TW201238596A - A cancer vaccine and the process of producing the same - Google Patents

Abstract

This invention relates to a cancer vaccine and the process of producing the cancer vaccine. The cancer vaccine comprises epidermal growth factor receptor 1 (ERBB1), epidermal growth factor receptor 2 (ERBB2), and mucin protein type 1 (MUC1) epitopes. The cancer vaccine can overcome the limitation of immune tolerance, exhibit cytotoxic effect on various kinds of tumor cells, and prevent tumor cells from relapsing.

Description

201238596 VI. Description of the Invention: [Technical Field] The present invention relates to a tumor vaccine and a method for synthesizing the same, which use a bioinformatics calculation method to design a sequence of a tumor-associated antigen, and then synthesize a tumor antigen to form A tumor vaccine. [Prior Art] Cancer, or malignant tumor, is a worrying health problem in all countries of the world. Despite years of development, new diagnostic techniques and various treatment methods, such as surgical resection and radiotherapy, have been developed. And chemotherapeutic 'but cancer is still the leading cause of death compared to common diabetes, heart disease, infectious diseases, and mental illness. Therefore, the primary task of the medical community today is to develop newer, more effective, and preventive treatments for tumor recurrence. For these reasons, scientists have shifted some of their efforts to study the relationship between the immune system and cancer, so tumor immunotherapy has evolved over the past decade into a promising approach to cancer treatment. Another treatment option. The so-called cancer immunotherapy refers to the use of the body's immune system to fight against tumors, and to kill cancer cells by stimulating or regulating the body's specific immunity to achieve cancer treatment. Dendritic cel 1, DCs are specialized antigen-presenting cells (CAntigen-pfesenting cells), which have recently been used in immunotherapy. Dendritic cells can present antigens to unactivated T cells, which in turn initiates cellular and humoral immunity. The cells express a large number of major histocompatibility complexes on the cell surface (Ma j 〇rs 3 201238596 his Tocompat ibi 1 i ty comp 1 ex, hereinafter referred to as MHC ) molecules and recognize a variety of antigens from bacteria, viruses, other pathogens and endogenous tumors. Dendritic cells carrying tumor antigens may be referred to as dendritic cell vaccines (DC vaccines) A dendritic cell vaccine can elicit an antigen-specific immune response against tumors. However, dendritic cells have limitations in immunotherapy for cancer. In the known art, dendritic cells are usually isolated from patients. After the tumor antigens of the tumor cells are co-cultured for a period of time, the dendritic cells can present the tumor antigen to the surface of the tau lymphocytes to induce an immune response against the tumor antigen in the human body. Since the dendritic cells of different individuals express differently MHC molecule, an MHC molecule can only recognize one or a certain class of antigenic determinants (Epitope), which is called M The mhc restricted response of HC. Therefore, immunotherapy is only performed with one tumor antigen, and its effect and poisoning are limited. Further, known dendritic cell immunotherapy can only be targeted at individual patients at the time. The cancer is tailor-made, and the process and preparation process is tedious and cost-effective. It is not easy to implement in terms of cost considerations. Because the immune system has the ability to recognize and destroy non-autologous antigens, the antigen expressed by the tumor (or Tumor antigens have become a tool for the development of cancer immunotherapy. Tumor antigens for immunotherapy can be distinguished into two types, one is tumor-specific antigen that only tumor cells will express (tumor-speci fic ant i gen , hereinafter referred to as TSA); another type of antigen is a tumor-associated antigen whose tumor cell expression is significantly higher than that of normal, 'field cell expression (additional traits such as (9) antigen, hereinafter referred to as TAA). It is not easy to find a TSA to prepare a tumor vaccine.

