WO2014015831A1 - Tumor antigenic peptide and application of same as tumor vaccine - Google Patents

Abstract

The present invention provides a human mucin-1 tumor antigenic peptide having an amino acid sequence shown in SEQ ID NO:1, or a variant thereof, which can be combined with HLA I and recognized by CD₈ ⁺T cell. Also disclosed are an antigen presenting cell capable of presenting the peptide on a cell surface and an immunological effector cell capable of recognizing the peptide or antigen presenting cell. Also disclosed is application of the peptide, or the variant, nucleic acid, antigen presenting cell, or immunological effector cell thereof in preparation of a vaccine or pharmaceutical composition for treating or preventing cancer.

Description

 Tumor antigenic polypeptide and its use as a tumor vaccine

[0001] This application claims the application No. 201210265422.2, filed on July 27, 2012, entitled "Tumor antigenic polypeptide and its use as a tumor vaccine" and submitted on July 27, 2012, the application number is 201210265629.X, the name of the invention is "mucin -

Priority of Chinese Patent Application for "Antigenic Polypeptides and Their Use as Tumor Vaccines", the entire contents of which are incorporated herein by reference.

 The present invention relates to the field of cancer immunity. In particular, the invention relates to human mucin-1 tumor antigen polypeptides and their use as tumor polypeptide vaccines. Background technique

 [0003] Among diseases that endanger human health, tumors have become the main cause of human death. The number of cancer-affected people worldwide is more than 10 million per year. According to the World Health Organization's World Cancer Report, by 2020, there will be 15 million cases each year and 10 million deaths. Domestic cancer mortality has risen to the top in the city. This means that the situation facing cancer treatment is very serious. At present, the clinical treatment of cancer is still based on surgery, supplemented by chemotherapy and radiotherapy, but the specificity of treatment is insufficient, often causing damage to normal tissues and organs, and there is still a problem of high recurrence and easy transfer after surgery. . Immunotherapy of tumor vaccines can produce specific responses to tumors, even remove residual tumor lesions and produce immune memory. It has become the fourth treatment for cancer after surgery, chemotherapy and radiotherapy, and is clearly complementary to the three conventional therapies.

 [0004] Tumor vaccines are a means of immunotherapy. Among them, the use of polypeptides for the production of vaccines is the use of tumor-specific antigens and other immunoregulatory cells to treat and prevent tumors. In the process of anti-tumor immune response, antigen-presenting cells (APCs) are processed and presented to T cells for activation. Dendritic Cell (DC), the most powerful full-time APC in human body, has a typical dendritic morphology and high expression of HLA I and HLA 11 molecules on the membrane surface, which can efficiently ingest, process and present antigens. Initiating a T cell-mediated immune response is a central part of the anti-tumor immune response. The application of tumor-loaded DCs can stimulate tumor-specific T cells in vivo, thereby effectively killing tumor cells and establishing a durable anti-tumor-specific immune response.

[0005] Human mucin 1 (MUCl) is a highly 0-glycosylated and contains a contiguous repeat peptide sequence. The high molecular weight glycoproteins, which are present in a variety of epithelial tissues, have multiple functions. Mucin is abnormally expressed in cancer tissues and is abundant in expression. Human mucin 1 (MUC1) is a tumor-associated antigen (TAA), a molecule present on the surface of 90% cancer cells. Muscle 1 is also contained in healthy human cells, but it is small in number and cannot trigger an immune response. Cancer cells with high concentrations of mucin 1 in the immune system may trigger an immune response, and activated toxic lymphocytes can attack and kill cancer packets.

 [0006] Human mucin 1 has a signal peptide. A signal peptide is often referred to as the N-terminal amino acid sequence of a newly synthesized polypeptide chain that directs transmembrane transfer of a protein. It usually consists of 15 ~ 30 amino acids. The signal peptide consists of three regions: a positively charged N-terminus, called the basic amino terminus: an intermediate hydrophobic sequence, mainly neutral amino acids, capable of forming a helical structure, which is the main functional region of the signal peptide; The longer negatively charged C-terminus, containing small molecular amino acids, is the signal sequence cleavage site, also known as the processing region.

[0007] In tumor immunotherapy, the elimination of tumor cells relies on the immune function of T cells. The recognition of antigen by T cells is not an intact antigen molecule. In antigen-presenting cells (APCs), tumor-associated antigens are broken down into polypeptides by enzymes, which are then transported to the endoplasmic reticulum and newly synthesized HLA molecules (human leukocyte antigen, human white) with the participation of the peptide chain transporter. The fine-packed antigen, including HLA I molecule or HLA II molecule, binds to and moves to the surface of APC to form an HLA/antigen peptide complex, and the Τ cell recognizes the HLA/antigen peptide complex on the surface of the antigen presenting cell through its surface-specific TCR. A co-stimulatory signal is received (the Β7 molecule interacts with the CD28 molecule). Under dual-signal stimulation, sputum cells are activated and proliferate, and most of them differentiate into effector cells. Among them, CD ₈ ⁺ T cells have lethality, causing foreign cells to rupture and die. CD ₈ ⁺ T cells secrete interleukin and other cytokines to cause CD ₈ ⁺ T cells, Μ φ and various phagocytic leukocytes to concentrate around tumor cells and destroy tumor cells. Some of the sputum lymphocytes become memory cells, and when they encounter the same antigen stimulation again, they will proliferate and differentiate into effector cells more rapidly. A small number of memory cells divide again into memory cells, which perform long-term specific immune functions.

[0008] In 1991, Rotzschke et al. revealed by X-ray crystal diffraction that the antigen peptide binding region composed of αα and α2 chains on the top of HLA class I molecules has a groove-like structure and can accommodate short peptides composed of 8 to 12 amino acid residues. The amino acid composition in the groove is highly conserved among different types of HLA I molecules, and forms stable and strong hydrogen bonds with the epitope peptide residues and C-terminal residues, ensuring that HLA I molecules can recognize specific epitopes. Amino acids can vary in the groove of different genotypes of HLA I, which is the basis for the formation of HLA I polymorphisms. Although the binding of antigenic peptides to HLA class I molecules is selective, the mechanism of binding to antibodies and antigens is different, as long as the bound polypeptide has 2 to 3 key amino acid residues with similar chemical properties (anchor mapping) Point ;), can be properly connected To the corresponding position of the polypeptide binding motif within the groove. The polypeptide is bound to it and transported to the surface of the cell for presentation to CD ₈ ⁺ T cells. CD ₈ ⁺ T cells specifically recognize the HLA/antigen peptide complex on the surface of antigen presenting cells through the T cell receptor (TCR) on the surface, and then activate and proliferate, and then differentiate into effector cells. It is this structural basis that each HLA I molecule can bind to a certain number of different polypeptides. This provides a theoretical basis for epitope prediction of different types of molecular binding peptide sequences, thereby enabling computer-aided means to predict CTL epitopes.

[0009] HLA class II molecules such as HLA-DP, DQ, DR, etc. are composed of a glycoprotein in which an alpha chain and a beta chain encoded by an HLA class II gene are non-covalently linked. After the α chain and the β chain are synthesized in the endoplasmic reticulum, they are each wound into an α-helix and a β-sheet to form a groove. There are also anchor points in the groove, and the groove combines with the antigen peptide to form an HLA/polypeptide complex. Because of its open end, it can hold longer polypeptides (about 12-20 amino acids). When the groove of HLA II binds to the antigenic peptide to form an MHC/polypeptide complex, it is transported to the cell surface and presented to CD ₄ ⁺ T cells. CD ₄ ⁺ T cells specifically recognize and recognize HLA/antigen peptide complexes on the surface of antigen-presenting cells through their surface TCR, and then proliferate and differentiate into effector cells. CD ₈ ⁺ T cells must have a signal that stimulates CD ₄ ⁺ T cells to produce an effective immune response. Activated CD ₄ ⁺ T cells can effectively promote the production of memory cells by CD ₈ ⁺ T cells, thus allowing the body to produce long-term anti-tumor CTL responses. In addition, activated CD ₄ ⁺ T cells can also directly kill tumor cells.

 [0010] The structural differences between HLA different gene loci or different alleles of the same locus in the same locus can form different HLA molecular groove structures. This results in the selectivity of the different HLA allele coding molecules for the binding of various antigenic peptides. Moreover, an antigenic peptide capable of binding to the same type of HLA molecule has an anchor site and an anchor residue which are often identical or similar. This suggests that a particular HLA molecule can selectively bind to an antigenic peptide by virtue of the desired common motif, in the sense that the combination of the two is selective. In fact, different HLA molecules can selectively bind peptides with different anchor positions and residues, so different HLA allele products may present different epitopes of the same antigen molecule, resulting in different individuals (with different The MHC allele) differs in intensity in response to the same antigen. This is an important mechanism by which HLA participates in and regulates immune responses.

