WO2014075631A1

WO2014075631A1 - Method for preparing autologous tumor vaccine and use thereof

Info

Publication number: WO2014075631A1
Application number: PCT/CN2013/087266
Authority: WO
Inventors: 韩德平; 林建华; 张鲁榕; 张美�; 张震寰; 张亨山; 吕文龙; 洪金省
Original assignee: 厦门鹭佳生物科技有限公司
Priority date: 2012-11-15
Filing date: 2013-11-15
Publication date: 2014-05-22
Also published as: CN109876137A; CN103800896A

Abstract

Provided are a method for preparing an autologous tumor vaccine and uses thereof, specifically comprising: radiating and/or heating autologous tumor cells to obtain the tumor cells having enhanced antigenicity and antigen presentation, cultivating the obtained tumor cells, deactivating the cultivated tumor cells, and adding adjuvant to prepare the anti-tumor vaccine.

Description

Preparation method of autologous tumor vaccine and application thereof

Technical field

The present invention is in the field of biotechnology and immunology, and in particular, the present invention relates to a method for preparing an autologous tumor vaccine and an application thereof. Background technique

Tumors are one of the leading causes of death in humans. Although the level of diagnosis and treatment of tumors has been continuously improved and improved in recent years, the programs of chemotherapy and radiotherapy are constantly improving, but in the end, most patients still have difficulty to escape the fate of death.

In different treatment modes of malignant tumors, immunotherapy has attracted more and more attention because of its specific killing of tumor cells, high efficiency and low toxic side effects. In recent years, advances in molecular biology and further understanding of the function of the immune system have led to the development and development of biotherapeutics that has entered a stage of rapid development. The development of tumor vaccine is the most important direction of tumor biotherapy.

However, malignant tumors have multiple immune escape mechanisms, including low antigen content in tumor cells, destruction or constant mutation of tumor antigens, poor treatment of tumor antigens, or lack of expression of some immune cofactors, or low immune function. Etc., the clinical treatment effect of existing tumor tumor vaccines is not good. Because of the heterogeneity, diversity, and variability of malignant tumors, the individual tumor antigens of different individuals are very different when they are suffering from the same kind of tumor. Thus, tumor immunotherapy should be individualized to induce active immunity of individual-specific killing autologous tumor cells.

Whole cells are now used as tumor vaccines to provide unknown tumor antigens to activate immune system tumor antigens. In a mouse tumor model, mice are usually immunized with inactivated tumor cells to protect the mice from inoculation of the inoculated tumor. However, when the time of use of the tumor cell vaccine is delayed until one week after inoculation of the tumor cells, the vaccine loses the ability to protect the mouse. The current clinical response to tumor cell vaccines is relatively poor, and it is only suitable for preventing recurrence of tumor patients without special tumor antigens. Very few good results have been obtained in clinical studies for advanced patients.

An ideal tumor-specific antigen should be immunogenic, expressed by tumor cells but not expressed in normal cells. Unfortunately, most tumor antigens do not have sufficient immunogenicity to induce an effective immune response.

At present, there is still a lack of good immunogenicity and specific autologous tumor vaccine and preparation method thereof. The field urgently needs to develop methods and vaccines. Summary of the invention

It is an object of the present invention to provide a method of preparing an autologous tumor vaccine.

Another object of the present invention is to provide an autologous tumor vaccine and use thereof. In a first aspect of the invention, a method of preparing an autologous tumor vaccine is provided, comprising the steps of:

(1) obtaining isolated autologous tumor cells;

(2) treating the autologous tumor cells of step (1) by radiotherapy and/or heating to obtain tumor cells having enhanced antigenicity and antigen-promoting properties;

(3) inactivating the tumor cells obtained in the step (2) to obtain inactivated tumor cells;

(4) The inactivated tumor cells obtained in the step (3) were prepared as an autologous tumor vaccine.

In another preferred embodiment, the tumor is: a solid tumor or a non-solid tumor.

In another preferred embodiment, the solid tumor is derived from: brain, lung, head and neck, skin, pancreas, gastrointestinal tract, bladder, genital tract, spinal cord, spleen, kidney, liver, limbs, bones, tongue, Or throat.

In another preferred embodiment, the non-solid tumor is derived from blood or bone marrow.

In another preferred embodiment, the tumor is a primary tumor or a secondary metastatic tumor.

In another preferred embodiment, the tumor is derived from a tissue of a non-human mammalian tissue or a human.

In another preferred embodiment, the method for obtaining the isolated autologous tumor cells according to the step (1) is selected from the group consisting of: enzymatic digestion, mechanical shearing, or artificial grinding of tumor tissue to treat tumor tissue to be culturable. Isolated autologous tumor cells.

