New antigen polypeptide for treating or improving prostate cancer and preparation method and application thereof

Abstract

The present invention discloses novel antigenic polypeptides for treating or ameliorating prostate cancer, as well as methods for their preparation and application. The present invention performs whole-exome sequencing on tumor cell samples to obtain a gene variation spectrum. Highly mutated sites are then used to predict tumor neoantigen mutation sequences. Subsequently, reverse transcription-polymerase chain reaction (RT-PCR) experiments are performed on mutations with MHC / HLA affinity, and finally, immunogenic neoantigen polypeptides are screened. The novel antigenic polypeptides of the present invention can be used to prepare personalized cancer vaccines for treating or ameliorating prostate cancer, and have broad application prospects in the field of prostate cancer treatment.

Description

Technical Field

[0001] The present invention belongs to the field of tumor immunology technology, and in particular relates to a personalized neoantigen polypeptide vaccine for treating or improving prostate cancer, as well as a preparation method and application thereof. Background Art

[0002] Cancer has always been a major threat to human health, and its pathogenesis is complex and involves multiple factors. The high morbidity and mortality of cancer make it a major public health problem worldwide. Cancer has become one of the main causes of death, especially in men, and prostate cancer is one of the malignant tumors with the highest morbidity and mortality. Although traditional treatments 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 existing prostate cancer treatment options.

[0003] As an emerging treatment method, tumor immunotherapy inhibits tumor growth and metastasis by activating the human immune system. It has become another effective tumor treatment method after surgery, radiotherapy, chemotherapy and targeted therapy. Neoantigens are unique antigens produced by gene mutations in tumor cells. These antigens do not exist in normal cells and are therefore highly tumor-specific. Neoantigen vaccines are immunotherapy strategies based on tumor-specific mutations. They can activate T cell responses against tumor-specific antigens, thereby breaking the state of immunosuppression in the body, inducing the body to recognize and attack tumor cells carrying specific mutations, and causing the body to switch from passive anti-cancer to active anti-cancer, thereby specifically inhibiting or eliminating tumors and achieving the purpose of cancer treatment. Personalized neoantigen cancer vaccine treatment avoids attacks on normal cells, so it has fewer side effects than traditional chemotherapy or radiotherapy.

[0004] However, in preclinical research on therapeutic neoantigen vaccines for prostate cancer, no effective neoantigen vaccine formulations have yet been developed, which has seriously hindered the development of these vaccines. Therefore, there is an urgent need to develop a neoantigen vaccine formulation for the treatment or improvement of prostate cancer. Summary of the Invention

[0005] To address at least some of the technical problems in the above-mentioned prior art, the present invention performs whole-exome sequencing of DNA from tumor cell samples to obtain the spectrum of prostate cancer gene mutations, predict high-frequency antigen mutation sequences, and screen to obtain immunogenic neoantigen polypeptide sequences. These neoantigen polypeptides can be used to prepare personalized cancer vaccines for the treatment or improvement of prostate cancer, and have broad market application prospects. Specifically, the present invention includes the following contents.

[0006] In a first aspect of the present invention, a new antigen polypeptide or a fragment thereof for treating or ameliorating prostate cancer is provided, wherein the new antigen polypeptide or a fragment thereof comprises at least one of the following amino acid sequences:

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

[0008] (II) an amino acid sequence that is homologous to or has the same function as the amino acid sequence shown in (I);

[0009] (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.

[0010] The second aspect of the present invention provides a nucleic acid molecule encoding the new antigen polypeptide or a fragment thereof according to the present invention.

[0011] The third aspect of the present invention provides a prostate cancer vaccine, which includes the new antigen polypeptide or fragment thereof according to the present invention, or the nucleic acid molecule according to the present invention, or a cell presenting the new antigen polypeptide or fragment thereof or nucleic acid molecule.

[0012] In certain embodiments, the prostate cancer vaccine according to the present invention 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 comprises an adjuvant.

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

[0015] In certain embodiments, according to the prostate cancer vaccine of the present invention, the concentration of the polyinosinic-polycytosine (PI-PC) diluent is 0.1-5 mg / ml.