[0011] Further research has also found that HLA molecules do not exhibit a strict one-to-one relationship to the recognition of antigenic peptides. This flexibility can be expressed in different aspects: 1. The antigenic peptides that make up the common base sequence are variable in sequence and structure; 2. The same HLA molecule (such as HLA II molecule) requires more than one anchor residue. Amino acids, the result is that the number of peptide chains corresponding to a particular common base sequence can be quite large, resulting in an HLA molecule that can bind multiple antigenic peptides, activate multiple specific T Cell clones; Third, antigenic peptides accepted by different HLA molecules may have similar common motifs. For example, at least four families of A2, A3, B4, and B44 are known in HLA class I molecules, and members of these families (various allelic products) can selectively recognize that they have the same or similar anchor residues. Antigenic peptide. 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 facilitates the use of peptide vaccines, in vitro sensitized dendritic cell vaccines or T cell vaccines for immunoprophylaxis and immunotherapy. It also provides a basis for identifying and modifying the polypeptide sequence of tumor vaccines.

 [0012] Not only can each HLA I molecule bind to a certain number of different polypeptides, but T cells also have a cross-reactivity phenomenon, i.e., a single T cell can recognize a protein complex of two or more different polypeptide antigens with MHC. This, in turn, provides a basis for identifying and modifying the polypeptide sequence of a tumor vaccine.

 [0013] Studies have shown that overcoming HLA polymorphism barriers and finding effective tumor epitope vaccines covering most of the population has become an important prerequisite for tumor-specific peptide vaccine immunotherapy research.

[0014] According to statistics, only HLA A2 (including A*0201, A*0202, A*0204, A*0205, A*0206, A*0207, and A*0208) and HLA A3 (including A*0301, A*) 1101, A*3101 and A*6801) The total average distribution frequency of the two large Chinese people exceeds 85%. The same largely identified peptide sequences are very similar.

 [0015] There is also a need in the art for more effective tumor vaccines covering most populations. Summary of the invention

 [0016] The present invention provides an antigenic determinant of human mucin I that is recognized by HLA I and HLA II and thereby elicits tumor associated T cells. These antigenic determinants account for the majority (more than 50%) of Asians, especially Chinese, which occur at a higher frequency. The invention also provides polypeptides and related tumor polypeptide vaccines prepared according to the antigenic determinants of these human mucin I. These prepared tumor polypeptide vaccines have a good therapeutic effect in a large population.

[0017] Specifically, the invention provides an isolated polypeptide or variant thereof, the polypeptide comprising an amino acid sequence identical or substantially identical to the amino acid sequence of (a) or (b) below:

 (a) the amino acid sequence shown as SEQ ID NO: 1;

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

 The amino acid sequence shown as SEQ ID NO: 1 is MTPGTQSPFFLLLLLTVLTV VTGSo

[0019] A polypeptide of the invention, or a variant thereof, can bind to an HLA I or HLA II molecule and be finely recognized by CD ₈ ⁺ T cells or CD ₄ ⁺ T. [0020] In the present invention, the term "isolated" refers to a non-natural form.

 [0021] In the present invention, the term "variant" or "variant of a polypeptide" refers to a change in protein activity, such as antigenicity, epitope or immunologically equivalent or better activity of the polypeptide. body. 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, such as substitution, deletion, insertion, or addition of one or more amino acids in the amino acid sequence of the polypeptide. . In some embodiments, the variants can be obtained by subjecting the polypeptides to amino acid substitutions by identifying residues and portions of molecules that are conserved between similar polypeptides. Those skilled in the art will also be able to analyze three dimensional structures and amino acid sequences associated with structures in similar polypeptides. Based on such information, one skilled in the art can predict amino acid sequence alignments of the three dimensional structure of the antigen, thereby preparing experimental variants comprising a single amino acid substitution at each desired amino acid residue. These variants can then be screened using activity assays well known to those skilled in the art.

 [0022] In the present invention, the expression "include" in the phrase "polypeptide contains an amino acid sequence" includes the meaning of "having".

 [0023] The polypeptide provided by the present invention comprises a fragment (immunogenic fragment) of a polypeptide having the amino acid sequence of SEQ ID NO: 1, that is, a contiguous portion having the amino acid sequence of SEQ ID NO: 1, This portion produces an immune response that recognizes the polypeptide of the amino acid sequence set forth in SEQ ID NO: 1. Preferred fragments include, for example, a truncated polypeptide having a contiguous amino acid sequence of SEQ ID NO: 1.

 [0024] In one aspect of the invention, the above polypeptide is provided, wherein the fragment of (b) is a fragment comprising 8 or 9 amino acids in the amino acid sequence of SEQ ID NO: 1 . In one aspect of the invention, the above polypeptide is provided, wherein the fragment in (b) is a fragment comprising or having 10 consecutive amino acids in the amino acid sequence set forth in SEQ ID NO: 1.

 [0025] In yet another aspect of the invention, the above polypeptide is provided having 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), amino acid sequence of TQSPFFLLLL (SEQ ID NO: 7).

In one aspect of the invention, the polypeptide or derivative thereof, wherein the derivative has the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 1 above The amino acid sequence obtained after substitution, deletion, insertion, and addition of an amino acid occurs. Preferably, the amino acid sequence obtained after substitution, deletion, insertion, and addition of one to three amino acids. [0027] In one aspect of the present invention, the polypeptide may bind HLA I molecules and ₈ ⁺ T cells recognize CD. CD ₈ ⁺ T cells are also known as cytotoxic T cells (CTLs). In still another aspect of the invention, wherein the HLA I is HLA A2 or HLA A3 type.

 [0028] As used herein, "substantially identical amino acid sequence" refers to the substitution, deletion, addition or insertion of one to several (eg, 2, 3, 4 or 5) amino acids in an amino acid sequence, which is associated with the amino acid sequence. It has the same or similar or better activity. In the present invention, the activity may mean, for example, the activity recognized by HLA I and HLA II and thereby eliciting tumor-associated T cells. Methods include, for example, the methods described in Peptide Synthesis, Interscience, New York, 1966.

 [0030] The polypeptides of the invention may also be prepared by conventional genetic engineering. For example, the polypeptide encoding the polypeptide can be prepared using conventional DNA synthesis and genetic engineering methods to prepare the polypeptide. That is, the polypeptide is prepared by the following method: inserting the above polynucleotide into a usual expression vector; transforming the 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, by the method described in Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983).

 [0031] The present invention also provides an isolated nucleic acid consisting of the base sequence encoding the above polypeptide of the present invention. The nucleic acid of the present invention may be a cDNA, mRNA or DNA/RNA chimera, preferably DNA. The nucleic acid can be double stranded or single stranded. When the nucleic acid is double-stranded, it can be double-stranded

DNA, double-stranded RNA or DNA: RNA hybrids. When the polynucleotide of the present invention is double-stranded, it can be inserted into an expression vector to produce a recombinant expression vector for expressing the peptide of the present invention. Accordingly, 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-described 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 polypeptide of the present invention after translation. The base sequence encoding the amino acid sequence can be obtained by PCR or the like using genomic DNA or RNA containing a mucin sequence as a template. On the other hand, in view of the efficiency of expression in a host cell, it is generally preferred to select a codon which is very often used for the host cell used. The frequency of use of codons in various biological species can be obtained from the database of genetic code usage frequencies. The nucleic acid of the present invention can also be chemically synthesized by a DNA/RNA automatic synthesizer.

[0033] In one aspect of the invention, vaccines and pharmaceutical compositions comprising the polypeptides or nucleic acids of the invention described above are provided. The vaccines and pharmaceutical compositions provided herein are useful for treating or preventing cancer. The above polypeptides of the invention are useful as vaccines and pharmaceutical compositions for the treatment or prevention of cancer. On The nucleic acids of the invention are also useful as vaccines and pharmaceutical compositions for the treatment or prevention of cancer. The nucleic acid of the present invention can be expressed in a conventional manner to obtain a polypeptide of the present invention, which is further used for the above use.

