Composition and application of an improved polypeptide tumor vaccine

Abstract

The present invention discloses an improved polypeptide tumor vaccine composition and application, specifically relating to the field of polypeptide tumor vaccines, the vaccine comprises a nucleic acid sequence encoding a plurality of mutant tumor-associated antigens, the nucleic acid sequence including but not limited to: a nucleic acid sequence encoding a mutant WT1 antigen, selected from the amino acid sequence of SEQ ID NO: 1 or a variant thereof with at least 90% identity; a nucleic acid sequence encoding a mutant MUC1 antigen, selected from the amino acid sequence of SEQ ID NO: 2 or a variant thereof with at least 90% identity; a nucleic acid sequence encoding a mutant PD-1 antigen, selected from the amino acid sequence of SEQ ID NO: 3 or a variant thereof with at least 90% identity; and the vaccine composition further comprises one or more modified elements that enhance antigen expression or immune response, including a signal peptide, an adjuvant coding sequence or an immunostimulatory sequence. The vaccine composition of the present invention is intended to enhance the immune response to a plurality of tumor antigens and improve the effectiveness of tumor treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide tumor vaccines, and more specifically, to the composition and application of an improved polypeptide tumor vaccine. Background Art

[0002] Cancer remains the leading cause of death in the United States and worldwide. The cancer vaccine market is growing rapidly, with lymphoma vaccines accounting for approximately 0.5% of the market share. Effective tumor vaccines can be used to block tumor growth and / or can be used as a more effective, less toxic alternative to standard treatment for patients with advanced cancer. An antigen associated with cancer and therefore a target for anti-tumor vaccines is WT1.

[0003] The Wilms tumor suppressor gene 1 (WT1) has been identified as the cause of embryonal malignancies of the kidney, affecting approximately 1 in 10,000 infants, occurring in both sporadic and inherited forms. Inactivation of WT1 leads to the development of Wilms tumor and Denys-Drash syndrome (DDS), resulting in kidney disease and possible genital abnormalities. The WT1 protein has been found to interact with many cytokines, including the master tumor regulatory gene p53, which is also a tumor suppressor transcription factor.

[0004] The WT1 gene is expressed in many tumor types and has been more widely involved in many cancers. For example, the WT1 protein is localized in the nuclei of 75% of mesotheliomas (14,200 cases per year worldwide, with the highest incidence in the United States) and 93% of ovarian serous carcinomas (190,000 ovarian cancer cases worldwide in 2010). In addition, WT1 has been involved in pancreatic cancer, leukemia, lung cancer, breast cancer, colon cancer, glioblastoma, head and neck cancer, and benign mesothelioma and cervical and ovarian cancers. WT1 is a target for gene therapy or immunotherapy as a method of cancer treatment.

[0005] WT1 encodes a transcription factor containing four zinc finger motifs at the C-terminus and a proline / glutamine-rich DNA-binding domain at the N-terminus, which plays an indispensable role in the normal development of the urogenital system, however, it is less essential in adults, suggesting it as a target for immunotherapy. Multiple transcript variants resulting from alternative splicing at two coding exons have been well characterized, and utilizing the entire reading frame to maximize CTL coverage would be considered advantageous.

[0006] Due to the conservation of the WT1 antigen, most efforts to generate strong immunity against this gene target have been unsuccessful. Vaccines have previously been studied using DNA vaccine technology, poxvirus vaccine technology, adenovirus vaccine technology, peptide vaccine technology, and protein-based vaccine technology. The vaccines studied used real gene structures, i.e., natural "normal" genes. In these studies, only low-level or non-functional T cell immunity was achieved.

[0007] Vaccines for the treatment and prevention of cancer have received great attention. Existing vaccines targeting WT1 are limited by poor antigen expression in vivo. Therefore, there is still a need in the art to develop safe and effective vaccines suitable for tumors expressing WT1, thereby providing improved polypeptide tumor vaccines that can enhance the immune response to multiple tumor antigens and improve the effectiveness of tumor treatment. Summary of the invention

[0008] In order to overcome the above-mentioned defects of the prior art, the present invention provides an improved polypeptide tumor vaccine composition and application to solve the problems raised in the above-mentioned background technology.