[0016] In a fourth aspect, the present invention provides a kit comprising the neoantigen polypeptide or fragment thereof described in the present invention or the nucleic acid molecule described in the present invention.

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

[0018] A sixth aspect of the present invention provides a method for preparing a prostate cancer vaccine, comprising the following steps:

[0019] (1) Extracting DNA from prostate cancer tumor cell samples and performing whole-exome sequencing to obtain the gene mutation spectrum in the tumor cells, predicting the tumor neoantigen mutation sequence at the high-frequency mutation sites, obtaining mutations with MHC / HLA affinity, and performing expression verification by reverse transcription-polymerase chain reaction or RNA sequencing, screening the immunogenic mutant antigen 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;

[0020] (2) Optionally, further comprising scaling up the production of the new antigen polypeptide or fragment thereof, or the nucleic acid molecule, or cells presenting the new antigen polypeptide or nucleic acid molecule obtained in step (1) by chemical synthesis or biosynthesis;

[0021] (3) preparing an adjuvant solution and mixing it with the neoantigen polypeptide or fragment thereof, or the nucleic acid molecule, or cells presenting the neoantigen polypeptide or nucleic acid molecule to obtain the vaccine.

[0022] In certain embodiments, according to the preparation method of the present invention, wherein, 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 liquor using pyrogen-free deionized water; the mother liquor 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 PBS buffer to a concentration of 0.1-5 mg / ml to obtain a polyinosinic acid-polycytosine nucleotide dilution solution, i.e., the adjuvant solution.

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

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

[0025] The present invention performs whole exome sequencing on tumor cell samples to obtain a gene variation spectrum, predicts the tumor neoantigen mutation sequence at the mutation sites with high frequency mutations, and then screens mutations with MHC / HLA affinity and uses reverse transcription-polymerase chain reaction to finally screen and obtain immunogenic neoantigen polypeptides. The present invention further provides a vaccine preparation containing the neoantigen polypeptide and verifies it in 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 participate in anti-tumor immune responses, and therefore, the vaccine formulation of the present invention has significant therapeutic efficacy against prostate cancer. The neoantigen vaccine formulation of the present invention can provide a reliable evaluation model for preclinical neoantigen tumor vaccines and other types of tumor therapeutics, and has broad application prospects in the field of prostate cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0027] Figure 2 The results of the present invention's screening of new antigenic polypeptides with immunogenicity are shown.

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

[0029] Figure 4 The results of multiple fluorescent immunohistochemistry of tumor tissue in Example 2 of the present invention are shown.

[0030] Figure 5 The flow cytometric analysis of tumor-infiltrating lymphocytes in Example 2 of the present invention is shown. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present 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 the event of any conflict with any incorporated document, the content of this specification shall prevail.

[0034] Neoantigen polypeptide or fragment thereof

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

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

[0037] (II) an amino acid sequence that is homologous to or has the same function as the amino acid sequence shown in (I);

[0038] (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.

[0039] In the present invention, the term "neoantigen polypeptide" refers to a tumor-specific antigen that is produced only by tumor mutations (not present in normal cells) and has immunogenicity. A "fragment" refers to a portion of a polypeptide that has the function of a neoantigen polypeptide, for example, a fragment that 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 indicated, the neoantigenic polypeptides or fragments thereof described herein are isolated polypeptides or fragments thereof. The term "isolated" as used herein refers to a polypeptide or fragment thereof that has been extracted from its natural environment. "Isolated" polypeptides or fragments thereof thus include polypeptides or fragments thereof purified by standard purification methods. The term also includes polypeptides or fragments thereof prepared by recombinant expression in host cells and chemically synthesized polypeptides or fragments thereof.

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

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

[0043] Nucleic acid molecules

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

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

[0046] Once the coding sequence for the neoantigenic polypeptide or fragment thereof described herein is isolated, it can be obtained in large quantities using chemical or biosynthetic methods. An exemplary method involves cloning the coding gene into a vector, transfecting the vector into cells, and then isolating the polypeptide from the proliferated host cells using conventional methods.

