New antigen polypeptide for treating or improving prostatic cancer as well as preparation method and application of new antigen polypeptide

Abstract

The invention discloses a novel antigen polypeptide for treating or improving prostatic cancer as well as a preparation method and application of the novel antigen polypeptide. The preparation method comprises the following steps: carrying out whole exon sequencing on a tumor cell sample to obtain a gene variation spectrum, carrying out prediction on a tumor neoantigen mutation sequence on a variation site of high-frequency mutation, then carrying out reverse transcription-polymerase chain experiment on mutation with MHC / HLA affinity, and finally screening to obtain the neoantigen polypeptide with immunogenicity. The novel antigen polypeptide can be used for preparing personalized cancer vaccines for treating or improving prostatic cancer, and has a wide application prospect in the field of prostatic cancer treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor immunology, and particularly relates to a personalized neoantigen polypeptide vaccine for treating or improving prostate cancer, its preparation method and application. Background Art

[0002] Tumor has always been one of the major threats to human health, and its pathogenesis is complex and involves multiple factors. The high incidence and mortality of tumors make it a major public health problem worldwide. Tumors have become one of the main causes of death, especially in men, and prostate cancer is one of the malignant tumors with the highest incidence and mortality. Although traditional treatment methods such as surgery, radiotherapy, chemotherapy and hormone therapy are effective to a certain extent, these methods have limitations such as treatment resistance and side effects. Therefore, there is an urgent need to find new treatment strategies to improve the existing prostate cancer treatment regimens.

[0003] As an emerging treatment method, tumor immunotherapy inhibits tumor growth and metastasis by activating the human immune system, and has become another effective tumor treatment method after surgery, radiotherapy, chemotherapy and targeted therapy. Neoantigens are unique antigens generated by gene mutations in tumor cells, and these antigens do not exist in normal cells, so they have high tumor specificity. Neoantigen vaccines are immunotherapy strategies based on tumor-specific mutations, which can activate T cell responses against tumor-specific antigens, thus breaking the state of immunosuppression in the body, inducing the body to recognize and attack tumor cells carrying specific mutations, and changing the body from passive anti-cancer to active anti-cancer, so as to specifically inhibit or eliminate tumors and achieve the purpose of cancer treatment. Personalized neoantigen cancer vaccine treatment avoids attacking normal cells, so it has fewer side effects compared with traditional chemotherapy or radiotherapy.

[0004] However, in the preclinical research of therapeutic tumor neoantigen vaccines for prostate cancer, no tumor neoantigen vaccine preparation with good efficacy has been developed yet, which seriously hinders the R & D process of prostate cancer tumor neoantigen vaccines. Therefore, there is an urgent need to develop a neoantigen vaccine preparation for treating or improving prostate cancer. Summary of the Invention

[0005] To solve at least some of the above-mentioned technical problems in the prior art, the present invention performs whole exome sequencing on the DNA of tumor cell samples to obtain a prostate cancer gene mutation spectrum, predicts antigen mutation sequences with high frequency mutations, and screens out neoantigen polypeptide sequences with immunogenicity. This neoantigen polypeptide can be used to prepare a personalized cancer vaccine for treating or improving prostate cancer, and has broad market application prospects. Specifically, the present invention includes the following contents.

[0006] In a first aspect of the present invention, there is provided a neoantigen polypeptide or a fragment thereof for treating or improving prostate cancer, characterized in that the neoantigen polypeptide or the fragment thereof comprises at least one of the following amino acid sequences:

[0007] (I) The amino acid sequences shown in SEQ ID NO.1-4;

[0008] (II) An amino acid sequence having homology with the amino acid sequence shown in (I) or having the same function;

[0009] (III) An amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (I) or (II) and having the same function.

[0010] In a second aspect of the present invention, there is provided a nucleic acid molecule encoding the neoantigen polypeptide or a fragment thereof according to the present invention.

[0011] In a third aspect of the present invention, there is provided a prostate cancer vaccine, which comprises the neoantigen polypeptide or a fragment thereof according to the present invention, or the nucleic acid molecule according to the present invention, or a cell presenting the neoantigen polypeptide or a fragment thereof or the nucleic acid molecule.

[0012] In certain embodiments, the prostate cancer vaccine according to the present invention, wherein the vaccine comprises one or more of tumor vaccines such as polypeptide vaccines, nucleic acid vaccines, whole cell vaccines, genetically engineered vaccines and antibody tumor vaccines.

[0013] In certain embodiments, the prostate cancer vaccine according to the present invention, wherein the vaccine comprises an adjuvant.

[0014] In certain embodiments, the prostate cancer vaccine according to the present invention, wherein the adjuvant comprises polyinosinic acid-polycytidylic acid Poly(I:C).

[0015] In certain embodiments, the prostate cancer vaccine according to the present invention, wherein the concentration of the polyinosinic acid-polycytidylic acid diluent is 0.1-5 mg / ml.

[0016] In a fourth aspect of the present invention, there is provided a kit, which comprises the neoantigen polypeptide or a fragment thereof according to the present invention or the nucleic acid molecule according to the present invention.