[0034] The polypeptide of the present invention can be presented to an HLA antigen of an antigen presenting cell, thereby treating or preventing a tumor in a patient. The polypeptide of the present invention can be presented to the HLA of the antigen presenting cell, and specifically activates a T cell capable of recognizing the binding complex of the HLA antigen and the presenting polypeptide, particularly CD ₈ ⁺ T cells, which can proliferate to kill the tumor cell, It can therefore be used to treat or prevent tumors in patients.

[0035] The polypeptide of the present invention comprising as an active ingredient for inducing T cells, especially CD ₈ ⁺ T cells can, for example, an agent or a suitable adjuvant is administered in combination with a pharmaceutically acceptable carrier, to effectively establish cellular immunity . Examples of adjuvants that can be used include those described in the literature by Clin. Microbiol. Rev _.: 7:277-289, 1994, which is incorporated herein by reference.

[0036] The polypeptide of the present invention is capable of particularly efficiently presenting and inducing specific cytotoxic T cells (CTLs) in two large sizes of HLAA2 and HLAA3. HLA A2 models include A*0201, A*0202, A*0204, A*0205, A*0206, A*0207, and A*0208. HLA A3 includes A*0301, A*1101, A*3101, and A*6801. The total average distribution frequency of these two large Chinese people is over 85%. Therefore, the polypeptide of the present invention and the vaccine or pharmaceutical composition for preventing and treating tumors based on the polypeptide of the present invention can effectively cover most of the population. Preferably, the polypeptide of the present invention is capable of efficiently presenting and inducing specific cytotoxic T cells (CTLs) in the HLA class A2. More preferably, the polypeptide of the present invention is capable of efficiently presenting and inducing specific cytotoxic T cells (CTL) in the A*0201, A*0202 type.

 [0037] A vaccine for inducing a prophylactic or therapeutic immune response comprising a polypeptide or derivative of the present invention as an active ingredient can be administered in admixture or 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, microbial-derived components or derivatives thereof, cytokines, plant-derived components or derivatives thereof, marine-derived components or derivatives thereof, mineral gels such as Aluminium hydroxide, lysolecithin, surfactants such as polyols, polyanions, peptides, oily emulsifiers (emulsifier formulations) and the like. Further, a lipid system agent, a nanoparticle preparation linked to a bead having a diameter of several micrometers, a preparation having a linked lipid, a microsphere preparation, a microcapsule preparation or the like can also be considered.

The method of administration of the above vaccine or pharmaceutical composition of the present invention includes 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, the age and body weight of the patient, and the like. Generally, the dosage of the peptide of the invention in a formulation It is from 0.0001 to 1000 mg, preferably from 0.001 to 1000 mg, more preferably from 0.1 to 10 mg, preferably once every few days or from one to several months.

[0040] The vaccines and pharmaceutical compositions of the invention described above can also be used to treat tumor patients by in vitro methods. In other words, the polypeptide or nucleic acid of the present invention can be contacted with antigen presenting cells and/or immune effector cells in vitro to produce antigen presenting cells capable of recognizing the antigen or antigen complex of the present invention, thereby inducing specific T cells, particularly CDs. ₈ ⁺ T cells are then returned to the patient for prevention or treatment of cancer. The present invention provides T cells, particularly CD ₈ ⁺ T cells, which are induced by contacting peripheral blood lymphocytes derived from a tumor patient with a polypeptide or nucleic acid of the present invention in vitro, and a method of producing the above cells.

 [0041] In one aspect of the invention, methods of producing antigen presenting cells are provided. In one aspect of the invention, the method comprises the step of contacting a polypeptide of the invention described above with a cell having antigen presentation ability. In one aspect of the invention, the method comprises the step of contacting a nucleic acid of the invention described above with a cell having antigen presentation 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. The antigen-presenting cells can be produced by contacting the polypeptide or nucleic acid of the present invention with cells 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 produced by contacting the polypeptide or nucleic acid of the present invention with a cell having antigen-presenting ability in vitro. The polypeptides and nucleic acids of the invention can also be used in vivo for the prevention or treatment of tumors.

 [0042] In the present invention, a cell having an antigen-presenting ability is a cell which expresses an HLA antigen presenting a polypeptide on the surface of the cell. One of the cells having antigen-presenting ability is a dendritic cell.

[0043] The present invention provides an antigen presenting cell which expresses a cell which presents an HLA antigen of the polypeptide of the present invention on the surface of the cell. One of the cells having antigen-presenting ability is prepared by cells having a dendritic antigen-presenting ability.

 [0044] The antigen presenting cells of the present invention can be obtained by isolating cells having antigen presenting ability from tumor patients, stimulating cells in vitro with the polypeptide of the present invention, and allowing antigen presenting cells to present a complex of HLA antigen and polypeptide. Things. When dendritic cells are used, lymphocytes can be isolated from the peripheral blood of tumor patients, cells that cannot adhere to the cells are removed, adherent cells are cultured in the presence of GM-CSF and IL-4 to induce dendritic cells, and culture. The antigen presenting cells of the present invention are prepared by stimulating dendritic cells with the peptide of the present invention.

[0045] The antigen presenting cells of the present invention are prepared by adding the nucleic acid of the present invention to a cell having antigen presenting ability. The nucleic acid can be in the form of DNA or RNA. The nucleic acid can be The polypeptide of the present invention is expressed in a cell having antigen-presenting ability.

[0046] The present invention provides an immune effector cell which is capable of specifically recognizing a surface-presenting presenting cell which presents a polypeptide of the present invention and an HLA antigen complex, which is activated and proliferated, and differentiated into an effector cell. Various effector cells include T cells, such as CD ₈ ⁺ T cells and CD ₄ ⁺ T cells. In one aspect, the present invention provides an immune effector cells, which are cells in CD ₈ ⁺ T, lethal to cancer cells, the cancer cells rupture and death, has become a memory cell, encounter the same antigen stimulation again At the time, it will proliferate more rapidly into effector cells.

[0047] The immune effector cells of the present invention can be obtained by adding a polypeptide or nucleic acid of the present invention to a cell having antigen-presenting ability, and then contacting a presenting cell presenting the polypeptide of the present invention and an HLA antigen complex with an immunogenic cell. And prepared. Vaccine or combination of drugs ^. The antigen presenting cells of the present invention have an immunologically inducible activity, and can be used to prepare an agent for inducing antigen-specific effector cells. The induced CD ₈ ⁺ T cells are capable of exerting an anti-tumor effect through cytotoxicity and production of lymphokines. Therefore, the antigen presenting cell of the present invention may be an active ingredient of a vaccine or a pharmaceutical composition for treating or preventing a tumor. The vaccine for inducing CTL containing antigen presenting cells as an active ingredient may include saline, phosphate buffer (PBS), medium, or the like to stably maintain antigen presenting cells. Methods of administration include intravenous administration. An agent for inducing CD ₈ ⁺ T cells containing an antigen presenting cell as an active ingredient can be returned to a patient's body, thereby efficiently inducing specific CD ₈ ⁺ T cells in a patient responsive to the polypeptide of the present invention, and the result can be treated or Prevent cancer.

[0049] The immune effector cells of the present invention are useful as vaccines or pharmaceutical compositions for treating or preventing cancer. The effect fineness of the present invention includes various T fines such as CD ₈ + T fine. The CD ₈ ⁺ T cells of the present invention are capable of exerting an antitumor effect through cytotoxicity and production of lymphokines. because

Saline, phosphate buffer (PBS), medium, etc. are used to stably maintain immune effector cells. Methods of administration include intravenous administration. The agent containing the immune effector cells as an active ingredient can be returned to the patient's body, and as a result, the tumor can be treated or prevented.

[0050] In one aspect of the invention, there are provided vaccines and pharmaceutical compositions comprising the antigen presenting cells or immune effector cells of the invention described above. The vaccines and pharmaceutical compositions provided by the present invention are useful as vaccines and pharmaceutical compositions for treating or preventing cancer. The substance can be used to treat tumor patients by an in vitro method. The polypeptide or nucleic acid of the present invention can be contacted with antigen presenting cells and/or immune effector cells in vitro to produce antigen presenting cells capable of recognizing the antigen or antigen complex of the present invention, thereby inducing specific effector cells, such as T cells (especially For preventing or treating cancer. In one aspect of the invention, the patient is of HLA type I, preferably HLA A2 or HLA A3 type, more preferably HLA A2 type, most preferably A*0201 or A*0202 type.

[0052] The immune effector cells of the present invention, such as T cells (particularly CD ₈ ⁺ T cells), can be used as an active ingredient of a vaccine or pharmaceutical composition for treating or preventing tumors.