In another preferred embodiment, the radiation method described in the step (2) is selected from the group consisting of: selected from α, β, γ, proton, or heavy ion radiation, optionally selected from the range of 0.1-50 Gy, optionally The autologous tumor cells of step (1) were treated at a dose rate of 0.1-10 Gy/min to obtain antigenic and antigen-presenting tumor cells.

In another preferred embodiment, the radiation is derived from a natural or artificial source of radiation.

In another preferred embodiment, the heating method described in the step (2) is selected from the group consisting of: heating temperature is selected from the group consisting of

37.5 ° C -50 ° C (preferably 38 ° C -45 ° C), the heating time is selected from 1 min to 2 hours (preferably 30 min - l hour).

In another preferred embodiment, between the step (2) and the step (3), the method further comprises the step of: culturing the tumor cells obtained by the step (2) to obtain antigenicity and antigen-promoting enhancement. In another preferred embodiment, the culture treatment time is selected from the group consisting of: 6 hours to 2 weeks.

In another preferred embodiment, in the culture treatment, a basic cell culture solution is used, and/or a growth factor or a small molecule compound is added.

In another preferred embodiment, the inactivation treatment described in the step (3) is selected from the group consisting of microwave cell inactivation, electromagnetic wave cell inactivation, laser cell inactivation, high temperature cell inactivation, sonication and cell disruption. Live method, repeated cell freeze-thaw, homogenate, centrifugation, pellet cell inactivation, enzymatic digestion cell inactivation, hypotonic rupture, or a combination thereof.

In another preferred embodiment, in step (4), the inactivated tumor cells obtained in the step (3) are mixed with an adjuvant to obtain an autologous tumor vaccine.

In another preferred embodiment, the adjuvant is Freund's complete adjuvant or incomplete adjuvant.

In another preferred embodiment, the adjuvant is selected from the group consisting of liposomes, cytokines (eg, GM-CSF, G-CSF,

CSF, IFN, IL-2, etc.), various plant proteins, DNA, deactivated or inactivated viruses, bacteria, fungi, microorganisms and their extracts, or combinations thereof.

In a second aspect of the invention, there is provided an autologous tumor vaccine prepared by the method of the first aspect.

In another preferred embodiment, the vaccine further comprises an adjuvant.

In a third aspect of the invention, there is provided the use of the autologous tumor vaccine of the second aspect, which is used for the preparation of a composition for preventing or treating a tumor.

In another preferred embodiment, the composition is a pharmaceutical composition or a vaccine composition.

In another preferred embodiment, the composition is in the form of an injection. Preferably, the injection is administered by a route selected from the group consisting of intradermal single/multiple injection, subcutaneous single/multiple injection, intralymphatic or peripheral injection, intramuscular injection, intrathoracic/peritoneal injection, Intravenous, peritumoral normal tissue injection. Preferably, it is the distal end of the intradermal limb.

In another preferred embodiment, the injection site of the injection is selected from the group consisting of: primary tumor, metastases, lymphatic drainage area around the primary tumor, lymphatic drainage area around the metastasis, distal extremities, proximal extremities, chest Back, abdomen, neck, locally accessible lymph nodes or lymphatic drainage areas.

In another preferred embodiment, the vaccine can be used in combination with a drug selected from the group consisting of cytokines, electromagnetic wave therapy, or a combination thereof. Preferably, the drug is selected from the group consisting of IFN, TNF, IL-2, or a combination thereof.

In another preferred embodiment, the inactivation treatment described in the step (3) is selected from the group consisting of microwave cell inactivation, electromagnetic wave cell inactivation, laser cell inactivation, high temperature cell inactivation, sonication and cell disruption. Live method, repeated cell freeze-thaw, homogenate, centrifugation, pellet cell inactivation, enzymatic digestion, cell inactivation, hypotonic rupture, chemical covalent Combined method, chemical denaturing method, or a combination thereof.

In a fourth aspect of the invention, a method of preventing or treating a tumor comprising administering an effective amount of the tumor vaccine of the second aspect of the invention to a subject is provided.

In another preferred embodiment, the administration time is selected from the group consisting of: postoperative, pre-chemotherapy, or WBC rebound to >3000 per ul.

In another preferred embodiment, the number of administrations may be 4 courses (3-4 weeks per course) to life.

In another preferred embodiment, the method further comprises: monitoring the extent of the immune response using an immunological assay.

In another preferred embodiment, the immunological assay is selected from the group consisting of a cellular immune response, a humoral immune response, an immunomodulatory response, or a combination thereof.