[0017] In a fifth aspect of the present invention, there is provided a method for constructing an animal model, which comprises the steps of immunizing an animal with the neoantigen polypeptide or a fragment thereof according to the present invention and obtaining a corresponding anti-tumor effect.

[0018] In a sixth aspect of the present invention, there is provided a method for preparing a prostate cancer vaccine, which comprises the following steps:

[0019] (1) Extract the DNA of prostate cancer tumor cell samples, perform whole-exome sequencing to obtain the gene mutation spectrum in tumor cells, predict the tumor neoantigen mutation sequences for the frequently mutated variant sites, obtain mutations with MHC / HLA affinity, and perform expression verification by reverse transcription-polymerase chain reaction or RNA sequencing to screen for immunogenic mutant antigen sequences as neoantigen polypeptides or their fragments, or obtain nucleic acid molecules encoding the neoantigen polypeptides or their fragments, or cells presenting the neoantigen polypeptides or nucleic acid molecules;

[0020] (2) Optionally, further include expanding production by chemical synthesis or biosynthesis to obtain the neoantigen polypeptides or their fragments, or the nucleic acid molecules, or cells presenting the neoantigen polypeptides or nucleic acid molecules obtained in step (1);

[0021] (3) Prepare an adjuvant solution and mix it with the neoantigen polypeptides or their fragments, or the nucleic acid molecules, or cells presenting the neoantigen polypeptides or nucleic acid molecules to obtain the vaccine.

[0022] In certain embodiments, according to the preparation method of the present invention, in step (3), the adjuvant solution is prepared by the following method: Prepare a stock solution of polyinosinic acid-polycytidylic acid at a concentration of 10 - 40 mg / ml using pyrogen-free deionized water; Dissolve the stock solution in a water bath at a temperature of 65 - 70 °C for 5 - 15 minutes, and place it at room temperature for 0.5 - 5 hours for cooling; Then dilute it with PBS buffer to a concentration of 0.1 - 5 mg / ml to obtain a polyinosinic acid-polycytidylic acid dilution, which is the adjuvant solution.

[0023] The seventh aspect of the present invention provides the use of a neoantigen based on prostate cancer gene mutations or a personalized neoantigen vaccine containing the same in the preparation of a drug for treating or improving prostate cancer.

[0024] In certain embodiments, according to the use of the present invention, the use includes a combined use, which includes a neoantigen based on prostate cancer gene mutations or a personalized neoantigen vaccine containing the same in combination with other drugs.

[0025] The present invention performs whole-exome sequencing on tumor cell samples to obtain a gene mutation spectrum, predicts the tumor neoantigen mutation sequences for the frequently mutated variant sites, then for mutations with MHC / HLA affinity, and finally by reverse transcription-polymerase chain reaction, and finally screens to obtain immunogenic neoantigen polypeptides. The present invention further provides a vaccine preparation containing neoantigen polypeptides, and verifies it through a mouse model. The results show that the vaccine preparation of the present invention produces a very effective T cell response in tumor-bearing mice, with an increase in the number of T cells, an increase in activated T cells, and the production of CD4 +T cells and CD8 + T cells are involved in anti-tumor immune responses. Therefore, the vaccine preparation of the present invention has significant efficacy against prostate cancer. The neoantigen vaccine preparation of the present invention can provide a reliable evaluation model for preclinical tumor neoantigen vaccines and other types of tumor treatment drugs, and has broad application prospects in the field of prostate cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Shows the flow chart of the preparation method, immunogenicity identification and anti-tumor application of the tumor neoantigen polypeptide vaccine of the present invention.

[0027] Figure 2 Shows the results of screening neoantigen polypeptides with immunogenicity in the present invention.

[0028] Figure 3 Shows the tumor growth curves of the tumor-bearing mice in Example 2 of the present invention after administration of the neoantigen vaccine preparation of the present invention. Among them, the curves from top to bottom are the PBS group, the G1 group, the G2 group, the G4 group, and the G3 group respectively.

[0029] Figure 4 Shows the results of multiplex fluorescence immunohistochemistry of tumor tissues in Example 2 of the present invention.

[0030] Figure 5 Shows the flow cytometry analysis of tumor-infiltrating lymphocytes in Example 2 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0031] Now, various exemplary embodiments of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Each intermediate value within any stated value or stated range and each smaller range between any other stated value or intermediate value within the stated range are also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described in this invention, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0034] Neoantigen polypeptide or its fragment

[0035] In one aspect of the present invention, there is provided a neoantigen polypeptide or a fragment thereof for treating or improving prostate cancer, wherein the neoantigen polypeptide or the fragment thereof comprises at least one of the following amino acid sequences:

[0036] (I) The amino acid sequences shown in SEQ ID NO.1-4;

[0037] (II) An amino acid sequence having homology with the amino acid sequence shown in (I) or having the same function;

[0038] (III) An amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (I) or (II) and having the same function.

[0039] In the present invention, the term "neoantigen polypeptide" refers to a tumor-specific antigen that is only produced by tumor mutations (not present in normal cells) and has immunogenicity. "Fragment" refers to a part of the polypeptide and a fragment having the function of the neoantigen polypeptide. For example, it can comprise at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the total length of the polypeptide.