The polypeptide or nucleic acid of the present invention described above, as well as the above-described antigen presenting cells of the present invention or the immunological effect, can be used for the prevention or treatment of cancer. The cancer includes blood cancer, solid tumor, and the like, and more specifically, includes lung cancer, malignant lymphoma (eg, reticulum sarcoma, lymphosarcoma, Hodgkin's disease, etc.), digestive organ cancer (eg, gastric cancer, biliary fistula) Cancer, cholangiocarcinoma, pancreatic cancer, liver cancer, colon cancer, rectal cancer, etc.), breast cancer, ovarian cancer, musculoskeletal sarcoma (such as osteosarcoma (osteosarcoma), bladder cancer, leukemia (such as acute) Leukemia, including acute exacerbation of chronic myeloid leukemia, kidney cancer, prostate cancer, etc., preferably digestive organ cancer, such as gastric cancer, biliary cancer, cholangiocarcinoma, pancreatic cancer, liver cancer, colon cancer, rectal cancer, and the like. Preferably, it is liver cancer.

 [0054] The invention also provides a polypeptide of the invention, or a variant thereof, as described above, or a nucleic acid of the invention as described above, or an antigen presenting cell of the invention as described above, or as previously described The use of the immune effector cells of the invention in the preparation of a vaccine or pharmaceutical composition for the treatment or prevention of cancer. The cancer includes blood cancer, solid tumor, and the like, and more specifically, includes lung cancer, malignant lymphoma (eg, reticulum sarcoma, lymphosarcoma, Hodgkin's disease, etc.), digestive organ cancer (eg, gastric cancer, biliary fistula) Cancer, cholangiocarcinoma, pancreatic cancer, liver cancer, colon cancer, rectal cancer, etc.), breast cancer, ovarian cancer, months and bones! ^Musculoskeletal sarcoma (such as osteosarcoma, etc.), bladder cancer, leukemia (such as acute leukemia, including acute exacerbation of chronic myeloid leukemia), kidney cancer, prostate cancer, etc., preferably digestive organ cancer, such as gastric cancer , biliary cancer, cholangiocarcinoma, pancreatic cancer, liver cancer, colon cancer, rectal cancer, etc. Preferably, it is liver cancer. DRAWINGS

 [0055] Figure 1 a Cell line MUC 1 protein expression Western immunoblot assay.

 [0056] Figure 1 b Cell line HLA-A2 protein expression Western immunoblot assay.

[0057] Figure 2a T cell proliferation stimulated by dendritic cells presenting a polypeptide of the invention. [0058] Figure 2b T fine form of the control group.

 [0059] Figure 2c shows the T cell morphology stimulated by DCs of the antigenic peptide.

 [0060] Figure 3. T cell killing tumor cell effect.

 [0061] Figure 4. T cell killing tumor cell effect.

 [0062] Figure 5. T cell killing tumor cell effect.

 [0063] Figure 6. T cell killing tumor cell effect.

 [0064] Figure 7. T cell killing tumor cell effect.

 [0065] Figure 8 T cells secrete the cytokine IFNy.

 [0066] Figure 9. Mature DC cell surface markers.

 [0067] Figure 10a T cell killing tumor cell effect.

 [0068] Figure 10b T cell killing tumor cell effect.

 [0069] Figure 11 Cell line MUC 1 protein expression Western immunoblot assay.

 [0070] Figure 12a Antigen peptides treat tumor effects in mice.

 [0071] Figure 12b Antigenic tumors prevent tumor effects in mice. detailed description

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

 Example 1 Synthesis of Antigen Peptides

 [0074] Synthetic polypeptide MTPGTQSPFFLLLLLTVLTVVTGS (SEQ ID NO :

1), the following cartridge is called an antigen peptide. The synthesized polypeptide was purified to 97%. The lyophilized antigen peptide was dissolved in disulfoxide (25 mM) and stored at -80 °C.

 [0075] The polypeptide 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), respectively referred to as antigen peptide 1, antigen peptide 2, antigen peptide 3, antigen peptide 4, antigen peptide 5 and antigen peptide 6.

[0076] Example 2 Preparation of Dendritic Cells (DCs)

075左右。 [0077] Peripheral blood mononuclear cells (PBMC) including lymphocytes and monocytes, the volume and shape specific gravity of about 1. 075 or so. Density gradient centrifugation was performed using a Ficoll-Hypaque separation solution with a specific gravity of 1.077 to separate cells of a certain specific gravity according to a corresponding density gradient, and various blood cells were separated. [0078] Obtain white blood cell concentrate from healthy volunteers from Hong Kong Red Cross Blood Center, dilute them with PBS in appropriate multiples, then add to the surface of Ficoll-Hypaque separation solution, centrifuge at 18-20 ° C, 800 g density gradient for 30 min, centrifuge At the end, the contents of the centrifuge tube are divided into three layers, the upper layer is plasma (containing platelets), the middle layer is separation liquid, the bottom layer is red blood cells and granulocytes, and the milky white turbid PBMC layer is visible at the upper and middle liquid interface. The PBMC layer was taken out, resuspended in PBS, centrifuged at 400 g for 10 min, and the above steps were repeated. The cell pellet was resuspended in RPMI 1640 containing 10% fetal calf serum, and cultured at 37 ° C for 2 hours, and non-adherent cells were taken out for use. T cells were isolated, and RPMI 1640 medium containing lOOOU/ml hGM-CSF and 500 U/ml hIL-4 was added to the adherent cells, and cultured at 37 ° C for 1 week to induce differentiation of monocytes into DCs, two every two days. Change the medium and cytokines to harvest non-adsorbed and loosely adsorbed cells, which are DCs.

Example 3 Preparation of T cells (Nylon cotton method)

 [0080] The surface of T cells is short and the fluff is short, and the B cell villi are long and long. Due to the different smoothness of the cell surface, B cells are easily attached to nylon cotton fibers at 37 ° C, but T cells do not have this ability. Using this property, T cells and B cells can be isolated.

[0081] Preparation of nylon cotton column: After lg nylon cotton/column sterilization treatment, RPMI 1640 medium was added, and it was allowed to stand at 37 ° C for 2 hours, and RPMI 1640 was allowed to flow out. The above non-adherent cells were resuspended in an appropriate volume of RPMI 1640 to a cell concentration of 0.5-1.0× ^{10 8} /ml, and then added to the prepared nylon cotton column, 2 ml/column, and incubated at 37 ° C for 1 hour. Add RPMI 1640, 10 ml/column to a nylon cotton column, elute the cells that are not adsorbed onto the nylon cotton, and collect the eluate, which is a T cell suspension. After centrifugation at 400 g for 5 min, the cell pellet was resuspended in the cell cryopreservation solution and stored frozen for use.

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

 [0083] The premise of cell proliferation is the replication of cytoplasm and nucleus. Generally, one cell cycle is roughly divided into four periods, namely, G1 phase, S phase, G2 phase, and M phase. Among them, the S phase is a DNA synthesis phase, and its main function is to perform DNA synthesis. 3H-TdR (mercapto-3H) Thymine nucleotide is a precursor of DNA synthesis. When it is added to a cell culture solution, it is taken up by cells and used as a 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.

[0084] To the culture solution of DCs, an appropriate concentration of antigen such as each antigen peptide of the experiment was added, and the cells were incubated at 37 ° C for 4 hours, DCs were harvested, and washed once with PBS, according to a certain cell ratio, from the same volunteer. DCs and T cells were added to a 96-well flat-bottomed plate. After co-cultivation at 37 °C for 5 days, 3H-TdR, luCi/well was added and incubated at 37 °C for 18 hours. Using a multi-head cell harvester, The cells were collected on glass fiber filter paper and washed three times with PBS, 5% trichloroacetic acid and absolute ethanol, and the filter paper was dried, and the filter paper was placed in a scintillation fluid, and the cpm value was measured on a β liquid scintillation counter.

[0085] Example 5 Determination of cytotoxic T lymphocytes (CTLs) (LDH method)

[0086] Lactate dehydrogenase (LDH) is one of the cytosolic enzymes of living cells, which normally cannot penetrate the cell membrane, but when the target cells are damaged by effector cells, the permeability of the cell membrane changes. The LDH is released into the media. LDH can convert oxidized coenzyme I (NAD) into reduced coenzyme I (NADH) in the process of catalyzing the production of pyruvic acid from lactic acid, which in turn reduces the iodonitro group by the hydrogen donor, pyridazine dioxime sulphate (PMS). Tetraoxazole chloride (INT), INT accepts a ruthenium compound in which H2+ is reduced to a purplish red color. The compound has a high absorption peak at 490 nm. Using the read A490nm as an index, the extent to which target cells are killed by effector cells is known.