S 4 201238596 Another aspect: Since normal cells also exhibit TAA, the immune system treats TM as an autologous molecule. Therefore, the immune response and tumor rescue effect that can be induced by using TAA to prepare a tumor vaccine is very low. Tumor-associated antigens do not elicit a potent immune response, because the immune system is resistant to autoantigens such as TAA. A feature of the immune system is its tolerance, which plays a very important role in assisting the immune system to distinguish between autologous and foreign molecules. The tolerance of the immune system can be divided into two types, one is central tolerance and the other is peripheral tolerance. Central tolerance is manifested in the thymus (which produces tau lymphocytes) and bone marrow (which produces axillary lymphocytes). In the case of sputum lymphocytes, the affinity between the complex of autoantigen and MHC and the T cell receptors (TCR) is very important for the immune tolerance of tau cells. The autologous antigen recognized by the MHC and the T cell receptor with high affinity for the T cell receptor are eliminated by the negative screening effect, leaving only the T cells with low affinity to prevent the immune system from attacking the autologous cell tissue. Although this mechanism can protect the autologous cell tissue under normal conditions, when cancer occurs, it is no different than giving the cancer cell a protective umbrella, allowing the cancer cells to escape the human body's defense system and growing into a tumor in the human body. Ultimately endangering health. In the well-known technique of using tumor antigen as immunotherapy, only a single type of tumor can be treated' or only a cellular immune reaction can be triggered without stimulating a humoral immune response, and the effect is limited and cannot prevent cancer cell recurrence, and It is impossible to break through the tolerance mechanism of the human immune system, so the immune effect that can be triggered is not strong enough, resulting in vain treatment. In summary, immunotherapy, for the treatment of cancer, provides a new 5 201238596 but this is a well-known cancer immunotherapy, but there is a specific swelling in the mouth (four) can not break through the (four) disease resistance Responsibility, even the use of new (f) is time-consuming and the problem still needs to be overcome. And the inability to prevent recurrence, etc. [Explanation] The object of the present invention is to describe that "ΜΗΓ" ° is not limited to the tolerance of the immune system and the limitation of MHC, and can be used for fine attack by cancer cells and can prevent A tumor vaccine for cancer recurrence. Another object of the present invention is to provide a synthetic method for preparing a tumor vaccine which is not limited to immune system tolerance, capable of being attacked against a plurality of different cancer cells, and which can prevent cancer from recurring. Although the T cell receptor has a strong specificity for the identification of Teng, there is a preliminary study, and the Ur's stomach has different antigens and one or more molecules will drive the TCR antigen recognition ability to decline. It leads to the variability of the ability of tau cells to cross-react with autoantigens. A hypothesis is that infectious antigenic antigens such as parasites, mites and viruses may cause the immune system to reduce immunity to autoantigens due to some minor differences between pathogens and host autoantigens. Literary restrictions. Therefore, it is estimated that as long as the amino group 1 sequence of the antigen produced by sputum is changed, the dendritic cells can be assisted to activate the cytotoxic sputum cells, thereby triggering an autoimmune reaction against tumor cells expressing sputum. β~ 6 201238596 According to this theory, the inventors decided to design a tumor vaccine based on the amino acid sequence to cause the immune system to attack cancer cells. Most tumor vaccines are designed based on their binding to the major histocompatibility complex (HLA) of humans. A single-peptide epitope containing 8 to 1 amino acid can bind to the first type of cell surface major histocompatibility complex (leg CI) on dendritic cells to initiate cytotoxic T The reaction of cells (Cytotoxic T lymphocyte, CTL), and the peptide antigenic determinant that binds to the major histocompatibility complex (MHCn) on the surface of the second