[0047] It is understood that vectors or host cells containing nucleic acid molecules encoding the new antigen polypeptides or fragments thereof of the present invention also fall within the scope of protection of the present invention. The vectors of the present invention are not limited and may be expression vectors, viral vectors, and the like. The vectors may use known vectors or self-constructed vectors, wherein known vectors include but are not limited to plasmid vectors, lentiviral vectors, adenoviral vectors, AAV viral vectors, and the like. The host cell of the present invention refers to any cell type suitable for transformation, transfection, transduction, and the like using 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 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 fragment thereof or nucleic acid molecule are also within the scope of protection of the present invention. Such cells include those naturally occurring cells or artificially synthesized cells (e.g., engineered or modified cells), examples of which include but are not limited to dendritic cells, lymphocytes, macrophages, chimeric artificial cells, viral vector-transduced cells, LNP-modified autologous cells, attenuated bacteria, cell mimics, and the like.

[0050] Currently, the development of tumor vaccines is primarily based on tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs), whose core mechanism is to activate the body's specific immune response to 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, peptide vaccines, and nucleic acid vaccines. However, most current prostate cancer vaccine designs are based on TAAs, which can lead to nonspecific attacks on normal cells (off-target effects) caused by the induced immune response and may not effectively target all prostate cancer cells. Tumor-associated antigens may also be expressed in normal cells, which can cause the immune system to mistakenly attack normal tissues, thereby triggering autoimmune toxicity. In contrast, the tumor-specific antigens of the present invention are only expressed in tumor cells and not in normal cells, thus stimulating a highly specific immune response and effectively avoiding "off-target" damage to normal tissues. Because TSAs are antigens unique to tumor cells, the immune system is more efficient in recognizing and attacking them and is less likely to induce immune tolerance. However, due to their expression in normal cells, TAA vaccines may cause the immune system to develop tolerance to them, thereby reducing the therapeutic effect. The tumor-specific antigen vaccines of the present invention can be customized based on the mutational characteristics of the patient's tumor cells, offering the advantages of high precision and individualization. This customized approach enables vaccine design tailored to each patient's unique tumor characteristics, significantly improving treatment efficacy.

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

[0052] The administration dose of the vaccine of the present invention is generally 0.01-500 mg / kg, preferably 0.5-500 mg / kg, further preferably 0.5-250 mg / kg, more preferably 0.5-100 mg / kg, more preferably 0.5-50 mg / kg, more preferably 0.5-40 mg / kg, more preferably 0.5-30 mg / kg, and most preferably 0.5-25 mg / kg. Exemplary administration dosages include, 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.

[00145] The present invention can be administered in any of the following doses: 1) a single dose, 2) a single dose, 3) a single dose, 4) a single dose, 5) a single dose, 6) a single dose, 7) a single dose, 8) a single dose, 9) a single dose, 10) a single dose, 11) a single dose, 12) a single dose, 13) a single dose, 14) a single dose, 15) a single dose, 16) a single dose, 17) a single dose, 18) a single dose, 19) a single dose, 20) a single dose, 21) a single dose, 22) a single dose, 23) a single dose, 24) a single dose, 25) a single dose. It will be appreciated that the vaccine of the present invention can be administered once daily, can be administered multiple times daily, or can be used at intervals.

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

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

[0055] Reagent test kit

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

[0057] In the present invention, the term "test kit" refers to a combination of reagents and other materials. The test kit is expected to include reagents, such as buffers, protein stabilizing agents, signal generating systems (e.g., fluorescent signal generating systems), antibodies or their antigen binding fragments, control proteins, and test containers (e.g., microtiter plates, etc.). The term "test kit" is not limited to a specific combination of reagents and / or other materials. For example, the test kit may also include instructions for using the reagents. The test kit can be packaged in any suitable manner, typically having components in a single container or (if necessary) in multiple containers and instructions for detecting the instructions. The test 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 cleaning solution, a substrate solution, a diluent, and a calibration solution. The composition of the cleaning solution is not particularly limited, and examples thereof include, but are not limited to, a buffer, a surfactant, and a preservative. A known substrate may be used for the substrate solution, and examples thereof include, but are not limited to, chromogenic substrates, fluorescent substrates, and luminescent substrates. The composition of the diluent is not particularly limited, and examples thereof include, but are not limited to, a buffer, a surfactant, and the like. The composition of the calibration solution is not particularly limited, and examples thereof include, but are not limited to, BSA solution, trehalose solution, and animal serum.