[0087] To the culture solution of DCs, an appropriate concentration of antigen such as each antigen peptide of the experiment was added, and the cells were incubated at 37 ° C for 4 hours, DCs were harvested, and washed once with PBS, DCs from the same volunteers were used according to a certain ratio. T cells were added to a 24-well culture plate and co-cultured for 7-10 days at 37 ° C. 20 U/ml hIL-2 was added to the culture solution, and T cells were collected by Ficoll-Hypaque density gradient centrifugation to obtain effector cells. 5 mM EDTA digests tumor cell line (e.g., SMMC-7721 or K562) cells to serve as target cells.

 [0088] The effector cells and target cells were separately resuspended in a volume of assay medium (RPMI 1640 containing 1% BSA), and effector cells (100 ul) and target cells (100 ul) were added to 96 at different ratios. In a round bottom culture plate, incubate at 37 ° C for 5 hours, remove the supernatant lOOul / well, and add to the 96-well microplate, add lOOul / well LDH reaction solution, reflect at room temperature for 30min in the dark, and add 1M HCL to terminate Liquid, 50 ul / well, A490nm, A630nm was determined as the reference wavelength. Calculation of cytotoxicity: Cytotoxicity (%) = [(killing test well - spontaneous release of target cells - spontaneous release of effector cells) I (maximum release pore - spontaneous release of target cells)] χ ΐοο%.

 [0089] Note: the killing test well is the effector cell + target cell; the target cell spontaneous release hole is the target cell + assay medium; the effector cell spontaneous release hole is the effector cell + assay medium; the maximum release hole is the target cell + 2% TritionX 100.

[0090] Example 6 ELISA-spot (ELISPOT) method for the determination of IFNy

[0091] The cytokine ELISPOT method is used to determine the number of cytokine-secreting cells in a single cell suspension, which is fast, highly sensitive, and easy to handle. The principle is to first coat the high-affinity anti-cytokine antibody on the ELISPOT plate, when adding the cells to be tested. After ELISPOT plate, the cytokine secreted by it will be captured by the coated antibody, so after removing the cell suspension to be tested, add another labeled anti-cytokine antibody, and then add the corresponding display. After the color reagent, spots representing the amount and location of cytokine secretion can be produced on the ELISPOT plate.

 [0092] Adding an appropriate concentration of antigen such as each antigen peptide of the experiment to the culture solution of DCs, incubating at 37 ° C for 4 hours, harvesting DCs, and washing once with PBS, according to a certain ratio, DCs from the same volunteers and T cells were added to 24-well culture plates and co-cultured for 7-10 days at 37 °C. 20 U/ml hIL-2 was added to the culture medium, and Ficoll-Hypaque density gradient was used to centrifuge and collect T cells as effector cells, using 5 mM EDTA. SMMC-7721 was digested and treated with γ-ray radiation as target cells. Effector cells and target cells were resuspended in a certain volume of medium, and effector cells (100 ul) and target cells (lOOul) were added at a certain ratio. To a 96-well ELISPOT reaction plate pre-coated with IFNy antibody, incubated at 37 ° C for 18 hours, remove the cell suspension, wash 10 times with PBST, add biotin-labeled IFNy antibody, incubate for 2 hours at 37 ° C, PBST Wash 10 times, add enzyme-labeled avidin antibody, incubate for 2 hours at 37 °C, wash 10 times with PBST, add substrate solution, react at room temperature for 15-30 min in the dark, using ELISPOT automatic reader Count.

Example 7 Flow Cytometry (FACS)

 [0094] Preparation of single cell suspension: The cells to be tested were harvested, and the cells were washed with cold PBS, the cells were resuspended in 50 ul/tube PBS, the corresponding antibodies were added, placed on ice for 1 hour, and washed twice with cold PBS. FITC-labeled secondary antibody was added, placed on ice for 30 min, washed twice with cold PBS, and the cells were resuspended in 2% polyfurfural for flow cytometry.

[0095] Example 8 Western blotting of tumor cell lines with MUC1 protein and HLA-A2 protein.

 [0096] SDS-PAGE and Western immunoblotting are commonly used in the art, that is, the protein is separated by one-way electrophoresis and then transferred to a nitrocellulose filter, and then the specific antigen is detected by radioactive or enzyme-labeled specific antibody. The method to identify proteins contained in tumor cells.

[0097] The tumor cell lines SMMC-7721 and SMMC-7721-0201 were all used by Shima. SMMC-7721 is a commercially available liver cancer tumor cell line provided by the Shanghai Institute of Cell Biology (see Lu J., Xu RB and Doung RC (1985) The glucocorticoid receptors and the induction induction tyrosine aminotransferase by glucocorticoid in the human liver cancer Cell line (SMMC-7721) in vitro. Shi Yan Sheng Wu Xue Bao 18: 231-238 ). SMMC- 7721-0201 is a cell line that transfects and stably expresses the human HLA-0201 gene in SMMC-7721.

 [0098] The tumor cells SMMC-7721 and SMMC-7721 - 0201 were first digested with 0.25% trypsin, and then the cells were blown off with 1640 complete medium (containing 10% FBS and 1% cyan-streptomycin) and collected in 15 ml centrifugation. In the tube. Centrifuge at 1000 rpm for 5 min and discard the supernatant. Resuspend the cells in 80μl PBS (the amount of cells can be increased by PBS volume), then add 5xSDS-PAGE to load Buffer, mix and boil for 5 minutes in boiling water bath.

 [0099] After sample treatment, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblotting experiments were carried out according to a conventional method. The primary antibodies in Western blotting were anti-Mucl monoclonal antibody and anti-HLA-A2 monoclonal antibody (BB7.2 cell supernatant;), and the secondary antibody was goat anti-mouse IgG-HRP.

[00100] Figures la and lb are the results of SDS-PAGE and Western immunoblot experiments.

[00101] wherein the first lane of the map la is a sample of the SMMC-7721-0201 cell line, and the second lane is a sample of the SMMC-7721 cell strain.

 [00102] wherein the first lane of Figure lb is a sample of SMMC-7721-0201 cell line and the second lane is a sample of SMMC-7721 cell line.

 [00103] As shown in Figure la, the tumor cell lines SMMC-7721 and SMMC-7721-0201 both expressed the MUC1 protein.

 [00104] As shown in Figure lb, the tumor cell line SMMC-7721 is HLA-A2 negative; SMMC-7721-0201 is HLA-A2 positive.

 [00105] Additional experiments also tested the expression of MHC I in SMMC-7721 and SMMC-7721-0201, demonstrating that the cell lines SMMC-7721 and SMMC-7721-0201 are MHC I positive.

[00106] Example 9 DCs loaded with antigenic peptides stimulate T cell proliferation

[00107] The antigenic peptide prepared in Example 1, ie, MTPGTQSPFFLLLLLTVLTVVTGS (SEQ ID NO: 1) was used to stimulate DCs, and then the obtained antigen-loaded DCs were used to stimulate the T cells of the antigen, and the DCs loaded with the antigen peptide were determined. Can stimulate the proliferation of its own T cells.

[00108] DCs were loaded with 10 ug/ml antigen peptide under load conditions for 4 hours at 37 ° C, then DCs were at different ratios to self T cells (DCs: T = 1:10 and DCs: T = 1:3) After co-cultivation, 3H-TdR was added after 5 days, and the uptake of 3H-TdR was measured 18 hours later to measure the proliferation of T cells. As a control, T cells are co-cultured with DCs that are not loaded with antigenic peptides, or T cells. Cultured separately. As shown in Figure 2a, the proliferation levels of T cells in each group were significantly different. At DCs: T = 1:10, the proliferation of T cells stimulated by DCs loaded with antigenic peptides was stimulated by unloaded DCs. The cell proliferation level was 10 times, and the cultured T cells were 15 times. When the DCs:T was increased to 1:3, the proliferation level of T cells was also significantly increased. These experimental results show that DCs loaded with antigenic peptides can effectively stimulate the proliferation of autologous T cells, while DCs without antigenic peptides can not stimulate the proliferation of autologous T cells.

 [00109] In addition, as shown in Figures 2b and 2c, in morphology, the T cells stimulated by the antigen peptide-loaded DCs are significantly different from the T cells of the control group, showing a significant activation state, such as an increase in the volume of the cells. Increase and so on.

 [00110] The same test was carried out on antigen peptide 1, antigen peptide 2, antigen peptide 3, antigen peptide 4, antigen peptide 5 and antigen peptide 6, and DCs loaded with antigen peptides were effective in stimulating the proliferation of autologous T cells.