[0009] To achieve the above object, the present invention provides the following technical solution: an improved polypeptide tumor vaccine composition, the vaccine comprises a nucleic acid sequence encoding a plurality of mutant tumor-associated antigens, the nucleic acid sequence includes but is not limited to:

[0010] A nucleic acid sequence encoding a mutant WT1 antigen, selected from the amino acid sequence of SEQ ID NO: 1 or a variant thereof having at least 90% identity;

[0011] A nucleic acid sequence encoding a mutant MUC1 antigen selected from the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least 90% identity;

[0012] A nucleic acid sequence encoding a mutant PD-1 antigen, selected from the amino acid sequence of SEQ ID NO: 3 or a variant thereof having at least 90% identity;

[0013] Furthermore, the vaccine composition may further comprise one or more modification elements that enhance antigen expression or immune response, including a signal peptide, an adjuvant coding sequence or an immunostimulatory sequence.

[0014] Preferably, the nucleic acid sequences are present in a tandem arrangement in a single vector, the vector is a plasmid or a viral vector, and the vector also contains a promoter sequence and a transcription terminator sequence to regulate the expression of the antigen gene.

[0015] Preferably, the vaccine composition also includes one or more fusion proteins, which contain a tandem arrangement of amino acid sequences of the above-mentioned multiple tumor-associated antigens, and the fusion proteins are obtained by transforming cells with a recombinant plasmid, culturing, and inducing expression with IPTG.

[0016] Preferably, the vaccine composition also includes a nucleic acid sequence encoding an inhibitor of the PD-1 receptor to further enhance the intensity and durability of the anti-tumor immune response.

[0017] Preferably, the vaccine further comprises one or more adjuvants selected from but not limited to: CpG DNA, MPL, aluminum salts, liposomes or DC cell activators to enhance the immunogenicity of the vaccine and / or improve the efficiency of the immune response.

[0018] Preferably, the isolated nucleic acid molecule comprises one or more nucleic acid sequences, wherein the nucleic acid sequences are selected from the group consisting of a nucleic acid sequence encoding SEQ ID NO:1, a nucleic acid sequence encoding a fragment comprising at least 90% of the length of SEQ ID NO:1, a nucleic acid sequence encoding a protein having at least 98% identity with SEQ ID NO:1, a nucleic acid sequence encoding a fragment comprising at least 90% of the length of a protein having at least 98% identity with SEQ ID NO:1, a nucleic acid sequence encoding SEQ ID NO:3, a nucleic acid sequence encoding a fragment comprising at least 90% of the length of SEQ ID NO:3, a nucleic acid sequence encoding a protein having at least 97% identity with SEQ ID NO:3, and a nucleic acid sequence encoding a fragment comprising at least 90% of the length of a protein having at least 97% identity with SEQ ID NO:3.

[0019] Preferably, the present invention also relates to an isolated nucleic acid molecule comprising one or more nucleic acid sequences, wherein the nucleic acid sequence is selected from the group consisting of: SEQ ID NO:2, a fragment comprising at least 85% of the length of SEQ ID NO:1, a nucleic acid sequence having at least 85% identity with SEQ ID NO:2, a fragment comprising at least 85% of the length of a nucleic acid sequence having at least 98% identity with SEQ ID NO:2, SEQ ID NO:3, a fragment comprising at least 85% of the length of SEQ ID NO:3, a nucleic acid sequence having at least 95% identity with SEQ ID NO:3, and a fragment comprising at least 80% of the length of a nucleic acid sequence having at least 98% identity with SEQ ID NO:3.

[0020] Preferably, the present invention also relates to a vaccine comprising a nucleic acid molecule, which may comprise a nucleic acid sequence having at least about 90% identity over the entire length of the nucleic acid sequence shown in SEQ ID NO:1, and the nucleic acid molecule may comprise a nucleic acid sequence having at least about 85% identity over the entire length of the nucleic acid sequence shown in SEQ ID NO:3, and the vaccine may also comprise a peptide, which may comprise an amino acid sequence having at least about 90% identity over the entire length of the amino acid sequence shown in SEQ ID NO:2, and the peptide may comprise an amino acid sequence having at least about 90% identity over the entire length of the amino acid sequence shown in SEQ ID NO:3.

[0021] Preferably, the vaccine composition is used in the preparation of a medicament for treating and preventing various types of cancer, in particular for tumor types expressing WT1, MUC1 or PD-1.