[0040] Unless otherwise specified, the neoantigen polypeptide or the fragment thereof described herein is an isolated polypeptide or the fragment thereof. The term "isolated" used herein refers to a polypeptide or a fragment thereof that has been extracted from its natural environment. The "isolated" polypeptide or the fragment thereof thus includes a polypeptide or a fragment thereof purified by standard purification methods. The term also includes a polypeptide or a fragment thereof prepared by recombinant expression in a host cell and a polypeptide or a fragment thereof synthesized chemically.

[0041] In the present invention, the terms "homology" and "identity" are used interchangeably. To determine sequence identity, sequence alignment can be performed, which can be carried out in various ways known to those skilled in the art, for example, using software such as BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR). Those skilled in the art can determine the appropriate parameters for alignment, including any algorithms required to achieve optimal alignment in the full-length sequences being compared.

[0042] In the present invention, the polypeptides obtained by modification also fall within the scope of protection of the present invention. The term "modification" refers to any chemical modification of the amino acid sequence, "substitution" refers to the replacement of one or more amino acids by different amino acids, "deletion" refers to the reduction of one or more amino acids in the amino acid sequence, and "insertion" or "addition" refers to a change in the amino acid sequence resulting in an increase in one or more amino acids compared to the naturally occurring molecule.

[0043] Nucleic acid molecule

[0044] In one aspect of the present invention, there is provided a nucleic acid molecule encoding the novel antigen polypeptide or a fragment thereof according to the present invention.

[0045] In the present invention, "nucleic acid" is intended to include polymeric forms of nucleotides of any length, which contain deoxyribonucleotides, ribonucleotides and / or their analogs, which include DNA, RNA and DNA / RNA hybrids, and which also include DNA or RNA analogs, such as those containing modified backbones (e.g., peptide nucleic acid (PNA) or phosphorothioate) or modified bases. Thus, the nucleic acids of the present invention include DNA, cDNA, mRNA, recombinant nucleic acids, etc.

[0046] Once the coding sequence of the novel antigen polypeptide or a fragment thereof according to the present invention is isolated, the novel antigen polypeptide or a fragment thereof can be obtained in large quantities by chemical synthesis or biosynthesis methods. An exemplary method is to clone its coding gene into a vector, then transfer it into cells, and then isolate it from the proliferated host cells by conventional methods.

[0047] It can be understood that vectors or host cells containing the coding nucleic acid molecule of the novel antigen polypeptide or a fragment thereof according to the present invention also fall within the scope of protection of the present invention. The vectors of the present invention are not limited and can be expression vectors, viral vectors, etc. The vectors can use known vectors or self-constructed vectors, wherein the known vectors include but are not limited to plasmid vectors, lentiviral vectors, adenoviral vectors, AAV viral vectors, etc. The host cells of the present invention refer to any cell type suitable for transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector containing the nucleic acid molecule of the present invention.

[0048] Prostate cancer vaccine

[0049] In one aspect of the present invention, a prostate cancer vaccine is provided, which comprises a neoantigen polypeptide or a fragment thereof according to the present invention or a nucleic acid molecule according to the present invention. It is understood that cells for presenting the neoantigen polypeptide or a fragment thereof or the nucleic acid molecule are also within the scope of protection of the present invention. Such cells include those that occur naturally or are artificially synthesized (e.g., engineered or modified cells), and examples thereof include, but are not limited to, dendritic cells, lymphocytes, macrophages, chimeric artificial cells, virus vector-transduced cells, LNP-modified autologous cells, attenuated bacteria, cell mimics, etc.

[0050] Currently, the development of tumor vaccines is mainly based on tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs), and their core mechanism lies in activating the specific immune response of the body against cancer cells. With the continuous development and improvement of tumor vaccine technology, various types of prostate cancer vaccines have been successfully developed, including cell vaccines, polypeptide vaccines, and nucleic acid vaccines, etc. However, currently, most prostate cancer vaccines are designed based on TAAs, which may lead to the induced immune response attacking normal cells non-specifically (off-target effect), and may not be able to effectively target all prostate cancer cells. Tumor-associated antigens may also be expressed in normal cells, which may lead to the misattack of the immune system on normal tissues, thereby triggering autoimmune toxicity. In contrast, the tumor-specific antigens of the present invention are only expressed in tumor cells and do not exist in normal cells. Therefore, they can stimulate a highly specific immune response and effectively avoid "off-target" damage to normal tissues. Since TSAs are unique antigens of tumor cells, the immune system has a higher efficiency in recognizing and attacking them and is not easily induced to immune tolerance. While TAA vaccines may lead to immune tolerance of the immune system due to their expression in normal cells, thereby reducing the therapeutic effect. The tumor-specific antigen vaccine of the present invention can be customized according to the mutation characteristics of the patient's tumor cells, and has the advantages of high precision and individuation. This customized method can design vaccines according to the unique tumor characteristics of each patient, thereby significantly improving the therapeutic effect.

[0051] In a preferred embodiment, the vaccine comprises an adjuvant. In a preferred embodiment, the adjuvant is polyinosinic acid-polycytidylic acid Poly(I:C).