type of cell can activate helper T cells to assist CTL response and antibody production. MHCII differs from MHCI in that the antigen binding region of MHC II can accommodate peptide chains of different lengths, typically between 13 and 25 amino acid sequences. Since activated helper T cells can also assist in the activation of memory cytotoxic T cells, MHCII-related tumor vaccines can effectively prevent cancer from recurring after treatment. Therefore, the inventors decided to start research and development of a vaccine that is specific to MHCII, and to prepare a tumor vaccine that can be restricted to the tolerance of the immune system and capable of attacking a plurality of different cancer cells. In this month, Epidermai gr〇wth factor receptor (hereinafter referred to as EGFR or ERBB) and epithelial mucin 1 (MUChpr〇teintypel 'MUC1) were used as candidate genes for tumor vaccines. The epidermal growth factor receptor is a member of the Reie__t_inekinases family, which has an extracellular ligand binding domain, a transmembrane lipophilic fragment, and an intracellular protein f kinase. (prQteinkinases) ribbon. Η 7 201238596 There are currently four EGFR subtypes found in the human body, including ERBB1, ERBB2, ERBB3 and ERBB4. EGFR is highly expressed in a variety of different solid tumors, including colorectal cancer, breast cancer, bladder cancer, kidney cancer, lung cancer, sputum cancer, alternative prostate cancer, and head and neck cancer. Epithelial mucin 1 (hereinafter referred to as MUC1) is a glycosylated transmembrane protein, and MUC1 is abundantly expressed in some common epithelial malignancies, such as pancreatic cancer, breast cancer, prostate cancer, and colorectal cancer. Since MUC1 is involved in the rapid proliferation of malignant cells, MUC1 has been regarded as a functionally significant oncogene and is classified as a tumor-associated antigen for cancer immunotherapy. The present inventors have found a tumor antigen sequence having a higher affinity with a human leukocyte antigen (HLA) class II antigen (ie, a human cell surface major histocompatibility complex MHC II) by a bioinformatics method. The design was improved, and ERBB1, ERBB2 and MUC1 tumor antigen sequences which can specifically bind to HLA class II antigen molecules were developed, and 14 kinds were obtained, which were divided into wild type and mutant type, each group containing 7 kinds of antigens. These sequences are recognized by most HU class II antigens. The distribution of these HLA molecules can cover more than 99% of the Chinese population (^仙(3)丨 from the population). The 14 antigen sequences are shown as SEQ N〇.丨 to seq n〇. i4. An embodiment of the present invention provides a tumor vaccine comprising at least one tumor antigen, wherein the sequence of the tumor antigen is selected from the group consisting of SEQ NO. 1 to SEQ NO. 14, wherein each antigen sequence has a molecular weight in Daltons. They are: seq paper 1 : Chau (10); Qing 8 201238596 N〇. 2 : 4121 ; SEQ NO. 3 : 3729 ; SEQ NO. 4 : 3842 ; SEQ NO. 5 : 4003 ; SEQ NO , 6 . 3818 ; SEQ NO 7: 3901; SEQ NO. 8: 3954; SEQ NO. 9: 3885 'SEQ NO. l〇: 4010; SEQ NO. 11: 3927; SEQ NO. 12: 3866; SEQ NO-13: 3641; SEQ NO. 14 : 3629. As a preferred embodiment of the present invention, the tumor antigen sequence is composed of at least 30 amino acids. As a preferred embodiment of the present invention, the tumor antigen is synthesized by chemical or genetic engineering. As a preferred embodiment of the present invention, the tumor antigen has a specific epidermal growth factor receptor and an epithelial mucin determinant, and is a limiting antigen of the HLA class II antigen molecule. The present invention provides a method for synthesizing a tumor vaccine, which comprises the following steps: (a) selecting an epidermal growth factor receptor 1 and an epidermal growth factor according to the distribution of HLA class II antigen subtype molecules in a Chinese population; The epitope of the amino acid sequence of the body 2 and the epithelial mucin j can be recognized by these HLA class II antigen molecules; (b) bioinformatics analysis between the antigenic determinant and each HLA class II antigen molecule Affinity' and select the appropriate tumor antigen amino acid sequence; (c) According to the results of step (b), 14 tumor antigen sequences containing at least 30 amino acids were designed; 5 9 201238596 (d) by chemical method Or genetically