[00111] Example 10 DCs loaded with each antigenic peptide are capable of activating tumor cell-specific CTLs

 [00112] CTLs play an extremely important role in the body's anti-tumor immune response. The antigen peptide prepared in Example 1 was used to stimulate DCs, and then the prepared antigen-loaded DCs were used to stimulate the T cells, and whether the antigen peptide-loaded DCs could activate the tumor cell-specific CTLs.

 [00113] DCs were loaded with different concentrations of antigenic peptides, incubated at 37 °C for 4 hours, then DCs were co-cultured with T cells at a ratio of DCs: T = 1:5, and after 7-10 days, T was determined by LDH method. The lethality of cells to liver cancer cell SMMC-7721-0201 expressing MUC1 protein. In the experiment, T cells were co-cultured with DCs loaded with antigen peptides, and as a control, T cells were co-cultured with DCs not loaded with antigen peptides, or T cells were cultured alone. As shown in Fig. 3, T cells stimulated by antigen peptide-loaded DCs can effectively kill tumor cells expressing MUC1 protein, while T cells co-cultured with DCs not loaded with antigen peptides and T cells cultured alone cannot. Kill tumor cells. As shown in Figure 4, when the concentration of the antigenic peptide of the DCs is increased, the killing ability of the T cells stimulated by the DCs to the tumor cells is also enhanced.

[00114] To determine the lethal effect of effector cells on tumor cells due to the activation of tumor cell-specific CTLs rather than the presence of non-specific killer NK cells, commercially available NK cell-sensitive cells are utilized in cytotoxicity assays. K56 ² (ATCC. Cat. no.: CCL-243) As a target cell, as shown in Fig. 5, T cells stimulated by DCs loaded with antigen peptide can effectively kill liver cancer cell SMMC-7721 expressing MUC1 protein. , SMMC-7721 is the source cell of the antigen peptide. However, T cells stimulated by DCs loaded with antigen peptides cannot kill K562 fine Cell. It is indicated that the killing of liver cancer cells is specific CTLs rather than non-specific NK cells.

[00115] In order to further determine the specificity for killing activity of liver cancer cells, human hepatoma cell lines SMMC-7721-0201 and SMMC-7721 were used as target cells in the cytotoxicity assay, and in addition, in order to determine whether the killing activity is MHC I Restriction, MHC I on the surface of target cells was blocked with antibodies against MHC I, as described in the results of Example 8, wherein SMMC-7721-0201 and SMMC-7721 were both MHC I and antigen peptide positive. As shown in the experimental results in Figure 6, the killing activity of CTLs on SMMC-7721-0201 was significantly higher than that on SMMC-7721. When MHC I on the surface of SMMC-7721 -0201 was blocked, the lethality of CTLs disappeared. Conversely, when the MHC I on the surface of SMMC-7721 was blocked, the lethality of CTLs did not change significantly. This indicates that the lethality of CTLs to SMMC-7721 is MHC I restricted, indicating that the killing activity against SMMC-7721 is mediated by CTLs.

[00116] Since the cell SMMC-7721, which is an antigen source, and the T cell as an effector cell are not from the same individual, the killing activity of T cells against SMMC-7721 may be an allogeneic response. In order to rule out the possibility of this allotype reaction, in the cytotoxicity assay, DCs loaded with antigen peptides and unloaded DCs were used as target cells. As shown in Figure 7, since DCs and T cells are from the same individual, T cells have no killing activity against unloaded DCs. Conversely, T cells exhibit significant killing activity against DCs loaded with antigenic peptides. Since the antigen peptide-loaded DCs process and present the tumor peptide bound by the antigen peptide, the tumor antigen is expressed on the cell surface, which indicates that the killing activity of CTLs on SMMC-7721 is an anti-tumor immune response specific to the tumor antigen rather than the same Atypical reaction.

[00117] The above experimental results show that the antigenic peptide-bound tumor antigen is successfully processed by DCs and presented to T cells, thereby effectively activating tumor cell-specific CTLs.

[00118] The same test was performed on antigen peptide 1, antigen peptide 2, antigen peptide 3, antigen peptide 4, antigen peptide 5 and antigen peptide 6, which were effective in activating tumor cell-specific CTLs.

[00119] Example 11 DCs loaded with antigenic peptides can stimulate T cells to secrete cytokines IFN y

 [00120] In an anti-tumor immune response, cell-mediated anti-tumor responses play a key role, in which CTLs and secretion of IFN gamma are major features of cellular responses.

[00121] It was determined whether DCs loaded with antigenic peptides can stimulate autologous T cells to secrete cytokine IFN gamma. DCs were loaded with 40 ug/ml of antigenic peptides for 4 hours at 37 ° C according to the method described in Example 6, and then DCs were co-cultured with T cells at a ratio of DCs: T = 1: 5, after 7-10 days, The condition in which T cells secrete IFN γ was measured. As a control, sputum cells and DCs without antigenic peptides Co-culture, or T cells are cultured separately. Stimulated autologous T cells were harvested by gradient centrifugation and IFN gamma secretion was determined by ELISPOT. The self-cultured cells and the unloaded antigen peptide DCs were co-cultured and the self-T cells themselves were used as controls. As shown in Figure 8, T cells stimulated by antigenic peptide-loaded DCs were able to secrete high levels of IFNγ when exposed to target cell SMMC-7721, and T cells stimulated by DCs without antigenic peptides and alone. Cultured T cells did not have significant IFN gamma secretion when they encountered the target cell SMMC-7721. The results of this experiment are consistent with the results of the above CTLs experiments, indicating that DCs loaded with antigenic peptides can activate tumor cell-specific T cells, which can secrete high levels of cytokine IFN gamma.

[00122] Example 12 Antigen peptide stimulation DCs maturation

 [00123] As described above, DCs loaded with antigenic peptides are capable of activating tumor cell-specific CTLs, suggesting that DCs have been processed to present tumor antigens bound by antigenic peptides and elicit a specific anti-tumor immune response. Due to the phenotypic shift of DCs, that is, the transition from immature DCs to mature DCs is a prerequisite for DCs to function. Therefore, it is possible to introduce phenotypic shifts in DCs loaded with antigenic peptides. To test this inference, it was determined whether the antigenic peptide can stimulate the maturation of DCs.

 [00124] DCs were incubated for 48 hours after incubation with 40 ug/ml of antigenic peptide at 37 ° C for 48 hours, and then the expression of HLA-DR, CD80, CD86, CD83 and CD40 on the surface of DCs was determined by flow cytometry. situation. As shown in Figure 9, after stimulation with antigenic peptides, the expression levels of HLA-DR, costimulatory molecules CD80 and CD86, and cell maturation markers CD83 and CD40 on DCs were significantly increased. This indicates that the antigenic peptide can effectively stimulate the maturation of DCs and is an effective activating molecule of DCs.

 [00125] The results of the above experiments show that each antigen peptide isolated and purified from tumor cells binds to tumor antigen and can effectively stimulate the maturation of DCs; and DCs loaded with antigen peptide can effectively process and present tumor antigens and activate tumors. Cell-specific anti-tumor cellular immune responses, including stimulation of T cell proliferation, activation of tumor cell-specific CTLs, and stimulation of T cell secretion of cytokine IFN y.

[00126] Example 13 DCs loaded with antigenic peptides are capable of activating tumor cell-specific CTLs and effectively attacking tumor cells

 [00127] This experiment tested the HLA phenotype specificity of each antigenic peptide of the present invention.

[00128] The assay used a kit purchased from PerkinElmer (AD0116) for the cytotoxic T lymphocyte killing assay: [00129] Target cells were digested with 0.25% trypsin (tumor cells SMMC-7721 and SMMC-7721-020, in which both SMMC-7721 and SMMC-7721-0201 were positive for MUC1 protein; SMMC-7721 was negative for HLA-A2; SMMC -7721 -0201 is HLA-A2 positive) and then washed once with 1640 complete medium (containing 10% FBS and 1% cyan-streptomycin); resuspend the cells in 1640 complete medium, adjust the cell number to 1 x 10 ⁶ cells/ml. 1 ml of cells were added to Ιμΐ BATDA, placed in a 37 ° C, 5% C0 ₂ incubator for 15 min. Then, 200 g, centrifuged for 5 min, the supernatant was discarded, and washed 5 times with PBS containing 2% FBS, and centrifuged at 200 g for 5 min each time. Finally, the cells were resuspended in 1640 complete medium and the number of cells was adjusted to 5 x 10 ⁴ cells/ml.