[0052] The administration dosage of the vaccine of the present invention is generally 0.01 - 500 mg / kg, preferably 0.5 - 500 mg / kg, more preferably 0.5 - 250 mg / kg, still more preferably 0.5 - 100 mg / kg, even more preferably 0.5 - 50 mg / kg, even more preferably 0.5 - 40 mg / kg, even more preferably 0.5 - 30 mg / kg, and most preferably 0.5 - 25 mg / kg. Exemplary administration dosages are, for example, 0.5 mg / kg, 0.75 mg / kg, 0.95 mg / kg, 1 mg / kg, 1.25 mg / kg, 1.5 mg / kg, 1.75 mg / kg, 2 mg / kg, 2.5 mg / kg, 2.75 mg / kg, 3 mg / kg, 3.25 mg / kg, 3.5 mg / kg, 3.75 mg / kg, 4 mg / kg, 4.25 mg / kg, 4.5 mg / kg, 4.75 mg / kg, 5 mg / kg, 5.25 mg / kg, 5.5 mg / kg, 5.75 mg / kg, 6 mg / kg, 6.25 mg / kg, 6.5 mg / kg, 6.75 mg / kg, 7 mg / kg, 7.25 mg / kg, 7.5 mg / kg, 7.75 mg / kg, 8 mg / kg, 8.25 mg / kg, 8.5 mg / kg, 8.75 mg / kg, 9 mg / kg, 9.25 mg / kg, 9.5 mg / kg, 9.75 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg. It can be understood that the vaccine of the present invention can be administered as a single dose once a day, can be administered in multiple doses per day, or can be used at intervals.

[0053] In the present invention, the vaccine includes, but is not limited to, one or several of tumor vaccines such as polypeptide vaccines, nucleic acid vaccines, whole cell vaccines, genetically engineered vaccines, and antibody tumor vaccines.

[0054] There is no particular limitation on the administration method of the vaccine of the present invention. Representative administration methods include, but are not limited to, subcutaneous injection, oral administration, intratumoral injection, rectal administration, etc. Correspondingly, the drug of the present invention can be made into various clinically acceptable dosage forms, and its examples include, but are not limited to, oral dosage forms, injection dosage forms, etc.

[0055] Kit

[0056] In one aspect of the present invention, a kit is provided, which includes the neoantigen polypeptide or its fragment according to the present invention or the nucleic acid molecule according to the present invention.

[0057] In the present invention, the term "kit" refers to a combination of reagents and other materials. The kit is expected to contain reagents such as buffers, protein stabilizing reagents, signal generation systems (e.g., fluorescence signal generation systems), antibodies or their antigen-binding fragments, control proteins, and test containers (e.g., microtiter plates, etc.). The term "kit" is not limited to a specific combination of reagents and / or other materials. For example, the kit may also include instructions for using the reagents. The kit can be packaged in any suitable manner. Generally, it has components in a single container or (if necessary) in multiple containers and a manual for detection instructions. The kit can be prepared by various methods known in the art.

[0058] In certain embodiments, the kit may further include at least one of a washing solution, a substrate solution, a diluent, and a calibration solution. Among them, the composition of the washing solution is not particularly limited, and its examples include but are not limited to buffers, surfactants, and preservatives. Known substrates can be used for the substrate solution, and its examples include but are not limited to chromogenic substrates, fluorescent substrates, chemiluminescent substrates, etc. The composition of the diluent is not particularly limited, and its examples include but are not limited to buffers, surfactants, etc. The composition of the calibration solution is not particularly limited, and its examples include but are not limited to BSA solutions, trehalose solutions, animal sera, etc.

[0059] It can be understood that the kit of the present invention can be used for immunizing animal models, so that application scenarios such as drug screening, drug structure optimization, drug combination therapy, efficacy evaluation, immune mechanism research, and immunotherapy optimization can be carried out. Therefore, the kit of the present invention can contain any suitable reagents for the above applications, and no special limitation is imposed thereon.

[0060] Method for constructing animal model

[0061] In one aspect of the present invention, a method for constructing an animal model is provided, which includes the steps of immunizing an animal with the neoantigen polypeptide or its fragment described in the present invention and obtaining a corresponding anti-tumor effect. In the present invention, the animal is not particularly limited, and its examples include but are not limited to mice, rats, guinea pigs, rabbits, monkeys, cats, dogs, pigs, fish, etc.