In another preferred embodiment, the method for detecting cellular immune response comprises:

1. Intradermal injection of individualized tumor vaccine, the anti-tumor delayed allergic reaction can be seen as a cumulus 0.5 cm or more for a good immune response, <0.5 cm for poor immune response;

2. Lymphocyte transformation reaction: The lymphocytes of the patient one month after immunization are separated, and the vaccine of the second aspect of the invention is added to observe the conversion rate (preferably by BrdU method, MTT method), and the conversion rate is high. Good immune response;

3. Detection of lymphokines secreted by T/B cell immune response;

Preferably, the lymphokine is selected from the group consisting of IL-2, IL-4, IL-6, IL-10, IL-12, IL-17, IL-18, TNF-a, IFN-γ, Or a combination thereof;

Preferably, the detection method is selected from the group consisting of ELISPOT, ELISA, FCM, or a combination thereof.

4. Detection of immune cell subpopulations and surface killing markers;

Preferably, the subset of immune cells is selected from the group consisting of CD4+, CD8+, CD1 lb+, Grl+, or a combination thereof;

Preferably, the killer activation marker is selected from the group consisting of Fas L, LIGHT, CD25, TNF-a, IFN-γ, perforin or a combination thereof.

In another preferred embodiment, each subpopulation is detected by flow cytometry.

In another preferred embodiment, the humoral immunoassay method comprises:

1. Detection of anti-autoantigen antibody: The prepared tumor vaccine is used as an antigen coating to add plasma before and after autoimmunization, and the antibody titer against autoantitumor antigen is compared before and after immunization. The higher the titer, the stronger the immunoreactivity. In another preferred embodiment, the molecular weight of the autoantigen is determined by the WESTERN BLOT method. In another preferred embodiment, the immunomodulatory reaction detecting method comprises:

1. Detection of a subset of immunoregulatory cells; preferably, said subpopulation comprises: regulatory T cells: Treg: CD4 ⁺ CD25 ⁺ , myeloid-derived suppressor cells MDSC: CD14 - CD11b ⁺ , tumor-associated macrophages TAM: Ml/M2, regulatory dendritic cell DCreg: CD11b+/Gr-l+, or a combination thereof. Preferably, the detection method is flow cytometry.

2. Determination of changes in immunoregulatory factors in plasma. Preferably, the immunoregulatory factors include: IL-2, IL-4, IL-6, IL-10, IL-12, IL-17, IL-18 , TNF-a, IFN-γ, or a combination thereof; the assay comprises: ELISA, LUMINEX bead-array assay, or a combination thereof.

In another preferred embodiment, the immunological assay is performed within 15-60 days after the first immunization.

In another preferred embodiment, the method further comprises: determining the therapeutic effect and/or adjusting the treatment regimen based on the degree of immune response measured by the immunological experiment.

In another preferred embodiment, the adjusted treatment regimen refers to adjusting one or more therapeutic parameters selected from the group consisting of: immunoadjuvant binding, immunization dose, immunization site, immunization time, number of immunizations, low dose radiation, other physics Or biological methods to stimulate immune organs.

In another preferred embodiment, the immune organ is bone marrow.

In another preferred embodiment, the method includes:

(i) administering the vaccine to the patient on the third postoperative day;

(ii) administering the vaccine to the patient again 7-14 days after surgery;

(iii) administering the vaccine to the patient again 14-24 days after surgery;

(iv) Immunological tests were used to monitor the degree of immune response on days 31-34 after surgery, and the immunotherapy protocol was adjusted by the test results.

In another preferred embodiment, the administration of the vaccine and its effect observation time may vary flexibly due to individual differences. It is to be understood that within the scope of the present invention, the various technical features of the present invention and the technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here. DRAWINGS

The following figures are used to illustrate specific embodiments of the invention and are not intended to be limited by the scope of the claims The scope of the invention is intended.

Figure 1 shows the results of anti-tumor antibody IgG assay 3 weeks after injection of the auto-tumor vaccine.

Figure 2 shows an anti-tumor T cell killing experiment.

Figure 3 shows the experimental grouping and the operational flow.

Figure 4 shows the results of real-time dynamic measurement of anti-tumor effect fluorescence (Live Image).

Fig. 5 shows the results of the vaccine antigenic cancer (4T1-Luc) effect (test 1) prepared by the method of Example 1 of the present invention.

Fig. 6 shows the results of the vaccine antigenic cancer (4T1-Luc) effect (test 2) prepared by the method of Example 1 of the present invention.

Figure 7 is a graph showing the results of a comparison between the vaccine prepared by the method of Example 1 of the present invention and the anticancer effect of Taxol (Trial 3, compared with the chemotherapy drug Taxol).

Figure 8 shows the killing inhibition effect of the method of one embodiment of the present invention on experimental lung metastasis.

Figure 9 shows the flow of an autologous tumor vaccine prepared in accordance with one embodiment of the present invention.

Fig. 10 shows a tumor cell illuminator; wherein, the Y-ray is used to break the DNA and cause DNA error repair, and at the same time, the characteristics of the oncogenic gene which promotes growth in a harsh environment are highly expressed, and the tumor cells are treated with the Y illuminator of the exclusive invention. The oncogene is highly expressed, and the activation of tumor antigen is enhanced to increase the efficacy of the method.

