenic peptide and the antigenic peptide 2. Compared with experimental groups, the survival rate of mice in the immunized group is higher than that in the treated group.

The above experimental results show that the polypeptide provided by the invention comprises the amino acid sequence shown in SEQ ID NO: 1, including fragments of 8-10 contiguous amino acids, particularly 10 contiguous amino acids (e.g., polypeptide FLLLLLTVLT, LLLLLTVLTV, LLLLTVLTVV, FFLLLLLTVL, GTQSPFFLLL, TQSPFFLLLL), are effective in stimulating the maturation of tumor-specific DCs, and the DCs loaded with the antigenic peptide are effective in processing and presenting tumor antigens and activating tumor-cell-specific immune responses, including stimulating T cell proliferation, activating tumor-cell-specific CTLs, and stimulating T cell secretion of cytokine IFN γ, among others.

Without being bound by any theory, the inventors have explored the therapeutic and prophylactic effects of the antigenic polypeptides of the invention. Computational-assisted methods have become a mature, efficient and effective way to analyze protein or polypeptide structure and function in recent years, and have been widely used in protein analysis and prediction, including analysis of epitopes. One skilled in the art can summarize and follow a study of the structural function of a protein to identify residues in similar polypeptides that are important for activity or structure, to predict the effect of amino acid residues in a protein, and to analyze the activity and function of the amino acid sequence of the protein. Computational-assisted methods have become a relatively sophisticated, efficient and effective method of determining epitopes in recent years. The calculation-aided determination of antigen epitope includes sorting and analyzing available data, establishing epitope activity model based on the obtained rule, and analyzing and predicting the sequence of epitope with antigenicity and its combination activity with antigen or MHC.

Generally, the antigenic recognition region is hydrophilic and is easy to deform on the surface and structure of the protein. Because in most natural environments, hydrophilic regions tend to concentrate on the surface of the protein, while hydrophobic regions are often encapsulated within the protein, it is also reasonable that antibodies can only interact with recognition regions found on the surface of the protein, and that these recognition regions, when sufficiently structurally flexible to be transferred to a location accessible to the antibody, will have a high affinity for the antibody. Continuous and discontinuous recognition regions are recognition regions composed of continuous or discontinuous amino acid sequences (residues). Many antibodies are directed against epitopes of a continuous recognition domain, and antibodies bind to such epitopes with high affinity. A discontinuous recognition region is a recognition region that represents a polypeptide sequence that has some fold, or an antibody that links two separate polypeptides together. In some cases, antibodies can be raised against such discontinuous recognition regions, but these antigenic polypeptides have a secondary structure similar to that of the discontinuous recognition region, and the length of the sequence is required to meet the relevant requirements. Epitopes are typically between 8 and 20 amino acid residues in length.

Analyzing by bioinformatics method, and predicting websites such as http:// www.cbs.dtu.dk/services/Bepided/; http:// www.epitope-information.com/links.htm; http:// bio.dfci.harvard.edu/Tools/index. html, etc., and SYFPEITHI, BIMAS, IMMUNEEPITOPE, MHC2PRED, etc., were used to analyze the antigenic peptides of the present invention.

The difference in binding force between HLA-A, -B, -C and the antigen peptide of the present invention or a fragment thereof and epitope sequence variants thereof, particularly HLA A2 molecule and HLA A3 molecule, was analyzed by the above-mentioned means, and it was found that the antigen peptide of the present invention (SEQ ID NO: 1) has high CD-activating activity₈ ⁺Epitopes of T cells, including fragments of 8-10 contiguous amino acids thereof, particularly 10 contiguous amino acids (e.g., polypeptide FLLLLLTVLT, LLLLLTVLTV, LLLLTVLTVV, FFLLLLLTVL, GTQSPFFLLL, TQSPFFLLLL, etc.), and epitopes present in the antigenic peptide that have high activation efficiency for CD4+ T cells, including: MTPGTQSPFF LLLLLTVLTVVTGS, MTPGTQSPFF LLLLL, and SPFF LLLLLTVLTV VTGS, and the like. These epitopes are represented by HLA A2 (including A0201, A02)02. A.cndot.0204, A.cndot.0205, A.cndot.0206, A.cndot.0207 and A.cndot.0208) has high binding force with HLA molecules in HLA A3 (including A.cndot.0301, A.cndot.1101, A.cndot.3101 and A.cndot.6801).