[0062] In a preferred embodiment, the neoantigen polypeptide of the present invention is formulated with sterile PBS buffer to a polypeptide solution with a concentration of 0.1 - 5 mg / ml (preferably 0.1 - 4.5 mg / ml, more preferably 0.2 - 4 mg / ml, further preferably 0.5 - 3 mg / ml, such as 0.5, 1, 1.5, 2, 2.5, 3 mg / ml); a mother liquor with a concentration of 10 - 40 mg / ml, preferably 15 - 35 mg / ml, more preferably 20 - 30 mg / ml, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 mg / ml is prepared from polyinosinic acid - polycytidylic acid and deionized water, dissolved at 65 - 70 °C (such as 65, 66, 67, 68, 69, 70 °C) for 5 - 15 minutes (preferably 6 - 14 minutes, more preferably 7 - 13 minutes, even more preferably 8 - 12 minutes, such as 8, 9, 10, 11, 12 minutes), placed at room temperature for 0.5 - 5 hours (preferably 0.5 - 4 hours, more preferably 0.5 - 3 hours, such as 0.5, 1, 1.5, 2, 2.5, 3 hours), and diluted with phosphate buffer to a concentration of 0.1 - 1 mg / ml (preferably 0.2 - 1 mg / ml, more preferably 0.3 - 1 mg / ml, even more preferably 0.4 - 1 mg / ml, such as 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1 mg / ml) to obtain an adjuvant solution; the polypeptide solution and the adjuvant solution are mixed to obtain a vaccine; the C57BL / 6 mice are immunized in the inguinal region with the vaccine, and then boosted on the 7th day and the 14th day respectively to obtain a mouse model.

[0063] It can be understood that the application examples of the animal model constructed by the method of the present invention include, but are not limited to, evaluating the efficacy of neoantigen polypeptide vaccines or drugs, evaluating drug safety, studying immune mechanisms, optimizing immunotherapy, etc.

[0064] Preparation method

[0065] One aspect of the present invention provides a method for preparing a prostate cancer vaccine. In a preferred embodiment, the preparation method includes the following steps:

[0066] (1) Extract the DNA of prostate cancer tumor cell samples, perform whole - exome sequencing to obtain the gene mutation spectrum in tumor cells, predict the tumor neoantigen mutation sequences for the frequently mutated variant sites, obtain mutations with MHC / HLA affinity, and verify the expression through reverse transcription - polymerase chain reaction experiments, screen for immunogenic mutant antigen sequences to obtain the neoantigen polypeptide or its fragment, or obtain a nucleic acid molecule encoding the neoantigen polypeptide or its fragment, or a cell presenting the neoantigen polypeptide or nucleic acid molecule;

[0067] (2) Prepare an adjuvant solution, and mix it with the neoantigen polypeptide or its fragment, or the nucleic acid molecule, or the cell presenting the neoantigen polypeptide or nucleic acid molecule to obtain the vaccine.

[0068] It can be understood that those skilled in the art can further expand the production through chemical synthesis or biosynthesis to obtain the neoantigen polypeptide or its fragment, or the nucleic acid molecule, or the cell presenting the neoantigen polypeptide or nucleic acid molecule in step (1).

[0069] In the present invention, the adjuvant is not particularly limited, and its examples include but are not limited to Freund's adjuvant (complete or incomplete), Toll-like receptor agonists, NOD-like receptor agonists, Mda5 agonists, RIG-I, PKR agonists, STING agonists, saponins, saponins, etc.

[0070] In step (2) of the present invention, the adjuvant solution is prepared by the following method:

[0071] Prepare a stock solution of polyinosinic acid-polycytidylic acid at a concentration of 10 - 40 mg / ml, preferably 15 - 35 mg / ml, more preferably 20 - 30 mg / ml, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 mg / ml, using pyrogen-free deionized water; dissolve the stock solution in a water bath at a temperature of 65 - 70 °C (such as 65, 66, 67, 68, 69, 70 °C) for 5 - 15 minutes (preferably 6 - 14 minutes, more preferably 7 - 13 minutes, even more preferably 8 - 12 minutes, such as 8, 9, 10, 11, 12 minutes), and let it cool at room temperature for 0.5 - 5 hours (preferably 0.5 - 4 hours, more preferably 0.5 - 3 hours, such as 0.5, 1, 1.5, 2, 2.5, 3 hours); then dilute it with PBS buffer to a concentration of 0.1 - 5 mg / ml (preferably 0.2 - 1 mg / ml, more preferably 0.3 - 1 mg / ml, even more preferably 0.4 - 1 mg / ml, such as 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1 mg / ml) to obtain a polyinosinic acid-polycytidylic acid dilution, that is, the adjuvant solution.

[0072] In a preferred embodiment, the neoantigen polypeptide or its fragment is formulated with sterile PBS buffer to a polypeptide solution with a concentration of 0.1 - 5 mg / ml (preferably 0.1 - 4.5 mg / ml, more preferably 0.2 - 4 mg / ml, further preferably 0.5 - 3 mg / ml, such as 0.5, 1, 1.5, 2, 2.5, 3 mg / ml), and then mixed with the adjuvant solution to obtain the vaccine.

[0073] Application

[0074] One aspect of the present invention provides the use of neoantigens based on prostate cancer gene mutations or personalized neoantigen vaccines containing the same in the preparation of drugs for treating or improving prostate cancer.

[0075] In the present invention, the term "treating or improving" refers to therapeutic treatment or prophylactic measures, the purpose of which is to slow down (reduce) undesired physiological changes or disorders, such as the occurrence, development or deterioration of tumors. Beneficial or desired clinical outcomes include, but are not limited to, the following whether detectable or undetectable, including relief of symptoms, reduction in the degree of disease, stabilization of the disease state (i.e., no deterioration), delay or slowdown in disease progression, improvement or remission of the disease state, and alleviation (whether partial or complete). Those in need of treatment include those who already have prostate cancer or prostate cancer-related diseases or those who need to prevent or improve prostate cancer or prostate cancer-related diseases.