Figure 11 shows a bone marrow growth stimulator; wherein, using medium/high frequency electromagnetic waves to stimulate cell growth, the bone marrow growth stimulator is placed in the humerus of tumor-free cells to stimulate hematopoietic stem cell proliferation and differentiation, so that autoantibodies The number of cancer immune cells is increased to increase the efficacy of this method. detailed description

After extensive and intensive research, the present inventors have discovered for the first time that the treatment of radiation and/or heating of autologous tumor cells can greatly stimulate the antigenicity of autologous tumor cells, and heat shock proteins enhance antigen presentation; The cells are inactivated and supplemented with adjuvant to make a vaccine. The vaccine has the special advantages of strong presentation and strong immunogenicity of the tumor, and can strongly stimulate the humoral and cellular immune responses of the tumor patients to the autologous tumor, so as to improve the antigenic and metastatic tumor effect and prolong the survival of the tumor patient. The purpose of time. On this basis, the invention of a novel autologous tumor vaccine was completed. Its application is also based on monitoring the patient's response to autologous tumor vaccines, changing the amount of vaccine, the location and time of injection, the length and interval of treatment, and truly Active immunotherapy. Preparation

The present invention provides a method of preparing an autologous tumor vaccine, and in a preferred embodiment, comprising the steps of: (1) obtaining isolated autologous tumor cells;

Specifically, it includes the following steps (Figure 9):

Obtaining tumor tissue by surgery or biopsy;

Prepare the tumor cells as follows: Obtain the tumor-free, non-necrotic part of the tumor under sterile conditions, store in DMEM or other culture medium, store at 4 ° C, rinse with PBS, and digest by enzyme. Purified tumor cells were obtained by method or / and mechanical separation method, transferred into a culture flask, subcultured at 37 ° C, 5% CO ₂ incubator, and reached the required number of cells to enter the tumor whole cell tumor preparation procedure;

The tumor cells in culture are treated by radiation and/or heating to excite the antigen, and the increased heat shock protein enhances antigen presentation.

Further, the treated tumor cells are inactivated by the following methods: microwave cell inactivation method, electromagnetic wave cell inactivation method, laser cell inactivation method, high temperature cell inactivation method, ultrasonic disruption cell inactivation method, repeated cells At least one of freezing and thawing, homogenization, centrifugation, sedimentation cell inactivation, enzymatic digestion, cell inactivation, hypotonic rupture, and the like;

After inactivation, a tumor whole cell vaccine is obtained.

The invention is supplemented by the above tumor whole cell vaccine with an adjuvant, and the adjuvant may be a complete Freund's adjuvant or an incomplete adjuvant, after a water-in-oil adjuvant, a liposome, a cytokine (such as GM-CSF, various Plant protein DNA), attenuating or inactivating an adjuvant such as a virus, a bacterium, a fungus, a microorganism or an extract thereof. After adjuvanting, it can better enhance the active immune response, reduce the negative regulation of immunity, and enhance the stimulation and memory of anti-tumor immune cells.

The method of the invention is to treat the tumor cells by antigenic stimulation treatment and enhance the antigen presenting ability, and then inactivate the treatment to lose the tumorigenicity, and retain the immunogenicity after the stimulation, and simultaneously combine the immunosupsis; As an individualized tumor vaccine, we will establish an individualized treatment method and keep track of individual immunity. State and tumor conditions continue to adjust individualized immunotherapy programs to achieve optimal anti-tumor efficacy. Immune effect monitoring

One of ordinary skill in the art can determine the anti-tumor immune response of a vaccine using conventional methods. Including: anti-tumor humoral immune response evaluation, anti-tumor cell immune response evaluation, anti-tumor clinical response evaluation.

The anti-tumor humoral immune response was evaluated by using an individualized tumor vaccine as an antigen-coated ELISA method to detect the anti-autoantibody antibody titer in the serum of the individual before and after immunization of the individualized tumor vaccine to objectively judge the anti-tumor humoral immune response.

Anti-tumor cell immune response evaluation, generally, including the new individualized tumor vaccine prepared by intradermal injection, observe the patient's own pimple size within 1-3 days (late-type individualized tumor vaccine immune response); or self-tumor stimulation specific Sexual lymphocyte transformation experiment: including steps: taking anticoagulation, separating lymphocytes, serum-free culture, adding individualized tumor vaccine or control vector, culturing, judging lymphocyte transformational proliferation level (BrdU incorporation method or MTT method); Individualized tumor vaccine immunomodulatory response. Immunomodulatory responses to individualized tumor vaccines include: detection of leukocyte populations (eg, CD4/CD8/CD1 lb/Gr-l, etc.) and their surface killing or activating markers (eg, FasL/LIGHT/CD25/TNF-a/IFN-) y, etc., according to the results of these in vitro and in vivo experiments can be divided into two types: good response and poor response: high antibody titer, pyel is greater than 0.5 cm, so can maintain the original individualized immunization program; poor response antibody titer Low, pipi is less than 0.5 cm, so you can change the use of individualized tumor vaccine and use different means to enhance the immune response. The subset of immunoregulatory cells includes: regulatory T cell Treg (CD4+CD25+), myeloid-derived suppressor cell MDSC (CD14-CD1 lb+), tumor-associated macrophage TAM (M1/M2), regulatory dendritic The cells were DCreg (CDl lb+/Gr-l+) and each subpopulation was detected by flow cytometry.