[0059] It is understandable that the kit of the present invention can be used for immunization of animal models, thereby enabling drug screening, drug structure optimization, drug combination therapy, drug efficacy evaluation, immune mechanism research, immunotherapy optimization and other application scenarios. Therefore, the kit of the present invention may contain any suitable reagents for the above-mentioned applications, without particular limitation.

[0060] Methods for constructing animal models

[0061] One aspect of the present invention provides a method for constructing an animal model, comprising the steps of immunizing an animal with the novel antigenic polypeptides or fragments thereof described herein and obtaining a corresponding anti-tumor effect. In the present invention, the animals are not particularly limited, and examples include, but are not limited to, mice, rats, guinea pigs, rabbits, monkeys, cats, dogs, pigs, fish, and the like.

[0062] In a preferred embodiment, the neoantigen polypeptide of the present invention is prepared using sterile PBS buffer to a concentration of 0.1-5 mg / ml (preferably 0.1-4.5 mg / ml, also preferably 0.2-4 mg / ml, further preferably 0.5-3 mg / ml, for example 0.5, 1, 1.5, 2, 2.5, 3 mg / ml) of the polypeptide solution; a mother solution of 10-40 mg / ml, preferably 15-35 mg / ml, also preferably 20-30 mg / ml, for example 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 mg / ml is prepared from polyinosinic acid-polycytosine nucleotides and deionized water and dissolved at 65-70° C. (for example 65, 66, 67, 68, 69, 70° C.) for 5-15 minutes (preferably 6-14 minutes, also preferably 7-15 minutes). 3 minutes, more preferably 8-12 minutes, for example 8, 9, 10, 11, 12 minutes), leaving at room temperature for 0.5-5 hours (preferably 0.5-4 hours, further preferably 0.5-3 hours, for example 0.5, 1, 1.5, 2, 2.5, 3 hours), and diluting with phosphate buffer to a concentration of 0.1-1 mg / ml (preferably 0.2-1 mg / ml, further preferably 0.3-1 mg / ml, more preferably 0.4-1 mg / ml, for example 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; mixing the polypeptide solution and the adjuvant solution to obtain a vaccine; immunizing C57BL / 6 mice in the inguinal region with the vaccine, and then performing booster immunizations on days 7 and 14 to obtain a mouse model.

[0063] It is understood that application examples of the animal model constructed by the method of the present invention include but are not limited to evaluating the efficacy of new antigen 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 comprises the following steps:

[0066] (1) Extracting DNA from a prostate cancer tumor cell sample and performing whole-exome sequencing to obtain the gene mutation spectrum in the tumor cells, predicting the tumor neoantigen mutation sequence at the high-frequency mutation sites, obtaining mutations with MHC / HLA affinity, and performing expression verification through reverse transcription-polymerase chain reaction experiments, screening for immunogenic mutant antigen sequences, obtaining the neoantigen polypeptide or fragment thereof, or obtaining a nucleic acid molecule encoding the neoantigen polypeptide or fragment thereof, or a cell presenting the neoantigen polypeptide or nucleic acid molecule;

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

[0068] It is understood that those skilled in the art can further expand 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 in step (1) by chemical synthesis or biosynthesis.