[0076] The present invention further provides the use of neoantigens based on prostate cancer gene mutations or personalized neoantigen vaccines containing the same in the preparation of drugs for combined treatment of prostate cancer with other drugs. Examples of the other drugs include, but are not limited to, immune checkpoint inhibitors (such as, but not limited to, anti-PD-1 monoclonal antibodies, anti-PD-L1 monoclonal antibodies, anti-CTLA-4 monoclonal antibodies, etc.), small molecule targeted therapies (such as, but not limited to, enzalutamide, abiraterone, apalutamide, anlotinib hydrochloride, etc.), chemotherapeutic drugs (including, but not limited to, docetaxel, doxorubicin, mitoxantrone, cabazitaxel, estramustine, cisplatin, etc.).

[0077] In the present invention, "combined treatment" means that the vaccine of the present invention is part of a specific treatment regimen, aiming to produce beneficial (additive or synergistic) effects through the combined action of the vaccine and one or more other drugs. The beneficial effects of the combination include, but are not limited to, pharmacokinetic or pharmacodynamic synergistic effects produced by the combination of the vaccine and other drugs. The combined administration of the vaccine and other drugs is usually carried out within a specified time period (usually several minutes, hours, days or weeks, depending on the selected combination). "Combined treatment" includes sequential administration of the vaccine and other drugs (i.e., each drug is administered at different times), and substantially simultaneous administration of these vaccines and other drugs, or they can be formulated as a single co-formulated pharmaceutical composition containing the two compounds.

[0078] Example 1

[0079] This example shows the preparation process of neoantigen polypeptides.

[0080] 1. Prediction and expression verification of neoantigen polypeptides

[0081] First, extract the DNA of high-quality prostate tumor cells, randomly break it into fragments of 150-200 bp, and purify it by magnetic bead purification technology. Perform end repair on the purified DNA fragments to ensure that both ends of all fragments have complete sticky ends. Add A bases to the 3' ends to form sticky ends. Connect the adapters containing specific barcode sequences to the 3' ends of the DNA fragments. Use magnetic beads to screen and remove incompletely ligated products and adapter self-ligation products to ensure the purity of the library. Perform PCR amplification using universal primers complementary to the adapter sequences to form the total sequencing library. Hybridize using probes in hybridization buffer, capture the target fragments of hybridization, and use magnetic beads for separation and purification. Perform PCR amplification on the captured DNA fragments and purify the PCR products to obtain the target sequencing library. Detect the concentration of the library and the length of the library fragments. Finally, perform next-generation sequencing to obtain whole exome data.

[0082] As Figure 1 shown, analyze the whole exome data and find 252 mutant polypeptide fragments. Among them, there are 62 mutations with an allele frequency greater than or equal to 0.6 (Table 1). Use software to predict the binding affinity of these mutations to the major histocompatibility complex (MHC), and obtain 24 mutations with high binding affinity to MHC. Subsequently, perform reverse transcription-polymerase chain reaction experiments on these 24 mutations, and the expression of 10 mutations is verified. Use chemical synthesis methods to synthesize 10 verified polypeptide fragments.

[0083] Table 1 Prediction and expression verification of neoantigen polypeptides

[0084]

[0085] 2. Screening of neoantigen polypeptides with immunogenicity

[0086] Mix the above 10 synthesized mutant polypeptide fragments with the adjuvant polyinosinic acid-polycytidylic acid Poly(I:C) and immunize the bilateral inguinal regions of C57BL / 6 mice. Each group is immunized with a mixture of 2 different mutant polypeptide fragments on days 0 and 7, with 1 mutant polypeptide fragment mixture on each side. Perform a booster immunization on day 7. After 14 days of immunization, take the spleens of the mice, prepare single-cell suspensions of the spleens, and use the enzyme-linked immunospot method ELISPOT technology to screen for neoantigen polypeptides. Using the number of IFN-γ spots produced by mutant polypeptide fragments being more than that produced by wild-type polypeptide fragments and having statistical differences as the positive standard, a total of 4 immunogenic neoantigen polypeptides are screened out from 10 mutant polypeptide fragments (see Figure 2 and Table 2).

[0087] Table 2 Polypeptide sequences

[0088]

[0089]

[0090] Example 2

[0091] The preparation process and efficacy evaluation of the neoantigen polypeptide vaccine are shown below.

[0092] 1. Preparation of the vaccine

[0093] The 4 screened polypeptide fragments were formulated with sterile PBS buffer to a concentration of 1 mg / ml to obtain a polypeptide mixture. Poly(I:C) was prepared into a stock solution of 25 mg / mL with pyrogen-free deionized water. The stock solution was dissolved in a water bath at 65 - 70 °C for 10 minutes and then cooled at room temperature for 1 hour; then it was diluted with sterile PBS buffer to a concentration of 1 mg / ml to obtain a polyinosinic acid-polycytidylic acid dilution. The polypeptide solution with a concentration of 1 mg / ml was mixed with the adjuvant solution with a concentration of 1 mg / ml at a ratio of 100 μg peptide: 50 μg adjuvant to obtain a tumor polypeptide vaccine preparation.