Anti-tumor clinical response: According to the tumor image size evaluation, according to the experimental results, the patient's immune status, tumor condition and other anti-tumor treatment programs, decide when to strengthen anti-tumor immunity, adjust individualized treatment plan, continue 2, 3, 4 Or more treatments. Efficacy monitoring includes, but is not limited to, imaging criteria, tumor-free survival time, and overall survival time.

In short, continuous tracking of individual immune status and continuous adjustment of individualized immunotherapy according to the condition of the tumor, in order to achieve the best anti-tumor efficacy.

As mentioned above, the immune response effect of individualized tumor vaccine can be monitored by cellular immunity, humoral immunity, and clinical tumor response. The monitoring time can be about 15 days to 60 days after the first immunization, and the data can be evaluated if the reaction In the case of poor results, individualized immunotherapy regimens can be re-adjusted, such as by adjusting immune adjuvant binding, immunization dose, immune site, immunization time, number of immunizations, and low-dose radiation-stimulated immune organs such as bone marrow. Composition and mode of administration

The present invention also provides a composition, which may be a pharmaceutical composition or a vaccine composition. The compositions of the invention may be therapeutic or prophylactic. The compositions of the present invention comprise an effective amount of an autologous tumor vaccine of the invention, and at least one pharmaceutically acceptable carrier, diluent or excipient.

The pharmaceutical compositions of the present invention can be prepared into various conventional dosage forms including, but not limited to, injections, granules, tablets, pills, suppositories, capsules, suspensions, sprays and the like.

(i) pharmaceutical composition

The pharmaceutical compositions of the present invention comprise an effective amount of an autologous tumor vaccine prepared by the method of the present invention.

The term "effective amount" as used herein refers to an amount of a therapeutic agent that treats, alleviates or prevents a target disease or condition, or an amount that exhibits a detectable therapeutic or prophylactic effect. This effect can be detected by, for example, antigen level. Therapeutic effects also include a reduction in physiological symptoms. The precise effective amount for a subject will depend on the size and health of the subject, the nature and extent of the condition, and the combination of therapeutic and/or therapeutic agents selected for administration. Therefore, it is useless to specify an accurate effective amount in advance. However, for a given situation, routine experimentation can be used to determine the effective amount.

For the purposes of the present invention, an effective dose is from about 0.2 micrograms per kilogram to 2 micrograms per kilogram of the subject. The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration to a therapeutic agent, such as a protein, VLP or other therapeutic agent. The term refers to pharmaceutical carriers which do not themselves induce the production of antibodies harmful to the individual receiving the composition and which are not excessively toxic after administration. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid and the like. These vectors are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable carriers or excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

The pharmaceutically acceptable carrier in the composition may include liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers. In general, the composition can be formulated as an injectable, such as a liquid solution or suspension; it can also be formulated to be suitable for dissolution prior to injection. Solid form of liquid or suspension, liquid excipient. Liposomes are also included in the definition of pharmaceutically acceptable carriers.

(ii) Vaccine composition

The vaccine compositions of the invention may be prophylactic or therapeutic. The vaccine composition comprises an immunogenic antigen (autologous tumor vaccine of the invention) and is typically combined with a "pharmaceutically acceptable carrier" which includes antibodies which do not themselves induce the production of antibodies harmful to the individual receiving the composition. Any carrier. Suitable carriers are generally large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, amino acid polymers, amino acid copolymers, lipid agglutinates (e.g., oil droplets or liposomes), and the like. These vectors are well known to those of ordinary skill in the art. In addition, these carriers can function as immunostimulating agents ("adjuvants"). In addition, the antigen can be coupled to bacterial toxoids (such as toxoids of pathogens such as diphtheria, tetanus, cholera, Helicobacter pylori) or bacterial extracts (such as endotoxin or flagellin).