[0022] A method for preparing an improved polypeptide tumor vaccine, comprising:

[0023] a) constructing a recombinant plasmid or viral vector comprising a nucleic acid sequence encoding a plurality of mutant tumor-associated antigens;

[0024] b) transforming the vector obtained in step a) into a host cell;

[0025] c) culturing host cells and inducing antigen expression under appropriate conditions;

[0026] d) collecting and purifying the expressed polypeptide antigen or fusion protein;

[0027] e) Mixing the polypeptide antigen, fusion protein and pharmaceutically acceptable excipients and / or adjuvants to prepare a vaccine preparation.

[0028] Preferably, the improved polypeptide tumor vaccine is used to induce immune responses against multiple tumor-associated antigens in individuals, especially to improve the immunotherapy effect and survival rate of cancer patients.

[0029] Technical effects and advantages of the present invention:

[0030] 1. The vaccine consists of three target antigens: WT1, MUC1 and PD-1, which are expressed in many tumor types, such as mesothelioma, ovarian serous carcinoma, pancreatic cancer, leukemia, lung cancer, breast cancer, colon cancer, etc. Therefore, the vaccine has a broad spectrum and is expected to be used in the treatment and prevention of various cancers.

[0031] 2. By utilizing a combination of mutated WT1 antigen, MUC1 antigen, and PD-1 antigen, the vaccine is able to induce a stronger immune response. This multi-target strategy can more effectively activate the immune system and improve the ability to recognize and kill tumor cells.

[0032] 3. Compared with traditional chemotherapy and radiotherapy, this tumor vaccine, as a form of immunotherapy, has lower toxicity. It mainly fights tumors by enhancing the patient's own immune system and reduces damage to normal cells.

[0033] 4. By inducing an effective immune response, the vaccine is expected to improve the survival rate of cancer patients. Immunotherapy is gradually gaining popularity in cancer treatment, and combined with traditional treatments, it can significantly improve the prognosis of patients.

[0034] 5. The nucleic acid molecules in the vaccine composition can be flexibly incorporated into plasmids or viral vectors, which provides more options for vaccine production and application. At the same time, the design of the vaccine is also convenient for future optimization and improvement to adapt to different cancer types and the personalized needs of patients.

[0035] 6. In addition to the basic vaccine composition, the invention also involves products in various forms including fusion proteins, recombinant plasmid-transformed cells, etc. These products can further enrich the treatment methods of tumor vaccines and meet the treatment needs of different patients.

[0036] 7. The design of the vaccine composition is based on in-depth tumor biology and immunology research, ensuring the effectiveness and safety of the vaccine. By optimizing the antigen sequence and carrier system, the vaccine can more effectively induce immune responses against specific tumor antigens. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is a graph of the present invention showing the plotting of immunization groups versus plaque forming units (SFU) per 106 splenocytes;

[0038] Figure 2 is a graph of the present invention showing the SFU per 106 splenocytes plotted against the immunization groups;

[0039] Figure 3 The nucleotide sequence encoding WT1 of the present invention;

[0040] Figure 4 The nucleotide sequence encoding Muc1 is shown in the present invention;

[0041] Figure 5 The present invention shows a nucleotide sequence encoding PD-1. DETAILED DESCRIPTION

[0042] The following will be combined with the attached Figure 1-5The present invention is described in detail, and the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] 1. Definition

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as understood by one of ordinary skill in the art. In the event of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0045] As used herein, the terms "include / comprise", "including", "have", "have", "may", "contain" and variations thereof are intended to be open transitional phrases, terms or words that do not exclude the possibility of additional actions or structures, unless the context clearly indicates otherwise, the singular forms "a", "and", and "the" include plural references, and the present disclosure also encompasses other embodiments that "include / comprise" the embodiments or components set forth herein, "consist of" and "consist essentially of" the embodiments or components, whether explicitly shown or not.

[0046] As used herein, "adjuvant" may mean any molecule added to the DNA plasmid vaccines described herein to enhance the antigenicity of one or more antigens encoded by the DNA plasmid and encoding nucleic acid sequences described below.

