Novel treatment method of WT1 polypeptide antigen activated specific T cells in tumor targeting

Abstract

The invention belongs to the technical field of medical treatment, and discloses a novel treatment method of WT1 polypeptide antigen activated specific T cells in tumor targeting, and the method comprises the following steps: step 1, antigen preparation, step 2, vaccine construction, step 3, immune activation, step 4, combined treatment, and step 5, treatment monitoring and adjustment. After the WT1 polypeptide antigen is combined with the variant T cells, the immunocompetence of the variant T cells is activated, proliferation and differentiation of the variant T cells are promoted, cell factors are released, and tumor cells are directly killed or the immune microenvironment is adjusted; the new strategy of activating the variant T cells by the WT1 polypeptide antigen shows huge potential and wide application prospect in the field of tumor targeted therapy; with continuous progress and perfection of the technology, the method is expected to provide more effective and safer treatment choices for more tumor patients.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically is a new treatment method for tumor targeting by activating specific T cells with WT1 polypeptide antigen. Background Art

[0002] The development of personalized medicine has been accompanied by the development of tumor immunotherapy. The innovation of genomics has promoted the discovery of new immune targets and promoted the rational method of designing immunotherapy clinical trials. Since the approval of anti-CTLA-4 antibody (ipilimumab) for the treatment of advanced melanoma in 2011, personalized treatment of immune checkpoints has promoted the transformation of many cancer treatment modes. Anti-PD-1 antibody alone received 9 new FDA approvals in 2018. Sequencing cancer genomes is a powerful tool in precision medicine. The use of multi-platform technology can identify more potential targets, thereby increasing the probability of matching effective drugs. At present, as a disease that seriously threatens human health, its treatment has always been the focus of research in the medical field. With the continuous development of biotechnology, immunotherapy has gradually become a new strategy for tumor treatment. The method of activating variant T cells using WT1 peptide antigen in this application has brought new hope for tumor targeted therapy. Summary of the invention

[0003] The purpose of the present invention is to provide a new therapeutic method for activating specific T cells with WT1 polypeptide antigen in tumor targeting, so as to solve the problems raised in the above background technology.

[0004] In order to achieve the above object, the present invention provides the following technical solution: a new method for treating tumors by activating specific T cells with WT1 polypeptide antigens, the method comprising the following steps: Step 1: Antigen preparation: Using advanced genetic engineering technology, accurately design and construct an expression vector that can efficiently transcribe and translate the WT1 gene; select microorganisms as "biofactories", introduce the above expression vector into the microorganisms, and optimize the culture conditions to promote the large-scale and stable production of WT1 protein fragments. Then, mix the WT1 protein fragments with tumor cell lysate peptides or tumor neoantigens to prepare composite antigens to enhance immunogenicity; Step 2: Vaccine construction: using exosomes as carriers to transport antigens and carry antigens into antigen-presenting cells; or using targeted nanoliposomes to load tumor-associated antigen WT1 to prepare a vaccine that can induce antigen-specific T lymphocytes; Step 3: Immune activation: The constructed vaccine or antigen-loaded vector is injected into the patient. The antigen in the vaccine or vector is taken up and processed by antigen-presenting cells, and combines with its own major histocompatibility complex molecules to form an MHC-antigen peptide complex and is presented to the cell surface for T cell recognition, thereby activating specific T cell immune responses, inducing the production of a large number of WT1-specific cytotoxic T lymphocytes and helper T lymphocytes, and releasing cytokines to directly kill tumor cells or regulate the immune microenvironment. Step 4: Combination therapy: Combining immunotherapy based on WT1 gene polypeptide antigen protein with other cancer treatments; the combination therapy strategy aims to attack the tumor from multiple angles, both directly eliminating tumor cells and preventing tumor recurrence and metastasis by activating the immune system; Step 5. Treatment monitoring and adjustment: During the treatment process, closely monitor the patient's immune response, tumor marker levels, and imaging examinations to evaluate the treatment effect; adjust the treatment plan according to the patient's specific situation, including vaccine dosage, injection frequency, and combination therapy drug selection, to achieve personalized treatment.