Preferred adjuvants for enhancing the effect of the immunological composition include, but are not limited to: (1) aluminum salts (alum) such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, and the like; (2) oil-in-water emulsion formulations, for example, (a) MF59 (see WO 90/14837), (b) SAF, and (c) RibiTM Adjuvant System (RAS) (Ribi Immunochem, Hamilton, MT), (3) saponin adjuvant; (4^ 61111 ₍ 1 Complete adjuvant (?) and ?61111 ₍ 1 incomplete adjuvant (1?); (5) cytokines, such as interleukins (such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc., interferon (such as gamma interferon), macrophage colony-stimulating factor (M-CFS), mononuclear-macrophage colony-stimulating factor (GM-CFS), tumor necrosis factor ( TNF), etc.; (6) Detoxification variants of bacterial ADP-ribosylating toxins (such as cholera toxin CT, pertussis toxin PT or E. coli heat labile toxin LT), see for example WO93/13302 and WO92/19265; 7) Other substances that act as immunostimulating agents to enhance the effect of the composition.

Vaccine compositions, including immunogenic compositions (e.g., can include antigens, pharmaceutically acceptable carriers, and adjuvants), typically contain a diluent such as water, saline, glycerol, ethanol, and the like. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may be present in such carriers.

More specifically, vaccines, including immunogenic compositions, comprise an immunologically effective amount of an immunogenic polypeptide, as well as other desired components as described above. "Immunologically effective amount" means that the amount administered to a subject in a single dose or in a continuous dose is effective for treatment or prevention. The amount may be based on the health and physiological condition of the individual being treated, the type of individual being treated (eg, human), the ability of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the assessment of the medical condition by the treating physician, And other relevant factors. This amount is expected to be in a relatively wide range and can be determined by routine experimentation. In general, the vaccine composition or immunogenic composition can be formulated as an injectable preparation, such as a liquid solution or suspension; it can also be formulated as a solid form suitable for solution or suspension, liquid excipient prior to injection. The formulation may also be emulsified or encapsulated in liposomes to enhance the adjuvant effect.

(iii) route of administration and dosage

The composition can be administered directly to a subject. The subject can be a human or a non-human mammal, preferably a human. When used as a vaccine, it can be directly administered to an individual by a known method. These compositions are usually administered by the same route of administration as conventional vaccines.

Routes for administering a pharmaceutical composition or vaccine composition of the invention include, but are not limited to: Formulated pharmaceutical compositions can be administered by conventional routes, including but not limited to: such as intradermal single/multiple Point injection, subcutaneous single/multiple injection, intra- or peripheral lymphatic injection, intramuscular injection, intrathoracic/peritoneal injection, intravenous injection, peritumoral/intratumoral injection. The above approaches may be applied separately depending on the specific situation, and may be applied in combination if necessary. The patient's own site may be, but is not limited to, the primary tumor, metastases or surrounding lymphatic drainage areas and distal or proximal extremities, thoracodorsal, abdomen, neck, locally accessible lymph nodes or lymphatic drainage areas.

When using a pharmaceutical composition, dose control should be considered within a safe and effective range. The specific dose should also take into account the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.

In addition, the vaccine of the present invention may also be used in combination with other drugs or physical therapies, including but not limited to: various cytokines such as IFN, TNF, IL-2, GM-CSF, G-CSF, SCF, And electromagnetic waves, etc. The main advantages of the present invention are as follows - (1) The present invention actively stimulates tumor antigen expression and antigen processing presentation function by irradiation and/or heating, and then inactivates, supplements with adjuvant, and modulates immunotherapy according to individual immune response. Program, to achieve the purpose of truly individualized tumor immunotherapy;

(2) The present invention can promote immunity in the body, especially for cellular immunity and humoral immunity of tumors;

(3) The technical method for treating or preventing tumors according to the present invention is suitable for use in the medical field, particularly for preventing or treating tumors, and is also suitable for postoperative tumor residuals and patients with micrometastases, and provides a field in the art. The new choice has broad application prospects. The invention is further illustrated below in conjunction with specific embodiments. It should be understood that these embodiments are for illustrative purposes only. The invention is not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. The suggested conditions. Example 1

Preparation of individualized tumor vaccine

Take 1*10 ⁷ cells in Lewis logarithmic growth phase or Lewis lung cancer cells, 30-60 minutes after irradiation with γ-rays (4-10 Gy, 1.4 Gy/min), and heat for 20 minutes in 37-40 °C water bath. At 37 ° C, 5% CO ₂ incubator for 48 hours, then ultrasonically disrupted cells and then added Freund's adjuvant to prepare an emulsifier is a tumor whole cell vaccine. Example 2

Individualized tumor vaccine for treatment of subcutaneous breast cancer in mice

In the present example, it was confirmed by the relevant indexes of the cell and animal experiments that the tumor vaccine prepared by the method of the present invention has strong antigen presentation ability and ability to elicit an antibody immune response. The specific results are as follows:

1. Promote the production of anti-tumor antibodies

Experimental method: Conventional Balb/c mice were immunized 3 weeks after immunization with the tumor cell antigen prepared in Example 1, and the plasma was diluted and added to the ELISA plate coated with tumor cells to anti-mouse IgG-HRP and TMB substrates. The antibody titer is shown (this experiment is 1:100000).