[0069] In the present invention, the adjuvant is not particularly limited, and 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] The polyinosinic acid-polycytosine nucleotide is prepared into a mother solution of 10-40 mg / ml, preferably 15-35 mg / ml, and more preferably 20-30 mg / ml, for example, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mg / ml using pyrogen-free deionized water; the mother solution is dissolved in a water bath at a temperature of 65-70° C. (for example, 65, 66, 67, 68, 69, or 70° C.) for 5-15 minutes (preferably 6-14 minutes, more preferably 7-13 minutes, more preferably 8-12 minutes, for example, 8, 9, 10, 11, or 12 minutes); The mixture is placed at room temperature for 0.5-5 hours (preferably 0.5-4 hours, further preferably 0.5-3 hours, for example 0.5, 1, 1.5, 2, 2.5, 3 hours) to cool; then diluted with PBS buffer to a concentration of 0.1-5 mg / ml (preferably 0.2-1 mg / ml, further preferably 0.3-1 mg / ml, more preferably 0.4-1 mg / ml, for example 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-polycytosine nucleotide dilution, i.e., an adjuvant solution.

[0072] In a preferred embodiment, the neoantigen polypeptide or fragment thereof is prepared using sterile PBS buffer to a polypeptide solution with a concentration of 0.1-5 mg / ml (preferably 0.1-4.5 mg / ml, also preferably 0.2-4 mg / ml, further preferably 0.5-3 mg / ml, for example 0.5, 1, 1.5, 2, 2.5, 3 mg / ml), and then mixed with an 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 "treat or improve" refers to therapeutic treatment or preventive measures, the purpose of which is to slow down (reduce) undesirable physiological changes or disorders, such as the occurrence, development or worsening of tumors. Beneficial or desired clinical results include, but are not limited to, the following detectable or undetectable results, including alleviation of symptoms, reduction of disease extent, stabilization of disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of disease state and relief (whether partial or complete). Those in need of treatment include those who have already suffered from prostate cancer or a disease associated with prostate cancer or those who need to prevent or improve prostate cancer or a disease associated with prostate cancer.

[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 the combined treatment of prostate cancer with other drugs. Examples of the other drugs are 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.), targeted therapy small molecules (such as but not limited to enzalutamide, abiraterone, apalutamide, anlotinib hydrochloride, etc.), chemotherapy drugs (including but not limited to docetaxel, doxorubicin, mitoxantrone, cabazitaxel, estradiol mustard, cisplatin, etc.), etc.

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

[0078] Example 1

[0079] This example shows the preparation process of the new antigen polypeptide.

[0080] 1. Prediction and expression verification of new antigen peptides

[0081] First, high-quality DNA from prostate tumor cells is extracted, randomly broken into 150-200bp fragments, and purified using magnetic bead purification technology. The purified DNA fragments are end-repaired to ensure that both ends of all fragments have complete sticky ends. A base is added to the 3' end to form a sticky end. A connector containing a specific barcode sequence is connected to the 3' end of the DNA fragment. Magnetic beads are used to screen and remove incompletely connected products and connector self-connection products to ensure the purity of the library. PCR amplification is performed using universal primers complementary to the connector sequence to form a total sequencing library. The probe is hybridized in hybridization buffer to capture the hybridized target fragments, and magnetic beads are used for separation and purification. The captured DNA fragments are PCR amplified, and the PCR products are purified to obtain the target sequencing library. The concentration of the library and the length of the library fragments are detected. Finally, second-generation sequencing is performed to obtain whole-exome data.

[0082] like Figure 1 As shown, analysis of whole-exome data revealed 252 mutant polypeptide fragments, of which 62 had an allele frequency greater than or equal to 0.6 (Table 1). Software was used to predict the binding affinity of these mutations to the major histocompatibility complex (MHC), and 24 mutations were found to have high binding affinity to the MHC. These 24 mutations were subsequently subjected to reverse transcription-polymerase chain reaction experiments, and the expression of 10 of these mutations was verified. These 10 verified polypeptide fragments were then synthesized using chemical synthesis methods.