[0047] "Antigen" refers to: a protein having a WT1 amino acid sequence including a mutation of SEQ ID NO: 1; a fragment or variant having the length shown in this article, i.e., a protein having a sequence identical to SEQ ID NO: 1 as shown in this article, a fragment of a variant having the length shown in this article, SEQ ID NO: 3; a fragment or variant thereof having the length shown in this article, i.e., a protein having a sequence identical to SEQ ID NO: 3 as shown in this article, a fragment of a variant having the length shown in this article, and a combination thereof, and the antigen may optionally include a signal peptide such as those from other proteins.

[0048] The vaccine can be administered to a mammal to elicit an immune response in the mammal. The mammal can be a human, a primate, a non-human primate, a cow, a cattle, a sheep, a goat, an antelope, a bison, a buffalo, a bison, a bovine, a deer, a hedgehog, an elephant, a camel, an alpaca, a mouse, a rat, a chicken, and a pig.

[0049] "Fragment" may mean a polypeptide fragment of an antigen that is capable of eliciting an immune response in a mammal, and in each case with or without a signal peptide and / or a methionine at position 1, a fragment of an antigen may be 100% identical to the full length, except for the loss of at least one amino acid from the N- and / or C-terminus, and a fragment may comprise 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, More, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more percent of the length of a particular full-length antigen, excluding any added heterologous signal peptide, fragments may comprise fragments of a polypeptide that have 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the antigen, and additionally comprise an N-terminal methionine or a heterologous signal peptide that is not included when calculating the percent identity, fragments may also comprise an N-terminal methionine and / or a signal peptide, such as an immunoglobulin signal peptide, for example an IgE or IgG signal peptide, the N-terminal methionine and / or signal peptide may be attached to the fragment of the antigen.

[0050] In each case with or without the sequence encoding a signal peptide and / or a methionine at position 1, a fragment of a nucleic acid sequence encoding an antigen may be 100% identical to the full length, except for the loss of at least one nucleotide from the 5' and / or 3' end, and the fragment may contain 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more. Fragments may comprise fragments encoding a polypeptide that is 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more percent of the length of a particular full-length coding sequence, excluding any added heterologous signal peptide, and fragments may comprise fragments encoding a polypeptide that is 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the antigen, and additionally optionally comprises a sequence encoding an N-terminal methionine or a heterologous signal peptide that is not included when calculating the percent identity, and a coding sequence encoding an N-terminal methionine and / or a signal peptide may be linked to a fragment of the coding sequence.

[0051] Example 1

[0052] 1. Material Preparation

[0053] Antigenic polypeptide sequences: Based on the amino acid sequences provided by SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, nucleic acid sequences encoding these polypeptides are synthesized to ensure that the synthesized nucleic acid sequences have a high degree of accuracy and purity.

[0054] Expression vector: Select a plasmid vector suitable for mammalian cell expression, such as pcDNA3.1, to ensure that the vector can efficiently and stably express the target polypeptide.

[0055] Cell lines: Commonly used mammalian cell lines such as HEK293T were selected to express fusion proteins.

[0056] Other reagents: IPTG (isopropyl-β-D-thiogalactoside), culture medium, antibiotics and other conventional cell culture reagents.

[0057] 2. Construction of recombinant plasmid

[0058] The nucleic acid sequences encoding SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 are cloned separately or in combination into an expression vector to construct a recombinant plasmid. Ensure that the inserted nucleic acid sequence is in the correct direction and has no frameshift mutation.

[0059] The correctness of the recombinant plasmid was verified by PCR, sequencing and other methods.

[0060] 3. Cell Transfection and Fusion Protein Expression

[0061] The verified correct recombinant plasmid was transfected into HEK293T cells using conventional methods such as liposome transfection.

[0062] After transfection, the cells are cultured under appropriate culture conditions, such as a constant temperature incubator at 37° C. and 5% CO 2 .

[0063] After the cells grow to an appropriate density, IPTG is added to induce the expression of the fusion protein. The expression of the fusion protein can be optimized by adjusting the IPTG concentration and induction time.

[0064] 4. Purification of Fusion Protein

[0065] The induced cell culture medium is collected and cell debris is removed by centrifugation or other methods.

[0066] The fusion protein is purified from the culture medium using affinity chromatography, ion exchange chromatography and other protein purification techniques to ensure that the purified fusion protein has high purity and biological activity.

[0067] 5. Preparation of vaccine composition

[0068] The purified fusion protein is mixed with an appropriate adjuvant to prepare a vaccine composition. The choice of adjuvant should be determined based on the specific needs of the vaccine and the type of target immune response.