[0094] 2. Evaluation of the vaccine effect

[0095] A mouse RM-1 transplanted tumor model was established. Different candidate tumor neoantigen vaccine preparations were administered to tumor-bearing mice with an average body weight of 20 g on the 5th and 12th days after tumor injection. The dose was 200 μl of the tumor neoantigen vaccine preparation (containing 100 μg peptide and 50 μg adjuvant). The tumor volume was measured every 1 day. The formula for calculating the tumor volume was: V = 0.5a × b 2 , where a and b represent the long diameter and short diameter of the tumor respectively. The antitumor efficacy of the tumor neoantigen vaccine preparation was evaluated using the tumor volume. There were a total of 5 treatment groups: PBS control group (PBS group), M4 polypeptide vaccine (G1 group), M3 polypeptide vaccine (G2 group), M2 polypeptide vaccine (G3 group), M1 polypeptide vaccine (G4 group). According to the results of the drug efficacy, the tumor neoantigen vaccine preparation with antitumor effect was screened out. Figure 3 It is the tumor growth curve of tumor-bearing mice in a mouse RM-1 prostate cancer syngeneic transplanted tumor model after administration of the tumor neoantigen vaccine. Each data point was represented by the mean tumor volume ± standard error of the mean (SEM), n = 5. The therapeutic effects of the 4 neoantigen vaccines (G1 - 4) in treating the RM-1 model were all significantly better than the PBS group, and the difference was statistically significant. These results indicate that the neoantigen vaccine has good antitumor effects.

[0096] 3. Pharmacodynamic test

[0097] On the 20th day after tumor inoculation, tumor tissues of mice were collected, fixed with 4% PFA, embedded in paraffin, and sectioned at a thickness of 4 μm. The sections were dewaxed in xylene and then rehydrated with gradient ethanol. After microwave treatment, the tissue was circled with a PAP pen, blocked with blocking solution, and incubated for 10 minutes. After blocking non-specific background staining, the sections were incubated with rat anti-CD4 / CD3 at room temperature for 1 hour, then incubated with polymer HRP-anti-mouse / rabbit IgG for 10 minutes, and then stained with PPD-520DYE for 10 minutes. After elution by microwave treatment, the blocking solution was dropped and covered the tissue, and incubated for 10 minutes. After blocking non-specific background staining, the sections were incubated with rat anti-mouse CD8 / CD137 at room temperature for 1 hour, then incubated with the same secondary antibody for 10 minutes, and then stained with PPD-570DYE for 10 minutes. After microwave treatment again, the cell nuclei were counterstained with DAPI and observed under a fluorescence microscope. The number of positive cells in each 40× field of view was counted using software. Figure 4 is a representative picture of multi-color immunofluorescence of tumor tissues. Compared with the control group, the infiltration of CD8 + and CD4 + T cells in the tumor neoantigen vaccine treatment group increased significantly, and the activated T cells co-labeled with CD3 and CD137 (CD3 + CD137 + T cells) increased significantly.

[0098] Another tumor tissue of mice was digested into single-cell suspension, and the components of tumor-infiltrating lymphocytes (TIL) in the mouse tumor tissue samples were detected by flow cytometry (FCW). The tumor tissue was dissected into 1-2 mm fragments and digested in RPMI-1640 medium containing 1 mg / mL type IV collagenase and 20 mg / mL deoxyribonuclease at 37 °C with shaking at 80 r / min for 1 h. The digestion solution was filtered through a 70 μm nylon mesh to remove visible debris and subjected to Ficoll density gradient centrifugation to isolate tumor-infiltrating lymphocytes. The cells were resuspended in PBS buffer, centrifuged at 400×G for 5 minutes, and the supernatant was removed; the cells were resuspended in 100 μL PBS buffer, and then antibodies were added as shown in Table 3 and incubated at room temperature in the dark for 20 minutes. After the antibody incubation was completed, the cells were centrifuged at 400×G for 5 minutes, and the supernatant was removed; the cells were resuspended in 1000 μL PBS buffer, centrifuged at 400×G for 5 minutes, the supernatant was removed, and the process was repeated once. Finally, the cells were resuspended in 200 μL staining buffer and transferred to a flow tube for detection by flow cytometry. The flow data were analyzed to analyze the percentages of T cells, activated T cells, CD4 + T cells, CD8 + T cells, and CD45 + T cells in CD45 cells. Figure 5It is a schematic diagram of the proportion of activated T cells in tumor-infiltrating lymphocytes of tumor-bearing mice in the present invention. After the tumor neoantigen vaccine treatment, the proportions of T cells and activated T cells in the tumor showed an increasing trend compared with the control group. This indicates that the prostate cancer tumor neoantigen vaccine produced an effective T cell response in tumor-bearing mice.