engineering these tumor antigens; (e) mixing these synthetic 14 tumor antigens with dimethyl sulfoxide, respectively, so that the final concentration of each tumor antigen mixture is 20mg/mL to 40mg/mL, and These tumor antigen mixtures are divided into wild type and mutant type groups, each group includes 7 kinds of tumor antigen mixture; (f) The tumor antigen mixture in the same group is separately taken out of the same volume, and fully mixed in Together, a tumor vaccine containing 7 wild-type or mutant tumor antigens was obtained and the final concentration of each of the tumor antigens was 2 mg/mL to 4 mg/mL. As a preferred embodiment of the present invention, the antigenic determinants of these tumor antigens are recognized by HLA class II antigen molecules. The technical solutions of the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of them. Example. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts are within the scope of the present invention. 1. Tumor vaccine design and implementation First, human epidermal growth factor receptor 1 (ERBB1), epidermal growth factor receptor 2 (ERBB2) and epithelial mucin type KHuman mucin type 1 protein (MUC1) are used as candidate genes for cancer tumor vaccines. The amino acid sequence 201238596 translated by these candidate genes was aligned with the Han Chinese HLA class II antigen sequence. Next, bioinformatics methods were used to find tumor antigen amino acid sequences with high affinity to these Chinese HLA class II antigens. Among them, the bioinformatics method includes using a computer and software and other auxiliary tools to perform database search, using computer algorithms to compare and analyze the retrieved sequence data, and using computer algorithms to predict HLA class II antigen and ERBB丨, ERBB2. Affinity and the like with the MU C1 tumor antigen amino acid sequence. According to the results predicted by the aforementioned bioinformatics method, suitable tumor antigen sequences are selected and combined into a tumor vaccine containing at least 3 amino acids, and a preferred embodiment thereof contains 33 amino acids (33-mer). ). According to the above method, a better 14 tumor antigen sequences can be obtained, and finally these tumor antigens are synthesized by chemical method or genetic engineering method (the actual synthesis does not require the use of physical ERBB1, ERBB2, MUC1, etc., but bioinformatics) The method can be obtained). The above 14 ERBB1, ERBB2 and MUC1 tumor antigens can be synthesized by chemical methods or genetic engineering methods, and each synthesized tumor antigen is dissolved in an appropriate amount of dimercaptopurine (DMSO), and the final concentration is adjusted to 20 mg, respectively. /mL to 40mg/mL. Thereafter, a mixture of 14 tumor antigens was divided into two groups of wild type and mutant type, each group of 7 types. Each group (wild type or mutant group) 7 kinds of ERBB1, ERBB2 and MUC1 tumor antigen mixture were mixed together in an equal volume ratio to become a tumor vaccine, wherein the final concentration of each tumor antigen was adjusted to 2 mg/mL. Up to 4 mg/mL. 50/z 1 to 200 of 1 tumor vaccine was taken and mixed with dendritic cells. The preferred embodiment is 1 mL of dendritic cells (containing about 1 to 2 million dendritic cells per ml) plus 5//1-10/zl of tumor epidemic 201238596 w 'and the final concentration of tumor antigen. Please refer to Figure 1 for a dendritic cell tumor vaccine containing 2〇yg/mL to 4〇pg/mL' and 4 to 1* containing ERBB1, ERBB2 and MUC1. The tumor vaccine can be administered to the human body by means of dendritic cell culture in vitro or by subcutaneous injection, and the dendritic cells carrying the ERBB1 tumor antigen can activate the helper tau cells and induce immunity against ERBB1, ERBB2 and MUC1. Reactions include antibodies that secrete specific anti-ERBM, ERBB2 and MUC1 tumor antigens. Therefore, the tumor vaccine can be used to treat most solid tumors, and can trigger the immune repellent effect to prevent cancer recurrence, for example, to prevent tumor recurrence after initial treatment (such as surgical resection and radiotherapy). 2. In vitro culture of dendritic cells 2. 1. Preparation of dendritic cells All procedures are strictly in accordance with the standard operating procedures (s〇p). The patient was collected 100 ml of peripheral blood (containing 2-3×108 mononuclear cells) and heparin was anticoagulated. through