The results showed that the tumor antigen prepared in Example 1 was more potent against tumor antibody production than the tumor vaccine prepared by the control method (Fig. 1): After 3 weeks of immunization of Balb/c mice, Example 1 The prepared antigen stimulated more antibodies against 4T1 breast cancer cells (t test, p<0.05, n=10), NC: control; NS: adjuvant control; ORIGINAL: original method (added only after killing tumor cells) Adjuvant) prepared tumor vaccine (as a control); EW: a novel tumor vaccine prepared by the present invention.

2. Promote anti-tumor cell immune response

The method is the same as above. Balb/c mice were immunized with the tumor cell antigen prepared in Example 1 for 3 weeks, and lymphocytes (LN) and spleen cells (SP) were taken and the constructed Luciferase-containing 4T1 tumor cells (4T1-luc) were 10:1. 100: 1, 1000: 1 ratio mixed culture, 72 hours after the release of Luciferase released from dead 4T1 cells Activity (represented by RLu).

As a result, as shown in Fig. 2, mice immunized with the tumor cells prepared in Example 1 were able to produce T cell killing more strongly than adjuvant (p < 0.05, n = 5) _; Fig. 2A is an antitumor T in the adjuvant group. Cell killing experiment, Fig. 2 is the tumor cell antigen anti-tumor cell killing experiment prepared by the method group of Example 1, Rlu refers to the activity of Luciferase; NC refers to the normal control group; LN refers to lymph node cells; SP refers to spleen cells. Example 3

Inhibition of mouse tumors

According to the principle of immunology, the mouse anti-tumor immunity test is divided into two steps:

1. Tumor immunosensitization: The treated tumor antigen novel tumor vaccine was first inoculated subcutaneously in the test mice, and 14 days later, the immunization was boosted once again, and the homologous mouse tumor cells were inoculated 7 days later (Fig. 3).

2. Immunization against cancer: There are two categories:

a. Inhibition of carcinoma in situ: BALB/c mice and 4T1 breast cancer cell lines were used; b. Inhibition of metastatic tumors: C57BL/6 mice and Lewis lung cancer cell lines were used. Lewis and 4T1 cancer cells are highly malignant, grow fast, are difficult to control, and can form lung metastases.

Judging anti-tumor effect:

1. Antigenic cancer judgment:

a, living tumor fluorescence quantitative method: the above 4T1 cells with Luciferase gene, so 4T1-Luc tumor size can be perfused into the anesthesia tumor-bearing mice peritoneal injection of Luciferin substrate, 5 minutes after the determination of fluorescence intensity; b, vernier caliper to determine the tumor diameter; Both methods can objectively reflect tumor size.

2. Anti-pulmonary metastasis effect: Due to the rapid growth of experimental Lewis lung cancer, the cancer nodules are rapidly fused into a piece, and the number of nodules cannot be calculated. Therefore, the results are calculated by whole lung weighing.

The mice immunized with the tumor antigen prepared in Example 1 were subjected to intravenous or subcutaneous injection of live tumor cells to form metastatic cancer and carcinoma in situ for the antitumor effect.

The experimental results are as follows:

1. Killing inhibition of carcinoma in situ

As shown in Figure 4, the tumor-injected Balb/c mouse tumor fluorescence imaging showed that compared with the control group (adjuvant immunization) and the original method of preparation of tumor vaccine (adjuvant + inactivated tumor cells) group, The vaccine group prepared in Example 1 had a small number of tumors and a small volume. The tumor growth curve (Fig. 5) showed that the growth rate of the in situ carcinoma of the new tumor vaccine group was significantly slower than that of the original tumor vaccine preparation group, the adjuvant group alone, and the saline group (p<0.01). Similar results were obtained (Fig. 6, Fig. 7). It is worth noting that the fastest growing tumor of 4T1-Luc cells could not be inhibited by Taxol 5 mg/kg (daily injection) but was inhibited by the vaccine prepared by the method of the present invention.

2. Kill inhibition of lung metastases

Metastasis is the main cause of tumor death. To investigate the killing inhibition effect of the vaccine of the present invention on metastatic tumors, experimental lung metastasis of Lewis lung cancer cells on homologous C57BL/6 mice was used as a detection model.

Conventional commercially available C57BL/6 mice (8 weeks old, female, 5/group) were divided into four groups:

A, saline control group, subcutaneous injection of simulated saline immunization process;

B. Adjuvant control group, only receiving adjuvant without tumor cell antigen;

C, new method (new tumor vaccine) group, adjuvant + treated tumor antigen;

D, Taxol group (Paclitaxel group): Conventional chemotherapy drugs, as a positive control group.