[00130] The appropriate concentration of antigen, including antigenic peptide and antigenic peptide 2, was separately added to the culture solution of DCs by the method described in Example 9 to stimulate DCs, and then the obtained DCs loaded with the antigen peptide were used to stimulate themselves. T cells.

[00131] The stimulated CD8+ T cells were collected, centrifuged at 1000 rpm, the supernatant was discarded, CD8+ T cells were resuspended in 1640 complete medium, and the number of cells was adjusted to lx10 ⁶ cells/ml.

 [00132] Adding ΙΟΟμΙ CD8+ T cells and ΙΟΟμΙ tumor cells (BATDA-loaded) to

In a 96-well plate. At the same time, a blank control group (media control), tumor cell spontaneous release group (ΙΟΟμΙ tumor cells +100μ1 medium) and tumor cell maximum release group (ΙΟμΙ lysate +90μ1 medium +100μ1 tumor cells) were established, and Τ cells were established without stimulation. group. The experimental group needs to set up 3 duplicate holes.

 [00133] The 96-well plate was placed at 37 ° C, cultured in a 5% CO 2 incubator for 2 h, centrifuged at 200 g for 5 min, and 20 μl of the supernatant was aspirated into an Elisa plate (provided with the kit), and a 200 μl Europium Solution was used. Shake for 15 min at room temperature. The detection was performed using a PerkinElmer instrument ( Victor2 V multilabel counter ), and an excitation filter was selected at 615 nm.

[00134] Calculation of experimental results: Experimental group - spontaneous release group of tumor cells

Attack effect (%) = 100% tumor cell maximal release group - tumor cell spontaneous release group

[00135] The results are shown in Figures 10a and 10b. Figure 10a shows the results of attacking tumor cells using antigen peptides loaded with DCs, and Figure 10b shows the results of attacking tumor cells after loading DCs with antigenic peptide 2.

[00136] The experimental results show that DCs stimulated T-packages loaded with antigen (including antigenic peptide and antigenic peptide 2) can kill MUC1 protein-positive SMMC-7721 and SMMC-7721-0201. The ability to kill SMMC-7721-0201 cells (HLA-A2+, MUC 1 + ) was higher than that of SMMC-7721 cells (HLA-A2-, MUCl+). It was demonstrated that the antigenic peptide of the present invention is more effective against HLA-A2 positive packets.

[00137] Example 14 Animal experiments: DCs loaded with antigenic peptides inhibit tumors in mice

[00138] 1. Construction of B16 (for animal experiments) cell line stably expressing MUC1 protein

[00139] B16 is a commercially available mouse melanoma cell line (ATCC CAT. NO. CRL-6322). The plasmid containing the MUC1 gene was purchased from Origene (Cat. No. RC221390).

[00140] B16 cells were digested with 0.25% trypsin and cells were blown off with 1640 complete medium (containing 10% FBS and 1% cyan-streptomycin). Then, an appropriate amount of cells were plated in a 24-well plate (three wells were inoculated), and after 36 hours, the plasmid containing the MUC1 gene was transfected into B16 cells by a lipofection method (plasma 0.5 g/well). The transfected cells were digested with 0.25% trypsin the next day, and ligated into all 24 wells to continue the culture. On the third day, the medium was changed, and the culture was continued by adding 1640 complete medium containing 1.2 mg/ml of G418.

 [00141] The cell death was observed every day, and the fluid was changed every other day; after four days, the viable cells were gradually stabilized, and the cells were subjected to limited dilution, and seeded into a 96-well plate at a cell volume of 0.5 cells/well (10-20 cells 96). Orifice). Observe the growth of cells in 96-well plates, look for monoclonal cells, and label them. The monoclonal cells which have been filled with cell wells are expanded and cultured, and the cells which are positive for MUC1 are identified by the Western method as described above. The positively identified cells were expanded and frozen.

 [00142] In Figure 11, lane 1 is an untransfected B16 cell line, lane 2 is a MUC1-4 monoclonal cell, and lane 3 is a MUC1-6 monoclonal cell.

 [00143] From the results of Figure 11, the two monoclonal cells of MUC1-4 and MUC1-6 showed significant bands and were positive. Therefore, two monoclonal cells of MUC 1-4 and MUC 1-6 were identified as B16 cell lines stably expressing MUC1 protein.

[00144] 2. Animal experiment

 [00145] Experimental mice (C57BL/6 mice, 6-8 weeks old) were divided into two groups: the treatment group and the immunoprotective group. In the treatment group, mouse tumor cells were first administered, and then the polypeptide of the present invention (antigen peptide and antigen peptide 2, respectively) were administered. In the immunoprotective group, the polypeptide of the present invention (antigen peptide and antigen peptide 2, respectively) is administered to a mouse, and then administered to tumor cells.

[00146] Treatment group: A total of 32 experimental mice were divided into 4 groups, 8 in each group, and labeled as A, B, C, D. Among them, 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 was subcutaneous to the right limb of the forelimb, and each mouse was injected with 1 x 104 cells at a dose of 200 μl. One week later, the peptides were injected. A total of 16 mice from the sputum and sputum were injected with PBS. Two groups of peptides were injected into the C and D groups: antigen peptide and antigen peptide 2, each mouse was injected.

40μ8/100μ1. In the next two weeks, the same peptides were injected once a week in the same group at the same dose. The tumor growth of the mice was observed every day, and the data were recorded.

 [00147] Figure 12a shows the effect of antigenic peptides in treating tumors in mice.

 [00148] wherein,

 [00149] B16-g: Group A, injected with B16 cells;

[00150] B16-mg: Group B, injected with B16-MUC1-4 cells;

[00151] B16-mg53: Group C, injected with B16-MUC1-4 cells and antigenic peptide 2;

[00152] B16-mg64: Group D, injected with B16-MUC1-4 cells and antigenic peptides.

[00153] Since the results of the two sets of B16-g and B16-mg are the same, the lines overlap.

[00154] Figure 12b shows the effect of antigenic peptide 2 immunization against tumors in mice.

[00155] wherein,

 [00156] B16-g: Group E, injected with B16 cells;

 [00157] B16-mg: Group F, injected with B16-MUC1-4 cells;

 [00158] B16-mg53: Group G, injected with B16-MUC1-4 cells and antigenic peptide 2;

 [00159] B16-mg64: Group H, injected with B16-MUC1-4 cells and antigenic peptides.

[00160] From the experimental results, mice injected with the polypeptide group were able to prolong their lifespan for more than two weeks as compared with the control group. A comparison between the antigen peptide and the antigen peptide 2 was given to give a high survival rate to the antigen peptide group. From the experimental group, the survival rate of the mice in the immunized group was higher than that in the treatment group.

 [00161] The results of the above experiments indicate that the polypeptide provided by the present invention comprises the antigen peptide of the amino acid sequence shown in SEQ ID NO: 1 and a fragment thereof, including the fragment of 8-10 amino acids in succession, especially 10 consecutive amino acids. (eg peptides FLLLLLTVLT, LLLLLTVLTV, LLLLTVLTVV, FFLLLLLTVL, GTQSPFFLLL, TQSPFFLLLL), which can effectively stimulate the maturation of tumor-specific DCs, and DCs loaded with antigenic peptides can efficiently process and present tumor antigens and activate tumor cell-specific Anti-tumor cellular immune responses, including stimulation of T cell proliferation, activation of tumor cell-specific CTLs, and stimulation of T cell secretion of cytokine IFNy.

[00162] Without being bound by any theory, the inventors explored the treatment and prophylaxis of antigenic polypeptides of the invention. Preventive action. Computationally assisted methods have become a mature, efficient and efficient method for analyzing the structure and function of proteins or polypeptides in recent years, and have been widely used in protein analysis and prediction including analysis of antigenic epitopes. Those skilled in the art will be able to summarize and identify residues in similar polypeptides that are important for activity or structure based on structural structural studies of the protein, thereby predicting the action of amino acid residues in the protein, and then analyzing the activity and function of the amino acid sequence of the protein. Computational assisted methods have become a more mature, efficient and efficient method for determining epitopes in recent years. Computational aids to determine epitopes By organizing and analyzing existing data, an epitope activity model is established according to the obtained rules, and a sequence of an antigenic epitope and its binding activity to an antigen or MHC are analyzed and predicted by a computer method.