[0005] Preferably, the step 1 of preparing the antigen includes: isolating and purifying high-purity WT1 polypeptide antigen protein from the bacterial culture through purification steps such as centrifugation, dialysis and chromatography to ensure the effectiveness and safety of subsequent steps.

[0006] Preferably, the exosomes are made by hybridizing plant exosomes and tumor exosomes, have a higher dendritic cell targeting ability, can effectively enhance the dendritic cell's ability to take up antigens, and enable the antigen to undergo efficient lymphatic migration and lymph node homing.

[0007] Preferably, the step three of immune activation also includes: when the receptor on the surface of the T cell binds to the MHC-antigen peptide complex, the T cell is activated and differentiated into two major categories; One type is cytotoxic T lymphocytes, which can directly recognize and kill tumor cells expressing WT1 antigen; The other type is helper T lymphocytes, which regulate the immune response by secreting cytokines, further enhancing the activity and number of CTLs.

[0008] Preferably, the microorganism in the antigen preparation in step 1 specifically includes but is not limited to one of Escherichia coli, yeast, insect cells and mammalian cells.

[0009] Preferably, the processing of the antigen in the vaccine or carrier after being taken up by antigen presenting cells includes: cutting the antigen into small molecule peptide segments through processing by the endoplasmic reticulum and Golgi apparatus.

[0010] Preferably, the combined treatment in step 4 is combined with, but not limited to, chemotherapy, which can cause tumor cell death, activate dendritic cells, induce cancer antigen-specific CTL response, and enhance the expression of cancer antigens, adhesion molecules and MHC.

[0011] Preferably, the step three before immune activation also includes: conducting a comprehensive assessment of the tumor patient, including tumor type, stage, and WT1 gene expression level, to determine whether the treatment method is suitable. If the assessment is suitable, then continue with step three; if not, then interrupt the treatment process.

[0012] Preferably, it also includes regular CT and MRI imaging examinations to observe changes in the size and morphology of the tumor; Detect carcinoembryonic antigen and alpha-fetoprotein tumor markers to assess tumor activity; Monitor the patient's immune indicators such as T cell subsets and cytokine levels to understand the intensity and duration of the immune response.

[0013] The beneficial effects of the present invention are as follows: After the variant T cells bind to the WT1 polypeptide antigen, the immune activity is significantly activated, which triggers a series of reactions: the variant T cells begin to proliferate and differentiate, and release cytokines at the same time. These cytokines can not only directly attack and kill tumor cells, but also effectively regulate the immune microenvironment, further enhancing the anti-tumor effect.

[0014] This approach exhibits several significant advantages: High specificity: Since variant T cells target WT1, this precise identification greatly reduces potential damage to normal tissues during treatment, ensuring the safety and effectiveness of treatment. Personalized treatment: Based on each patient's unique tumor characteristics and immune status, a treatment plan that best suits their individual situation can be customized, thereby improving the targeting and effectiveness of treatment. Potential long-term efficacy: By activating the patient's own immune system, the method of activating variant T cells with WT1 peptide antigens is expected to achieve long-term tumor control and bring lasting health benefits to patients.

[0015] Therefore, the new strategy of activating variant T cells with WT1 peptide antigens has shown great potential and broad application prospects in the field of tumor targeted therapy. With the continuous advancement and improvement of technology, this method is expected to provide more effective and safe treatment options for more cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The figure is a simplified flow chart of the novel method for targeted tumor therapy of the present invention. DETAILED DESCRIPTION