Four groups of mice were injected with lx lO ⁵ live Lewis lung cancer cells 21 days after receiving different immunotherapy. After 23 days, the lung tumor weight of the mice was weighed to obtain the mean and standard deviation.

The results showed (Fig. 8): no difference in lung weight between the three control groups (no immunization, adjuvant, Taxol), Taxol

5mg/Kg did not inhibit the growth of Lewis lung tumors; however, the new method (new tumor vaccine) group reduced tumors by 400 mg, and t-test analysis showed statistically significant differences (p=0.0069). All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the the In addition, it is to be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims

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1. A method for preparing an autologous tumor vaccine, characterized by comprising the steps:

(1) Obtain isolated autologous tumor cells;

(2) Treat the autologous tumor cells in step (1) with radiation and/or heating to obtain tumor cells with enhanced antigenicity and antigen-presenting properties;

(3) Inactivate the tumor cells obtained in step (2) to obtain inactivated tumor cells;

(4) Prepare an autologous tumor vaccine from the inactivated tumor cells obtained in step (3).

2. The method of claim 1, wherein the tumor is: a solid tumor or a non-solid tumor. 3. The method of claim 1, wherein the method of obtaining isolated autologous tumor cells in step (1) is selected from the group consisting of enzymatic digestion, mechanical shearing, or manual grinding of tumor tissue. The method is to process tumor tissue into isolated autologous tumor cells that can be cultured.

4. The method of claim 1, wherein the radiation method in step (2) is selected from the following group: using radiation selected from α, β, γ, protons, or heavy ion radiation, optionally selected from 0.1 The autologous tumor cells in step (1) are processed in a dose range of -50 Gy and a dose rate optionally selected from 0.1-10 Gy/min to obtain tumor cells with enhanced antigenicity and antigen-presenting properties.

5. The method according to claim 1, characterized in that the heating method in step (2) is selected from the following group: The heating temperature is optionally selected from 37.5°C-50°C (preferably 38°C-45°C ), the heating time is optionally selected from 1 min to 2 hours (preferably 30 min to 1 hour).

6. The method according to claim 1, characterized in that, between step (2) and step (3), it further includes the step of: performing a test on the tumor cells with enhanced antigenicity and antigen-presenting properties obtained in step (2). Cultivation processing.

7. The method of claim 1, wherein the inactivation treatment in step (3) is selected from the group consisting of: microwave cell inactivation method, electromagnetic wave cell inactivation method, laser cell inactivation method, high temperature cell inactivation method Inactivation method, ultrasonic disruption cell inactivation method, repeated cell freezing and thawing, homogenization, centrifugation, sedimentation cell inactivation method, enzymatic digestion cell inactivation method, hypotonic rupture method, or a combination thereof.

8. The method of claim 1, wherein in step (4), the inactivated tumor cells obtained in step (3) are mixed with an adjuvant to obtain an autologous tumor vaccine.

9. An autologous tumor vaccine, characterized in that the vaccine is prepared by the method of claim 1.

10. The use of the autologous tumor vaccine according to claim 9, characterized in that it is used to prepare a composition for preventing or treating tumors.

1 1. The method of claim 1, wherein the inactivation treatment in step (3) is selected from the group consisting of: microwave cell inactivation method, electromagnetic wave cell inactivation method, laser cell inactivation method, high temperature Cell inactivation method, ultrasonic disruption cell inactivation method, repeated cell freezing and thawing, homogenization, centrifugation, sedimentation cell inactivation method, enzymatic digestion cell inactivation method, Hypotonic rupture method, chemical covalent cross-linking method, chemical denaturation method, or a combination thereof.

12. A method for preventing or treating tumors, characterized by comprising administering an effective amount of the tumor vaccine according to claim 9 to a subject.

13. The method of claim 12, wherein the administration time is selected from the following group: after surgery, before chemotherapy, or when WBC rises to >3000/ul.

14. The method of claim 12, wherein the number of administrations can range from 4 treatment courses (3-4 weeks per treatment course) to a lifetime.

15. The method of claim 12, wherein the method further includes: using immunological experiments to monitor the degree of immune response.

16. The method of claim 15, wherein the immunological experiment is selected from the group consisting of: cellular immune response, humoral immune response, immune regulatory response, or a combination thereof.

17. The method of claim 12, wherein the immunological experiment is performed within 15-60 days after the first immunization.

18. The method of claim 12, wherein the method further includes: determining the therapeutic effect and/or adjusting the treatment plan based on the degree of immune response measured by immunological experiments.

19. The method of claim 12, characterized in that, the method includes:

(i) on the third postoperative day, administer the vaccine to the patient;

(ii) 7-14 days after surgery, administer the vaccine to the patient again;

(iii) 14-24 days after surgery, administer the vaccine to the patient again;

(iv) Use immunological experiments to monitor the degree of immune response on days 31-34 after surgery, and adjust the immunotherapy plan based on the test results.