 [00163] Generally speaking, the antigenic recognition region is characterized by being hydrophilic, located on the surface of the protein, and structurally deformable. Because in most natural (natural) environments, hydrophilic regions tend to concentrate on the surface of the protein, and hydrophobic regions are often entrapped inside the protein. Similarly, antibodies can only interact with recognition regions found on the surface of the protein. When these recognition regions have sufficient structural deformability and are transferred to a position where the antibody can be contacted, there is a high affinity with the antibody. The continuous and discontinuous recognition regions are recognition regions consisting of continuous or discontinuous amino acid sequences (residues). Many antibodies are epitopes directed against successive recognition regions, and antibodies can bind to such epitopes with high affinity. The discontinuous recognition region is a segment of a polypeptide that represents a certain fold, or an identification region of an antibody that links two separate polypeptides together. In some cases, antibodies to such discrete recognition regions can also be produced, but these antigenic polypeptides have a secondary structure similar to the discontinuous recognition region, and the length of the sequence needs to meet the relevant requirements. The length of the epitope is usually between 8-20 amino acid residues.

 [00164] Applied bioinformatics analysis, using epitope prediction sites such as http:〃 www.cbs.dtu.dk I services/ BepiPred/; http:// www.epitope-informatics.com/ Links.htm; http: // Bio.dfci.harvard.edu/ Tools/index.html, etc., and various software such as SYFPEITHL BIMAS, IMMUNE EPITOPE, MHC2PRED, etc., analyzed the antigen peptide of the present invention.

[00165] The difference in binding ability of different HLA-A, -B, -C to the antigen peptide of the present invention or a fragment thereof and its epitope sequence variant, particularly HLA A2 molecule and HLA A3 molecule, was analyzed by the above means, It has been found that an epitope having a highly efficient activation of CD8+ T cells is present in the antigenic peptide (SEQ ID NO: 1) of the present invention, including fragments thereof which are consecutively 8-10 amino acids, particularly 10 consecutive amino acids (eg polypeptide FLLLLLTVLT) , LLLLLTVLTV, LLLLTVLTVV, FFLLLLLTVL, GTQSPFFLLL, TQSPFFLLLL, etc.), and epitopes present in the antigenic peptide with efficient activation of CD4+ T cells, including: MTPGTQSPFF LLLLLTVLTV VTGS, MTPGTQSPFF LLLLL and SPFF LLLLLTVLTV VTGS, etc. These epitopes are in HLA A2 (including A*0201, A*0202, A*0204, A*0205, A*0206, A*0207, and A*0208) and HLA A3 (including A*0301, A*1101, A). *3101 and A*6801) have higher binding to HLA molecules.

 [00166] The present invention unexpectedly discovered an antigenic peptide of the amino acid sequence set forth in SEQ ID NO: 1 and fragments thereof, including fragments thereof that are consecutively 8-10 amino acids, particularly 10 consecutive amino acids (eg, polypeptide FLLLLLTVLT, LLLLLTVLTV, LLLLTVLTVV, FFLLLLLTVL, GTQSPFFLLL, TQSPFFLLLL), can effectively stimulate the maturation of tumor-specific DCs, and DCs loaded with antigenic peptides can efficiently process and present tumor antigens and activate tumor cell-specific anti-tumor cellular immune responses It can effectively treat or prevent tumors in animals. Accordingly, the present invention provides the use of a tumor polypeptide antigen as a cancer vaccine. Meanwhile, the present invention provides a DC tumor vaccine obtained by efficiently sensitizing dendritic cells, or a T cell tumor vaccine prepared by activating dendritic cells sensitized with the above tumor polypeptide antigen, and directly The use of these polypeptides or compositions is a method and use for the preparation of tumor vaccines.

[00167] Unless otherwise indicated, the practice of the present invention will employ <RTIgt;conventional</RTI><RTIgt;</RTI><RTIgt;</RTI><RTIgt;</RTI><RTIgt;</RTI><RTIgt;</RTI><RTIgt;</RTI><RTIgt; Other aspects and modifications within the scope of the invention will be apparent to those skilled in the art. Many variations and modifications are possible in light of the teachings of the invention and are therefore within the scope of the invention. All patents, patent applications, and scientific papers referred to herein are hereby incorporated by reference.

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 <120> Tumor antigenic polypeptides and their use as tumor vaccines

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Claims

Rights request

An isolated polypeptide or variant thereof, the polypeptide comprising an amino acid sequence identical or substantially identical to the amino acid sequence of (a) or (b) below:

 (a) the amino acid sequence shown as SEQ ID NO: 1;

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

2. The polypeptide of claim 1, or a variant thereof, which binds to an HLA I or HLA II molecule and is recognized by CD ₈ ⁺ T cells or CD ₄ ⁺ T cells.

 3. The polypeptide of claim 1 or 2, or variant thereof, wherein the variant has SEQ ID NO:

The amino acid sequence shown in 1 or the amino acid sequence shown in the above SEQ ID NO: 1 shows an amino acid sequence obtained by substitution, deletion, insertion, and addition of an amino acid.

 The polypeptide according to claim 1 or 2, or the variant thereof, wherein the fragment described in (b) is a fragment comprising 8 to 10 amino acids in the amino acid sequence of SEQ ID NO: 1, preferably 10 consecutive amino acids. Fragment of.

 5. The polypeptide of claim 4, or a variant thereof, having the amino acid sequence of FLLLLLTVLT, LLLLLTVLTV, LLLLTVLTVV, FFLLLLLTVL, GTQSPFFLLL, TQSPFFLLLL.

Polypeptide of any one of 1-5 or a variant thereof as claimed in claim 6, said fragment may bind HLA I molecules and ₈ ⁺ T cells recognize CD.

 7. The polypeptide of claim 6, or variant thereof, wherein said HLA I is HLA A2 or HLA A3, preferably HLAA2, more preferably A*0201.

 An isolated nucleic acid consisting essentially of the base sequence encoding the polypeptide of any one of claims 1-7.

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

An antigenic effector cell, which can recognize the polypeptide of any one of claims 1 to 7 or a variant thereof, or an antigen presenting cell which presents the polypeptide of any one of claims 1 to 7 or a variant thereof on the cell surface, preferably of the immune effector cell is a T cell, for example, CD ₈ ⁺ T cells or CD ₄ ⁺ T cells.

A method of producing an antigen presenting cell, the method comprising the step of contacting the polypeptide of any one of claims 1 to 7 or a variant thereof with a cell having antigen presenting ability, or comprising the nucleic acid of claim 8. In the step of expressing in a cell having antigen-presenting ability, preferably, the cell having antigen-presenting ability is a dendritic cell.

12. A method of producing an immune effector cell, the method comprising the step of contacting the antigen presenting cell of claim 9 with an immunogenic effector cell, preferably, the antigen presenting cell is a dendritic cell, and, preferably, wherein the immune effector cell is a T cell, for example, CD ₈ ⁺ T cells or CD ₄ ⁺ T cells.

 A vaccine or pharmaceutical composition for treating or preventing cancer in a patient, comprising the polypeptide of any one of claims 1 to 7, or a variant thereof, or comprising the nucleic acid of claim 8, or comprising the method of claim 9. An antigen presenting cell, or comprising the immune effector cell of claim 10.

 The vaccine or pharmaceutical composition according to claim 13, wherein the patient has an HLA of HLA type I, preferably HLA A2 or HLA A3 type, preferably HLA A2 type, more preferably A*0201 or A*0202 type.

 The vaccine or pharmaceutical composition according to claim 13 or 14, wherein the cancer is blood cancer, solid tumor or the like, preferably lung cancer, malignant lymphoma (e.g., reticulum sarcoma, lymphosarcoma, Hodgkin's disease, etc.), digestion Organ cancer (eg gastric cancer, biliary cancer, cholangiocarcinoma, pancreatic cancer, liver cancer, colon cancer, rectal cancer, etc.), breast cancer, ovarian cancer, musculoskeletal sarcoma (eg osteosarcoma, etc.), bladder cancer, leukemia (eg acute leukemia) , including acute exacerbation of chronic myeloid leukemia), kidney cancer, and prostate cancer.

 16. The polypeptide of any of claims 1-7, or a variant thereof, or the nucleic acid of claim 8, or the antigen presenting cell of claim 9, or the immune effector cell of claim 10, for use in the preparation of a medicament for the treatment or prevention of cancer Use in a vaccine or pharmaceutical composition.

 Use of the polypeptide of any one of claims 1 to 7 or a variant thereof, or the nucleic acid of claim 8, or the antigen presenting cell of claim 9, or the immune effector cell of claim 10 for the treatment or prevention of cancer .