[0017] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0018] like Figure 1 As shown, the embodiment of the present invention provides a new method for treating tumors by activating specific T cells with WT1 polypeptide antigens, and the method comprises the following steps: Step 1: Antigen preparation: Using advanced genetic engineering technology, accurately design and construct an expression vector that can efficiently transcribe and translate the WT1 gene; select microorganisms as "biofactories", introduce the above expression vector into the microorganisms, and optimize the culture conditions to promote the large-scale and stable production of WT1 protein fragments. Then, mix the WT1 protein fragments with tumor cell lysate peptides or tumor neoantigens to prepare composite antigens to enhance immunogenicity; Step 2: Vaccine construction: using exosomes as carriers to transport antigens and carry antigens into antigen-presenting cells; or using targeted nanoliposomes to load tumor-associated antigen WT1 to prepare a vaccine that can induce antigen-specific T lymphocytes; Step 3: Immune activation: The constructed vaccine or antigen-loaded vector is injected into the patient. The antigen in the vaccine or vector is taken up and processed by antigen-presenting cells, and combines with its own major histocompatibility complex molecules to form an MHC-antigen peptide complex and is presented to the cell surface for T cell recognition, thereby activating specific T cell immune responses, inducing the production of a large number of WT1-specific cytotoxic T lymphocytes and helper T lymphocytes, and releasing cytokines to directly kill tumor cells or regulate the immune microenvironment. Step 4: Combination therapy: Combining immunotherapy based on WT1 gene polypeptide antigen protein with other cancer treatments; the combination therapy strategy aims to attack the tumor from multiple angles, both directly eliminating tumor cells and preventing tumor recurrence and metastasis by activating the immune system; Step 5. Treatment monitoring and adjustment: During the treatment process, closely monitor the patient's immune response, tumor marker levels, and imaging examinations to evaluate the treatment effect; adjust the treatment plan according to the patient's specific situation, including vaccine dosage, injection frequency, and combination therapy drug selection, to achieve personalized treatment.

[0019] Among them, step 1 antigen preparation includes: separating and purifying high-purity WT1 polypeptide antigen protein from bacterial culture through purification steps such as centrifugation, dialysis, and chromatography to ensure the effectiveness and safety of subsequent steps.

[0020] Among them, exosomes are made by hybridizing plant exosomes and tumor exosomes, which have higher dendritic cell targeting ability, can effectively enhance the dendritic cell's ability to take up antigens, and enable antigens to undergo efficient lymphatic migration and lymph node homing.

[0021] Among them, step three of immune activation also includes: when the receptor on the surface of T cells binds to the MHC-antigen peptide complex, the T cells are activated and differentiated into two major categories; One type is cytotoxic T lymphocytes, which can directly recognize and kill tumor cells expressing WT1 antigen; The other type is helper T lymphocytes, which regulate the immune response by secreting cytokines, further enhancing the activity and number of CTLs.

[0022] The microorganism in step 1 antigen preparation specifically includes but is not limited to one of Escherichia coli, yeast, insect cells and mammalian cells.

[0023] Among them, the processing process of the antigen in the vaccine or carrier after being taken up by the antigen presenting cells includes: cutting the antigen into small molecule peptide segments through processing by the endoplasmic reticulum and Golgi apparatus.

[0024] Among them, the combined treatment in step four adopts but is not limited to chemotherapy. Chemotherapy can cause tumor cell death, activate dendritic cells, induce cancer antigen-specific CTL response, and enhance the expression of cancer antigens, adhesion molecules and MHC.

[0025] Among them, step three before immune activation also includes: a comprehensive assessment of tumor patients, including tumor type, stage, and WT1 gene expression level, to determine whether the treatment method is suitable. If it is met, continue to step three, otherwise the treatment process will be interrupted.

[0026] This also includes regular CT and MRI imaging examinations to observe changes in the size and morphology of the tumor; Detect carcinoembryonic antigen and alpha-fetoprotein tumor markers to assess tumor activity; Monitor the patient's immune indicators such as T cell subsets and cytokine levels to understand the intensity and duration of the immune response.