[0069] Conduct quality tests on vaccine components, including sterility testing, purity testing, stability testing, etc., to ensure that the quality of vaccine components meets relevant standards.

[0070] 6. Vaccine effectiveness evaluation

[0071] The vaccine effect was evaluated on animal models such as mice. The vaccine composition was injected, including WT-1: 1.2mmol, MUC-1: 0.2mg, PD-1: 0.2mg, GM-CSF: 3000lU, IL-2: 3000lU, IL-9: 2000lU, IFN-γ: 1000lU as a polypeptide vaccine dose, and the changes in the immune response in the animals were observed by subcutaneous injection.

[0072] Evaluation indicators include specific antibody levels, T cell proliferation capacity, CTL activity, etc. At the same time, attention should also be paid to vaccine safety indicators, such as the incidence of adverse reactions.

[0073] in conclusion

[0074] This embodiment provides an improved polypeptide tumor vaccine composition. By optimizing antigen selection and vaccine preparation process, the vaccine composition can more effectively induce immune responses against multiple tumor cells. In the future, the vaccine composition is expected to play an important role in cancer treatment.

[0075] Example 2

[0076] In further implementation, based on the above embodiments, this embodiment further improves the composition of the polypeptide tumor vaccine, and improves the immunogenicity and stability of the vaccine by optimizing the antigen sequence and its combination, in the hope of achieving better results in cancer treatment.

[0077] Antigen sequence selection and optimization

[0078] Based on the original sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, sequence alignment and homology analysis were performed to screen out mutation sites with higher immunogenicity. Molecular simulation was used to predict the effects of these mutations on the three-dimensional structure of the antigen to ensure that the optimized antigen sequence can maintain the original antigenicity and improve the affinity with the T cell receptor.

[0079] The optimized antigen sequence was finally determined to be:

[0080] WT1 target antigen optimized sequence (SEQ ID NO:1_opt)

[0081] MUC1 target antigen optimized sequence (SEQ ID NO:2_opt)

[0082] PD-1 target antigen optimized sequence (SEQ ID NO:3_opt)

[0083] Construction of fusion proteins

[0084] Genetic engineering technology is used to connect the optimized antigen sequences in series to construct a fusion protein gene. In order to enhance expression efficiency and stability, appropriate linker peptides and tag sequences (such as His tags) are introduced to finally obtain a fusion protein gene sequence.

[0085] Construction of expression vector

[0086] The fusion protein gene is inserted into the plasmid vector to construct a recombinant plasmid. In this embodiment, pUC19 plasmid is selected as the expression vector, and the fusion protein gene is inserted into the multiple cloning site of the plasmid through PCR amplification, enzyme digestion, ligation and other steps.

[0087] Cell transfection and expression

[0088] The recombinant plasmid is transfected into an expression host cell (such as E. coli or a mammalian cell line) and the fusion protein is expressed by inducing IPTG. The cell lysate is collected and the expression of the fusion protein is detected by SDS-PAGE and Western Blot.

[0089] Purification and identification

[0090] The fusion protein is purified by an appropriate purification method (such as nickel column affinity chromatography) and identified by mass spectrometry, HPLC and other means to ensure the purity and structural correctness of the purified product.

[0091] Vaccine preparation

[0092] The purified fusion protein is mixed with a pharmaceutically acceptable excipient to prepare a vaccine preparation. If necessary, an adjuvant (such as CpG ODN, polyinosinic-polycytidylic acid, etc.) can be added to enhance the immune effect.

[0093] Sequence optimization results

[0094] The optimized antigen sequence improves its affinity with T cell receptors while maintaining its original antigenicity, and is expected to trigger a stronger immune response.

[0095] Fusion protein expression

[0096] The recombinant plasmid successfully expressed the fusion protein in the expression host cells with high expression level and good stability.

[0097] Purification and identification

[0098] The purity of the purified fusion protein reached over 95%, and the correctness of its structure and sequence was confirmed by mass spectrometry and HPLC.

[0099] Vaccine performance evaluation

[0100] Vaccine immunization experiments were conducted in animal models, and the results showed that the improved polypeptide tumor vaccine can significantly enhance the immune response of animals, induce the production of high levels of specific antibodies and cytotoxic T lymphocytes (CTLs), and have a good killing effect on tumor cells expressing WT1, MUC1 and PD-1.