The present invention unexpectedly found that SEQ ID NO: 1, including fragments of 8-10 contiguous amino acids, particularly 10 contiguous amino acids (e.g., polypeptide FLLLLLTVLT, LLLLLTVLTV, LLLLTVLTVV, FFLLLLLTVL, GTQSPFFLLL, TQSPFFLLLL), are effective in stimulating the maturation of tumor-specific DCs, and the DCs loaded with the antigenic peptide are effective in processing and presenting tumor antigens and activating tumor-cell-specific immune responses, and are effective in treating or preventing tumors in animals. Thus, the present invention provides the use of tumor polypeptide antigens as cancer vaccines. Meanwhile, the invention provides a DC tumor vaccine obtained by effectively sensitizing dendritic cells with the polypeptide, or a T cell tumor vaccine prepared by activating T cells with the dendritic cells sensitized with the tumor polypeptide antigen, and a preparation method and application of directly applying the polypeptide or the composition as the tumor vaccine.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of biotechnology, organic chemistry, inorganic chemistry and the like, and it will be apparent that the invention may be practiced otherwise than as specifically described in the foregoing description and examples. Other aspects and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains. Many modifications and variations are possible in light of the above teaching and are therefore within the scope of the invention. All patents, patent applications, and scientific articles mentioned herein are hereby incorporated by reference.

Claims (14)

1. An isolated polypeptide comprising an amino acid sequence that is identical or substantially identical to the amino acid sequence of (a) or (b) below:

(a) SEQ ID NO: 1;

(b) a fragment of the amino acid sequence of (a).

2. The polypeptide or variant thereof of claim 1, wherein the fragment in (b) is a fragment comprising the amino acid sequence of SEQ id no: 1, or a fragment of consecutive 10 amino acids of the amino acid sequence shown in seq id no.

3. The polypeptide of claim 2 or variant thereof, said fragment having the amino acid sequence of LLLLLTVLTV.

4. A polypeptide or variant thereof according to any of claims 1 to 3, said fragment binding to an HLA I molecule and being CD-encoded₈ ⁺T cell recognition.

5. The polypeptide or variant thereof according to claim 4, wherein said HLA I is HLA A2 or HLA A3 type, preferably HLA A2, more preferably A0201 or A0202.

6. An isolated nucleic acid consisting essentially of a base sequence encoding the polypeptide of any one of claims 1-5.

7. An antigen presenting cell which can present a polypeptide of any one of claims 1 to 5 or a variant thereof on the surface of a cell, preferably said antigen presenting cell is a dendritic cell.

8. An immune effector cell which recognizes the polypeptide of any one of claims 1 to 5 or a variant thereof or an antigen presenting cell which presents the polypeptide of any one of claims 1 to 5 or a variant thereof on the cell surface, preferably, the immune effector cell is a T cell, such as a CD₈ ⁺T cells.

9. A method for producing an antigen presenting cell, said method comprising the step of contacting a polypeptide or variant thereof according to any of claims 1-5 with a cell having antigen presenting ability, or comprising the step of expressing a nucleic acid according to claim 6 in a cell having antigen presenting ability, preferably said cell having antigen presenting ability is a dendritic cell.

10. A method of producing an immune effector cell, the method comprising administering an antibody of claim 7The step of contacting the antigen presenting cell with a cell capable of an immune effector, preferably, the antigen presenting cell is a dendritic cell, and, preferably, wherein the immune effector cell is a T cell, such as CD₈ ⁺T cells.

11. A vaccine or pharmaceutical composition for treating or preventing cancer in a patient comprising the polypeptide or variant thereof of any one of claims 1-5, or comprising the nucleic acid of claim 6, or comprising the antigen presenting cell of claim 7, or comprising the immune effector cell of claim 8.

12. Vaccine or pharmaceutical composition according to claim 11, wherein the patient's HLA is of HLA type I, preferably HLA type a2 or HLA type A3, preferably HLA type a2, more preferably type a x 0201 or a x 0202.

13. Vaccine or pharmaceutical composition according to claim 11 or 12, wherein the cancer is a blood cancer, a solid tumor or the like, preferably a lung cancer, a malignant lymphoma (e.g. reticulosarcoma, lymphosarcoma, hodgkin's disease or the like), a cancer of the digestive organs (e.g. stomach cancer, gall bladder cancer, bile duct cancer, pancreatic cancer, liver cancer, colon cancer, rectal cancer or the like), a breast cancer, an ovarian cancer, a musculoskeletal sarcoma (e.g. osteosarcoma or the like), a bladder cancer, a leukemia (e.g. acute leukemia including acute episodes of chronic myelogenous leukemia), a kidney cancer, a prostate cancer.

14. Use of a polypeptide or variant thereof according to any one of claims 1 to 5, or a nucleic acid according to claim 6, or an antigen presenting cell according to claim 7, or an immune effector cell according to claim 8, for the preparation of a vaccine or a pharmaceutical composition for the treatment or prevention of cancer.

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