[0099] Table 3 Flow cytometry staining protocol

[0100]

[0101]

[0102] The present invention uses the second-generation genome sequencing technology to perform exon sequencing on tumor cell samples to obtain the gene mutation spectrum in tumor cells. Then, software is used to predict the tumor neoantigen mutation sequences at the mutation sites with high frequency. Subsequently, mutations with MHC / HLA affinity are verified by reverse transcription-polymerase chain reaction, and the verified polypeptide fragments are synthesized by chemical synthesis. Finally, an in vitro ELISPOT experimental method for verifying the immunogenicity of tumor gene mutation sequences is established to screen out immunogenic polypeptide fragments.

[0103] Then, the screened polypeptide fragments are respectively prepared into a polypeptide mixture using sterile PBS buffer. Then, they are formulated with an adjuvant in a certain proportion to produce a tumor neoantigen vaccine preparation. Then, a mouse RM-1 transplanted tumor model is established to verify the drug efficacy. The results show that the tumor neoantigen vaccine preparation produced a very effective T cell response in tumor-bearing mice, with an increase in the number of T cells and activated T cells, and the production of CD4 + T cells and CD8 + T cells all actively participated in the anti-tumor immune response.

[0104] The neoantigen vaccine preparation of the present invention achieved significant therapeutic effects in the RM-1 tumor model. The tumor volume of the treatment group was significantly smaller than that of the control group. Moreover, the neoantigen vaccine preparation of the present invention is very suitable for combination therapy with other types of anti-tumor drugs, such as antibody drugs, targeted therapy small molecules, and chemotherapy drugs.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features. And these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A new antigen polypeptide or a fragment thereof for treating or improving prostate cancer, characterized in that: The neoantigen polypeptide or fragment thereof comprises at least one of the following amino acid sequences: (I) the amino acid sequence shown in SEQ ID NO. 1-4; (II) an amino acid sequence that is homologous to or has the same function as the amino acid sequence shown in (I); (III) An amino acid sequence obtained by modifying, replacing, deleting or adding one or more amino acids to the amino acid sequence shown in (I) or (II) and having the same function.

2. A nucleic acid molecule, characterized in that It encodes the new antigen polypeptide or a fragment thereof according to claim 1.

3. A prostate cancer vaccine, characterized in that It comprises the neoantigen polypeptide or fragment thereof according to claim 1, or the nucleic acid molecule according to claim 2, or a cell presenting the neoantigen polypeptide or fragment thereof according to claim 1, or the nucleic acid molecule according to claim 2; Preferably, the vaccine comprises one or more of tumor vaccines such as polypeptide vaccine, nucleic acid vaccine, whole cell vaccine, genetic engineering vaccine and antibody tumor vaccine.

4. The prostate cancer vaccine according to claim 3, characterized in that The vaccine may include an adjuvant; Preferably, the adjuvant comprises polyinosinic acid-polycytosine nucleotide Poly (I:C); Preferably, the concentration of the polyinosinic acid-polycytosine nucleotide dilution solution is 0.1-5 mg / ml.

5. A kit, characterized in that: It comprises the new antigen polypeptide or a fragment thereof according to claim 1 or the nucleic acid molecule according to claim 2.

6. A method for constructing an animal model, characterized in that: The method comprises the steps of immunizing an animal with the new antigen polypeptide or a fragment thereof according to claim 1 and obtaining a corresponding anti-tumor effect.

7. A method for preparing a prostate cancer vaccine, characterized in that: It includes the following steps: (1) Extracting DNA from prostate cancer tumor cell samples and performing whole exome sequencing to obtain the gene mutation spectrum in tumor cells, predicting tumor neoantigen mutation sequences at high-frequency mutation sites, obtaining mutations with MHC / HLA affinity, and performing expression verification by reverse transcription-polymerase chain reaction or RNA sequencing, screening immunogenic mutant sequences as neoantigen polypeptides or fragments thereof, or obtaining nucleic acid molecules encoding the neoantigen polypeptides or fragments thereof, or cells presenting the neoantigen polypeptides or nucleic acid molecules; (2) Optionally, further comprising amplifying the production of the new antigen polypeptide or fragment thereof, or the nucleic acid molecule, or the cell presenting the new antigen polypeptide or nucleic acid molecule obtained in step (1) by chemical synthesis or biological synthesis; (3) preparing an adjuvant solution and mixing it with the new antigen polypeptide or its fragment, or the nucleic acid molecule, or the cell presenting the new antigen polypeptide or nucleic acid molecule to obtain the vaccine.

8. The preparation method according to claim 7, characterized in that: In step (3), the adjuvant solution is prepared by the following method: polyinosinic acid-polycytosine nucleotide is prepared into a 10-40 mg / ml mother solution using pyrogen-free deionized water; the mother solution is dissolved in a water bath at a temperature of 65-70°C for 5-15 minutes, and cooled at room temperature for 0.5-5 hours; and then diluted with a buffer solution to a concentration of 0.1-5 mg / ml to obtain a polyinosinic acid-polycytosine nucleotide dilution, i.e., the adjuvant solution.

9. Use of neoantigens based on prostate cancer gene mutations or personalized neoantigen vaccines containing the same in the preparation of drugs for treating or improving prostate cancer.

10. The use according to claim 9, characterized in that: The applications include combined applications, which include combining neoantigens based on prostate cancer gene mutations or personalized neoantigen vaccines containing the same with other drugs.

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