Ficoll-Paque (GE Health care Life Sciences) isolated mononuclear cells, prepared autologous plasma, washed twice with centrifugation, resuspended with serum-free RpMn64〇, adjusted the cell concentration to 2-4×10 6 /ml, and placed in a 6-well plate. After incubating for 2 hours at 37 ° C, 5% (: 2 incubator), gently shake the 6-well plate, collect the suspension cells to another 5 μml centrifuge tube, and wash the plate 1-2 times with serum-free RPMI1640 medium. Add the AIM_V medium containing 丨% autologous plasma to continue the culture; add the dendritic cell culture solution 2_3ml/well (clais〇n, Shanghai, China) on the third day; add the tumor-specific antigen on the fifth day (the final concentration is 〇_〇2-〇.04mg/ml), adding dendritic cell maturity factor 12-16 hours after induction

S 12 201238596 (Claison, Shanghai, China); cells were collected on day 8, centrifuged, washed with physiological saline 3 times, and resuspended in saline containing 10% of patient autologous plasma in a total volume of 100 ml. 2. 2. Cytological analysis: 10 ml of peripheral blood of patients was collected on the day of cell collection and the first and fourth weeks after injection, and fluorescent double labeling was performed with CD3 'HLA class II antigen and CD4 monoclonal antibody, respectively (FITC and PE or APC), analysis and detection of the percentage value (%) of the tau cell subset. If the percentage values of DR+CD3+ and CD4+CD3+ T cells rise with time, it indicates that the tau cells have been activated by the tumor antigen. Natural killer cells (Natural “Η, NK cel Is” can be entered at this time. 2.3·Antibody analysis 5 ml of peripheral blood of patients was collected on the day of cell collection and at the end of 1, 3, 6, 12 and 24 months of injection, and plasma was separated by centrifugation. Or serum. Enzyme-linked immunosorbent assay (ELISA) is used to detect the antibody levels of anti-ERBB1, ERBB2 and MUC1 tumor antigens in the blood. According to the antibody level, it is necessary to re-inject the vaccine. 3. Subcutaneous injection 3.1 · Vaccine dose and method of use The tumor vaccine can be used by subcutaneous multiple injection. Each treatment requires injection of 700-1400 micrograms (#g / time) of tumor antigen mixture (each antigen amount is 13 201238596 100- 200 micrograms), should be mixed with 100 micrograms of adjuvant: such as Granulocyte-macrophage colony-stimulating factor (GM-CSF) before injection. Inject once a month for 4-6 consecutive injections. In the future, depending on the antibody levels of anti-ERBB1, ERBB2 and MUC1 tumor antigens in the blood, it is necessary to re-inject the vaccine. 3. 2 . Antibody level analysis 5 ml of peripheral blood of patients was collected every 6 months at the first injection and each re-injection and after the end of treatment, and the plasma or serum was separated by centrifugation. The blood was detected by enzyme-linked immunosorbent assay (ELISA). Anti-ERBB1, ERBB2 and MUC1 tumor antigen antibody levels. Determine whether re-injection of vaccine is required according to antibody level. The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited to this. Those skilled in the art will be able to cover changes or substitutions that are easily conceivable within the scope of the present invention. Therefore, the scope of protection of the present invention should be protected by the scope of the claimed patent. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a flow chart showing a method for synthesizing a tumor vaccine according to the present invention. [Explanation of main component symbols] 201238596 Sequence Listing <110> Li Guanghui <120> A tumor vaccine and its synthesis Method <160> 14 <210> 1 <211> 33 <212> PRT < 213 > 213 > artificial sequence <220><223 As a tumor antigen preparation < 400 > SEQ NO.1