[0027] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0028] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A new method for treating tumors by activating specific T cells with WT1 polypeptide antigens, characterized in that: The method steps include: Step 1: Antigen preparation: Using advanced genetic engineering technology, accurately design and construct an expression vector that can efficiently transcribe and translate the WT1 gene; select microorganisms as "biofactories", introduce the above expression vector into the microorganisms, optimize the culture conditions to promote the large-scale and stable production of WT1 protein fragments, and then mix the WT1 protein fragments with tumor cell lysate peptides or tumor neoantigens to prepare composite antigens to enhance immunogenicity; Step 2: Vaccine construction: using exosomes as carriers to transport antigens and carry antigens into antigen-presenting cells; or using targeted nanoliposomes to load tumor-related antigen WT1 to prepare a vaccine that can induce antigen-specific T lymphocytes; Step 3: Immune activation: The constructed vaccine or antigen-loaded vector is injected into the patient. The antigen in the vaccine or vector is taken up and processed by antigen-presenting cells, and combines with its own major histocompatibility complex molecules to form an MHC-antigen peptide complex and is presented to the cell surface for T cell recognition, thereby activating specific T cell immune responses, inducing the production of a large number of WT1-specific cytotoxic T lymphocytes and helper T lymphocytes, and releasing cytokines to directly kill tumor cells or regulate the immune microenvironment. Step 4: Combination therapy: Combining immunotherapy based on WT1 gene polypeptide antigen protein with other cancer treatments; the combination therapy strategy aims to attack the tumor from multiple angles, both directly eliminating tumor cells and preventing tumor recurrence and metastasis by activating the immune system; Step 5. Treatment monitoring and adjustment: During the treatment process, closely monitor the patient's immune response, tumor marker levels, and imaging examinations to evaluate the treatment effect; adjust the treatment plan according to the patient's specific situation, including vaccine dosage, injection frequency, and combination therapy drug selection, to achieve personalized treatment.

2. The novel therapeutic method of activating specific T cells with WT1 polypeptide antigen in tumor targeting according to claim 1, characterized in that: The step 1 of antigen preparation includes: separating and purifying high-purity WT1 polypeptide antigen protein from bacterial culture through purification steps of centrifugation, dialysis and chromatography to ensure the effectiveness and safety of subsequent steps.

3. The novel therapeutic method of activating specific T cells with WT1 polypeptide antigen in tumor targeting according to claim 1, characterized in that: The exosomes are made by hybridizing plant exosomes and tumor exosomes, and have a higher dendritic cell targeting ability, which can effectively enhance the dendritic cell's ability to take up antigens, allowing the antigens to undergo efficient lymphatic migration and lymph node homing.

4. The novel therapeutic method of activating specific T cells with WT1 polypeptide antigen in tumor targeting according to claim 1, characterized in that: The step three of immune activation also includes: when the receptor on the surface of the T cell binds to the MHC-antigen peptide complex, the T cell is activated and differentiated into two major categories; One type is cytotoxic T lymphocytes, which can directly recognize and kill tumor cells expressing WT1 antigen; The other type is helper T lymphocytes, which regulate the immune response by secreting cytokines, further enhancing the activity and number of CTLs.

5. The novel therapeutic method of activating specific T cells with WT1 polypeptide antigen in tumor targeting according to claim 1, characterized in that: The microorganism in the antigen preparation in step 1 specifically includes but is not limited to one of Escherichia coli, yeast, insect cells and mammalian cells.

6. The novel therapeutic method of activating specific T cells with WT1 polypeptide antigen in tumor targeting according to claim 1, characterized in that: The processing of the antigen in the vaccine or carrier after being taken up by the antigen presenting cells includes: cutting the antigen into small molecule peptide segments through processing by the endoplasmic reticulum and the Golgi apparatus.

7. The novel therapeutic method of activating specific T cells with WT1 polypeptide antigen in tumor targeting according to claim 1, characterized in that: The combined treatment in step 4 is but not limited to chemotherapy. Chemotherapy can cause tumor cell death, activate dendritic cells, induce cancer antigen-specific CTL response, and enhance the expression of cancer antigens, adhesion molecules and MHC.

8. The novel therapeutic method of activating specific T cells with WT1 polypeptide antigen in tumor targeting according to claim 1, characterized in that: The step three before immune activation also includes: conducting a comprehensive assessment of the tumor patient, including tumor type, stage, and WT1 gene expression level, to determine whether the treatment method is suitable. If the patient is suitable, then step three will be continued; if not, the treatment process will be interrupted.

9. The novel therapeutic method of activating specific T cells with WT1 polypeptide antigen in tumor targeting according to claim 1, characterized in that: It also includes regular CT and MRI imaging examinations to observe changes in the size and morphology of the tumor; Detect carcinoembryonic antigen and alpha-fetoprotein tumor markers to assess tumor activity; Monitor the patient's immune indicators such as T cell subsets and cytokine levels to understand the intensity and duration of the immune response.

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