[0083] Table 1 Prediction and expression verification of new antigen peptides

[0084]

[0085] 2. Screening of new antigenic peptides with immunogenicity

[0086] The above 10 synthesized mutant polypeptide fragments were mixed with the adjuvant polyinosinic acid-polycytosine nucleotide Poly (I: C) and immunized in the bilateral inguinal regions of C57BL / 6 mice. Each group was immunized with two different mutant polypeptide fragment mixtures on the 0th and 7th days, with one mutant polypeptide fragment mixture on each side. Another booster immunization was performed on the 7th day. 14 days after the immunization, the spleen of the mice was taken and a spleen single cell suspension was prepared. The new antigen polypeptides were screened using the enzyme-linked immunosorbent spot (ELISPOT) technique. The positive standard was that the number of IFN-γ spots produced by the mutant polypeptide fragment was greater than that produced by the wild-type polypeptide fragment and the difference was statistically significant. From the 10 mutant polypeptide fragments, a total of 4 immunogenic new antigen polypeptides were screened (see Figure 2 and Table 2 ).

[0087] Table 2 Peptide sequences

[0088]

[0089]

[0090] Example 2

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

[0092] 1. Vaccine Preparation

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

[0094] 2. Vaccine effectiveness evaluation

[0095] A mouse RM-1 xenograft tumor model was established. Tumor-bearing mice weighing an average of 20 g were treated with different candidate tumor neoantigen vaccine formulations on days 5 and 12 after tumor injection. Each dose was 200 μl of the tumor neoantigen vaccine formulation (containing 100 μg of peptide and 50 μg of adjuvant). Tumor volume was measured every other day. The tumor volume was calculated using the formula: V = 0.5a × b 2 , a and b represent the long and short diameters of the tumor, respectively. The tumor-suppressing efficacy of the neoantigen vaccine preparation was evaluated using tumor volume. Five treatment groups were divided: PBS control group (PBS group), M4 peptide vaccine (G1 group), M3 peptide vaccine (G2 group), M2 peptide vaccine (G3 group), and M1 peptide vaccine (G4 group). Based on the efficacy results, neoantigen vaccine preparations with demonstrated antitumor effects were screened. Figure 3 Figure 2 shows tumor growth curves in mice bearing the RM-1 prostate cancer homograft model after administration of neoantigen vaccines. Each data point is expressed as mean tumor volume ± standard error (SEM), n = 5. All four neoantigen vaccines (G1-4) demonstrated statistically significant superiority in the RM-1 model compared to the PBS group. These results demonstrate that neoantigen vaccines have a potent anti-tumor effect.

[0096] 3. Pharmacodynamics testing

[0097] On day 20 of tumor growth, tumor tissues were harvested from mice, fixed with 4% PFA, and embedded in paraffin. Sections were sliced ​​at 4 μm thickness. Sections were deparaffinized in xylene and rehydrated with graded ethanol. After microwave treatment, sections were circled with a PAP pen, covered with blocking solution, and incubated for 10 minutes. After blocking nonspecific background staining, sections were incubated with rat anti-CD4 / CD3 antibodies at room temperature for 1 hour, followed by HRP-anti-mouse / rabbit IgG for 10 minutes, and then stained with PPD-520DYE for 10 minutes. After microwave treatment and elution, sections were covered with blocking solution and incubated for 10 minutes. After blocking nonspecific background staining, sections were incubated with rat anti-mouse CD8 / CD137 antibodies at room temperature for 1 hour, followed by incubation with the same secondary antibody for 10 minutes, and then stained with PPD-570DYE for 10 minutes. After microwave treatment again, cell nuclei were counterstained with DAPI and observed under a fluorescence microscope. The number of positive cells per 40× field of view was counted using software. Figure 4 This is a representative picture of multicolor immunofluorescence of tumor tissue. Compared with the control group, the CD8 + and CD4 + T cell infiltration increased significantly, and activated T cells co-labeled with CD3 and CD137 (CD3 + CD137 + T cells) increased significantly.