Ser Gly Asp Leu His lie Asp Pro Val Ala Phe Arg Leu His lie Leu 15 10 15

Pro Val Ala Phe Ser Tyr Val Leu lie Ala Thr Asn Thr Val Glu Arg 20 25 30

Lys <210> 2 <211> 33 <212> PRT <213>Artificial sequence <400> SEQ NO.2

Arg Gin Arg Leu Arg lie Tyr Arg Gly Thr Gin Leu Phe Gin Asn Leu 15 10 15

Gin Val lie Arg Ser Tyr Val Leu His Ala His Asn Gin Val Arg Gin 20 25 30

Lys 15 201238596 <210> 3 <211> 33 <212> PRT < 213 > 213 > artificial sequence < 220 < 223 > 223 > used as tumor antigen preparation <400> SEQ N0.

Glu Thr Gin Phe Asn Gln Tyr Lys Ser Glu Ala Ala Ser Val Ser Asn 1 5 l〇 15

Val Pro Phe Pro Phe Tyr Val Ser Asp Val Pro Phe Pro Phe Ser Ala 20 25 30

Gin <210> 4 <211> 33 <212> PRT <213> artificial sequence <220><223> used as tumor antigen preparation <400> SEQ NO. 4

Arg Glu Asn Leu Gin lie lie Arg Gly Asn Met Tyr Tyr Val Leu lie 15 10 15

Ala Leu Asn Thr Val Leu His lie Leu Pro Val Ala Phe Arg Gly Asp 20 25 30

Lys 16 201238596 &lt;210&gt; 5 &lt;211&gt; 33 &lt;212&gt; PRT &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt; used as tumor antigen preparation <400> SEQ NO.

Arg Gin Arg Leu Arg lie Val Arg Gly Thr Gin Leu Phe Gin Asn Leu 1 5 10 15 Gin Val lie Arg Gly Tyr Val Leu lie Ala His Asn Gin Val Arg Gin 20 25 30 Lys &lt;210〉 6 &lt;211〉 33 &lt;212&gt; PRT &lt; 213 &gt; artificial sequence &lt;220&gt;&lt;223&gt; used as tumor antigen preparation &lt;400&gt; SEQ N0.6

Arg Ser Tyr Tyr Asn Leu Thr lie Ser Asp Val Ser Ser Val Ser Asp 15 10 15

Val Pro Phe Pro Phe Phe Asn Gin Tyr Lys Thr Glu Ala Ala Ser Arg 20 25 30

Lys 17 5 201238596 &lt;210> 7 &lt;211> 33 &lt;212&gt; PRT <213> artificial sequence &lt;220&gt;<223&gt; used as tumor antigen preparation &lt;400&gt; SEQ NO. 7

Ser Gly Asp Leu His lie Asp Pro Val Ala Phe Arg Tyr Gin Arg Met 15 10 15

Phe Asn Asn Cys Glu Tyr Val Leu lie Ala Thr Asn Thr Val Glu Arg 20 25 30

Lys &lt;210> 8 &lt;211> 33 &lt;212&gt; PRT &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt; used as tumor antigen preparation &lt;400> SEQ NO.

Arg Glu lie lie Arg Gly Arg Thr Lys Gin His Gin Tyr Val Leu His 15 10 15

Ala Leu Asn Thr Val Leu Gin Arg Met Phe Asn Asn Cys Ser Val Val 20 25 30

Lys s 18 201238596 &lt;210&gt; 9 &lt;211&gt; 33 &lt;212&gt; PRT &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt; used as tumor antigen preparation &lt;400&gt; SEQ NO. 9

Arg Gly Asp Leu His lie Leu Pro Val Ala Phe Ser Leu His Ala Phe 15 10 15

Glu Arg Leu Glu lie Tyr Val Leu Tyr Ala Leu Asn Thr Val Glu Arg 20 25 30

Lys &lt;210&gt; 10 &lt;211&gt; 33 &lt;212&gt; PRT &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt; used as tumor antigen preparation &lt;400&gt; SEQ NO. 10

Arg Glu Gin Tyr Gin Val Phe Glu Thr Leu Glu Glu Tyr Thr Leu lie 15 10 15

Asp Thr Asn Arg Ser Tyr Val Leu His Ala His Asn Gin Val Arg Gin 20 25 30

Lys

S 19 201238596 &lt;210&gt; 11 &lt;211> 33 &lt;212&gt; PRT &lt; 213 &gt; 213 > artificial sequence &lt;220 &lt; 223 &gt; 223 &gt; used as tumor antigen preparation &lt;400&gt; SEQ NO.