[0101] in conclusion

[0102] This example successfully prepared an improved polypeptide tumor vaccine by optimizing the antigen sequence and constructing a fusion protein. The vaccine improves immunogenicity and stability while maintaining the original antigenicity, providing a new option for cancer immunotherapy.

[0103] WT1 Application Range

[0104] Clinically, it can be used for patients with high expression of WT1 tumor neoantigens: acute lymphoblastic leukemia, acute myeloid leukemia, lung adenocarcinoma, breast cancer, digestive system colorectal cancer, pancreatic cancer, gastric cancer, esophageal cancer, head and neck cancer, soft tissue sarcoma, female ovarian cancer, cervical cancer, brain glioma, pediatric neuroblastoma and more than 20 other cancer types;

[0105] Peptide tumor vaccines are tumor polypeptide fragments containing tumor-specific antigens (TSA) or tumor-associated antigens (TAA). After entering the human body, they can activate the patient's own immune system, induce specific immune responses (CTL), overcome the immunosuppressive state, and improve resistance to specific tumors. It is an active immunization method.

[0106] Effective dose of vaccine and how to use it:

[0107] Clinical patient inoculation dose: 100μl / tube:

[0108] Directions: Subcutaneous injection.

[0109] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An improved polypeptide tumor vaccine composition, characterized in that: The vaccine comprises nucleic acid sequences encoding multiple mutant tumor-associated antigens, including but not limited to: A nucleic acid sequence encoding a mutant WT1 antigen, selected from the amino acid sequence of SEQ ID NO: 1 or a variant thereof having at least 90% identity; A nucleic acid sequence encoding a mutant MUC1 antigen selected from the amino acid sequence of SEQ ID NO: 2 or a variant thereof having at least 90% identity; A nucleic acid sequence encoding a mutant PD-1 antigen, selected from the amino acid sequence of SEQ ID NO: 3 or a variant thereof having at least 90% identity; Furthermore, the vaccine composition may further comprise one or more modification elements that enhance antigen expression or immune response, including a signal peptide, an adjuvant coding sequence or an immunostimulatory sequence.

2. The improved polypeptide tumor vaccine composition according to claim 1, characterized in that: The nucleic acid sequences are present in a single vector in the form of a tandem arrangement. The vector is a plasmid or a viral vector, and the vector also contains a promoter sequence and a transcription terminator sequence to regulate the expression of the antigen gene.

3. The improved polypeptide tumor vaccine composition according to claim 1, characterized in that: The vaccine composition also includes one or more fusion proteins, which contain the tandem arrangement of the amino acid sequences of the above-mentioned multiple tumor-associated antigens. The fusion proteins are obtained by transforming cells with recombinant plasmids, culturing them, and inducing their expression with IPTG.

4. The improved polypeptide tumor vaccine composition according to claim 1, characterized in that: The vaccine composition also includes a nucleic acid sequence encoding an inhibitor of the PD-1 receptor to further enhance the intensity and durability of the anti-tumor immune response.

5. The improved polypeptide tumor vaccine composition according to claim 1, characterized in that: The vaccine further comprises one or more adjuvants, which are selected from but not limited to: CpG DNA, MPL, aluminum salts, liposomes or DC cell activators to enhance the immunogenicity of the vaccine and / or improve the efficiency of the immune response.

6. The improved polypeptide tumor vaccine composition according to claim 1, characterized in that: The vaccine composition is used in the preparation of drugs for treating and preventing various types of cancer, especially for tumor types expressing WT1, MUC1 or PD-1.

7. A method for preparing the improved polypeptide tumor vaccine according to any one of claims 1 to 6, comprising: a) constructing a recombinant plasmid or viral vector comprising a nucleic acid sequence encoding a plurality of mutant tumor-associated antigens; b) transforming the vector obtained in step a) into a host cell; c) culturing host cells and inducing antigen expression under appropriate conditions; d) collecting and purifying the expressed polypeptide antigen or fusion protein; e) Mixing the polypeptide antigen, fusion protein and pharmaceutically acceptable excipients and / or adjuvants to prepare a vaccine preparation.

8. Use of the improved polypeptide tumor vaccine according to any one of claims 1 to 6 in inducing immune responses against multiple tumor-associated antigens in an individual, in particular for improving the immunotherapy effect and survival rate of cancer patients.

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