[0098] Tumor tissue from mice was digested into single-cell suspensions, and flow cytometry (FCW) was used to analyze the tumor-infiltrating lymphocyte (TIL) composition in the mouse tumor tissue samples. 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 80 rpm for 1 hour at 37°C. The digest 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 and centrifuged at 400 × g for 5 minutes. The supernatant was removed and the cells were resuspended in 100 μL of PBS buffer. Antibodies were then added, as indicated in Table 3, and incubated at room temperature in the dark for 20 minutes. After the antibody incubation is complete, centrifuge at 400×G for 5 minutes and remove the supernatant. Resuspend the cells in 1000μL PBS buffer and centrifuge at 400×G for 5 minutes. Remove the supernatant and repeat this process. Finally, resuspend the cells in 200μL staining buffer and transfer them to a flow cytometer for analysis. + T cells, activated T cells, CD4 + T cells, CD8 + The percentage of T cells. Figure 5This figure shows the proportion of activated T cells in tumor-infiltrating lymphocytes in tumor-bearing mice treated with the neoantigen vaccine. Compared to the control group, the proportion of T cells and activated T cells in the tumor increased after treatment with the neoantigen vaccine, indicating that the prostate cancer neoantigen vaccine generated an effective T cell response in the tumor-bearing mice.

[0099] Table 3 Flow cytometry staining protocol

[0100]

[0101]

[0102] This method utilizes second-generation genome sequencing technology to perform exon sequencing on tumor cell samples, obtaining a spectrum of genetic mutations in the tumor cells. Software is then used to predict tumor neoantigen mutation sequences at high-frequency mutation sites. Mutations with MHC / HLA affinity are then verified for expression using reverse transcription-polymerase chain reaction (RT-PCR). These verified peptide fragments are then synthesized using chemical synthesis. Finally, an ELISPOT in vitro assay is developed to verify the immunogenicity of tumor gene mutation sequences, allowing for the screening of immunogenic peptide fragments.

[0103] Then, the screened polypeptide fragments were prepared with sterile PBS buffer to obtain a polypeptide mixture. Then, they were mixed with adjuvants in proportion to prepare a tumor neoantigen vaccine preparation. Then, a mouse RM-1 transplant tumor model was established to verify the efficacy of the drug. The results showed 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, an increase in activated T cells, and the production of CD4 + T cells and CD8 + T cells are actively involved in anti-tumor immune responses.

[0104] The neoantigen vaccine formulation of the present invention achieved significant therapeutic effects in the RM-1 tumor model, with the tumor volume in the treatment group being significantly smaller than that in the control group. Moreover, the neoantigen vaccine formulation 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 intended only to illustrate the technical solutions of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may be modified or some of the technical features thereof may be replaced with equivalents. Such modifications or replacements do not deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A novel antigen polypeptide for treating or ameliorating prostate cancer, characterized in that: The amino acid sequence of the neoantigen polypeptide is at least one of the sequences shown in SEQ ID NO. 1-4.

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

3. A prostate cancer vaccine, characterized in that It comprises the neoantigen polypeptide according to claim 1, or the nucleic acid molecule according to claim 2, or a cell presenting the neoantigen polypeptide according to claim 1 or the nucleic acid molecule according to claim 2.

4. The prostate cancer vaccine according to claim 3, characterized in that The vaccine includes one or more of polypeptide vaccines, nucleic acid vaccines, whole cell vaccines, genetic engineering vaccines and antibody tumor vaccines.

5. The prostate cancer vaccine according to claim 3, characterized in that The vaccine includes an adjuvant.

6. The prostate cancer vaccine according to claim 5, characterized in that The adjuvant includes polyinosinic acid-polycytosine nucleotide Poly (I:C).

7. The prostate cancer vaccine according to claim 6, wherein The concentration of the polyinosinic acid-polycytosine nucleotide dilution solution is 0.1-5 mg / ml.

8. A kit, characterized in that It comprises the neoantigen polypeptide according to claim 1 or the nucleic acid molecule according to claim 2.

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

10. Use of the neoantigen polypeptide according to claim 1 or a personalized neoantigen vaccine comprising the same in the preparation of a medicament for treating or ameliorating prostate cancer.

11. The use according to claim 10, characterized in that The application includes combined application, which includes combining a personalized neoantigen vaccine based on or containing the neoantigen polypeptide with other drugs.

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