Gin Leu Ala Leu Thr Leu Met Asp Thr Asn Arg Ser Phe Gin Asn Lys 15 10 15

Gin Val lie Arg Gly Leu Arg lie Tyr Arg Gly Thr Gin Leu Phe Glu 20 25 30

Asp &lt;210&gt; 12 &lt;211&gt; 33 &lt;212&gt; PRT &lt;213>Artificial sequence &lt;220&gt;&lt;223&gt; Used as tumor antigen preparation &lt;400>SEQ NO. 12

Thr Gly Tyr Leu Tyr lie Ser Ala Lys Pro Asp Ser Leu Arg lie Val 15 10 15

Arg Ser Thr Gin Leu Phe Gin Asn Leu Gin Val lie Arg Ser Arg lie 20 25 30

Lys 20 201238596 &lt;210&gt; 13 &lt;211&gt; 33 &lt;212> PRT &lt; 213 &gt; 213 > artificial sequence &lt;220 &lt; 223 &gt; 223 &gt; used as tumor antigen preparation &lt;400&gt; SEQ NO. 13

Arg Gly Ser Val Val Val Asp Leu Thr Leu Ala Phe Val Val Val Arg 1 5 10 15

Leu Thr Leu Ala Phe Val Val Val Gin Leu Thr Leu Ala Gin Arg Glu 20 25 30

Lys &lt;210&gt; 14 &lt;211&gt; 33 &lt;212&gt; PRT &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt; used as tumor antigen preparation &lt;400>SEQ NO. 14

Arg Gly Ser Val Val Val His Leu Thr Leu Ala Phe Val Val Val Tyr 15 10 15

Leu Thr Leu Ala Phe Val Val Val Gin Leu Thr Leu Ala Ser Arg Glu 2〇 25 30

Lys 21

Claims (1)

201238596 VII. Patent application scope: 1. A tumor vaccine comprising at least one tumor antigen, wherein the sequence of the tumor antigen is selected from the group consisting of SEQ Ν〇·1 to SEQ N.14, wherein each antigen sequence is in Dow The molecular weights of the units are: SEQ Ν〇·1: 3693; SEQ N0. 2: 4121; SEQ N0. 3: 3729; SEQ N0. 4: 3842; SEQ N0. 5: 4003; SEQ NO. 6: 3818 SEQ NO. 7 : 39〇ι ; SEQNO. 8 : 3954 ; SEQ NO. 9 : 3885 ; SEQ NO. 10 : 4010 ; SEQ NO. 11 : 3927 ; SEQ NO · 12 : 3866 ; SEQ NO · 13 : 3641 ; SEQ NO. 14 : 3629. The tumor vaccine of claim 1, wherein the tumor antigen sequence is composed of at least 30 amine guanidines. The tumor vaccine according to claim 1, wherein the tumor antigen is synthesized by a chemical method or a genetic engineering method. The tumor vaccine according to claim 1, wherein the tumor antigen has a specific epidermal long-number receptor and an epithelial mucin determinant, and is a human leukocyte tissue type 1 original molecule. Restriction antigen. 3 - A method for synthesizing a tumor vaccine, comprising the steps of: (a) selecting an epidermal growth factor receptor 1 and a epidermis according to the distribution of a human leukocyte tissue antigen DR, DQ and DP subtype molecules in a Chinese population Growth factor receptor 2 and an amino acid sequence of epithelial point protein 1 can be recognized by these human leukocyte tissue type II antigen molecules; bioinformatics analysis of antigenic determinants and human leukocyte tissue The affinity between the antigens + and the appropriate tumor antigen amino acid sequence were selected; (c) 14 tumor antigens 22 201238596 sequences containing at least 30 amino acids were designed according to the results of step (b); d) synthesizing these tumor antigens by chemical or genetic engineering; (e) mixing these synthesized 14 tumor antigens with dimercaptosulfoxide, respectively, so that the final concentration of each tumor antigen mixture is 20 mg/mL to 40 mg /inL, and these tumor antigen mixture is divided into wild type and mutant type, each group includes 7 kinds of tumor antigen mixture; (f) the same group of tumor antigen mixture Do not remove the same volume, and thoroughly mixed together 'to give a tumor vaccine containing 7 Wild-type or mutant tumor antigen, and wherein the final concentration of each of tumor antigens 2mg / mL to 4mg / mL. 6. The method of synthesizing a tumor vaccine according to claim 5, wherein the antigenic determinant of the tumor antigen is recognized by a molecule of a human white blood cell type II antigen. S 23