WO2018033164A1 - 一种肿瘤免疫抗原及其制备方法和应用 - Google Patents

一种肿瘤免疫抗原及其制备方法和应用 Download PDF

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WO2018033164A1
WO2018033164A1 PCT/CN2017/105727 CN2017105727W WO2018033164A1 WO 2018033164 A1 WO2018033164 A1 WO 2018033164A1 CN 2017105727 W CN2017105727 W CN 2017105727W WO 2018033164 A1 WO2018033164 A1 WO 2018033164A1
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tumor
cells
heating
cooling
tumor cells
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French (fr)
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徐学敏
刘苹
张爱丽
白景峰
孙建奇
邹金成
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上海交通大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/48—Automatic or computerized control

Definitions

  • the present invention is in the field of medical engineering, and in particular, the present invention relates to a method for stimulating tumor immune antigens, particularly heat shock protein 70 (HSP70) levels in tumor tissues, by thermophysical methods.
  • HSP70 heat shock protein 70
  • Malignant tumor cells can reduce the immunogenicity of their own cells, down-regulate the expression of costimulatory molecules on the cell surface, release a large number of immunosuppressive factors, and recruit a variety of immunosuppressive cells to form a stable pattern of tumor immune tolerance and protect immune cells from tumor cells. And promote the occurrence and development of tumors. It is also this tumor-induced immune tolerance that results in poor treatment of tumors.
  • most malignant tumors will cause different degrees of immunosuppression, in order to facilitate tumors to evade immune surveillance and attack, and promote tumor progression. Numerous studies have shown that immunotherapy for malignant tumors not only eliminates tumors in situ but also inhibits killing of distantly metastatic tumor cells. Therefore, a new tumor treatment strategy is getting more and more attention: by relieving immunosuppression, it can stimulate the body's anti-tumor effect, so as to obtain better therapeutic effect.
  • HSP70 heat shock protein 70
  • HSP70 heat shock protein 70
  • the HSP70 protein in the tumor cells binds to the antigen of the tumor to form an immune complex, which is recognized by the antigen-presenting cells (dendritic cells or macrophages) of the body to recognize phagocytosis; at the same time, the dendritic cells are activated to promote the maturation of the dendritic cells and obtain the immune response of the body.
  • the antigen-presenting cells dendritic cells or macrophages
  • the present invention provides a method for stimulating tumors to produce heat shock proteins to activate human immunity and to resist tumors.
  • a method for promoting tumor tissue and/or tumor cells to release tumor immune antigen heat shock protein 70 comprising the steps of:
  • cooling comprises cooling the tumor tissue and/or tumor cells to obtain cooled tumor tissue and/or a tumor cell; wherein the cooling comprises cooling the tumor tissue and/or tumor cells to T1, and -50 ° C ⁇ T1 ⁇ 0 ° C, preferably, -30 ° C ⁇ T1 ⁇ -10 ° C, more preferably Ground, -25 ° C ⁇ T1 ⁇ -15 ° C; optimally, -20 ° C ⁇ T1 ⁇ -18 ° C;
  • step I) is a stepwise cooling step
  • Step II) is a segmented heating step.
  • the time required for cooling is S1 ⁇ 15 min, preferably S1 ⁇ 10 min, more preferably S1 is 5-8 min; and/or
  • step I) after the temperature reaches T1, it lasts for 5-30 min at T1; preferably, for 10-25 min; more preferably for 15-20 min.
  • the time required for heating is S2 ⁇ 15 min, preferably S2 ⁇ 10 min, more preferably S2 is 5-8 min; and/or
  • the temperature reaches T2 in step II) After the temperature reaches T2 in step II), it lasts for 5-30 min at T2; preferably, for 10-25 min; more preferably for 15-20 min.
  • the cooling and/or heating is linear or non-linear cooling and/or heating.
  • step II) further comprises the following sub-steps of segmented heating:
  • step i) after the temperature of step i) reaches T2a, it is maintained at 0-2 min under T2a; and/or in step ii), after reaching T2, it is maintained at T2 for 5-30 min; 10-25 min; Good place, lasts 15-20min.
  • the method further comprises an optional step of:
  • steps I) and/or II) further comprise temperature monitoring steps for tumor tissue and/or tumor cells;
  • steps I) and/or II) each further comprise a quantitative and/or qualitative detection step for a tumor immune antigen.
  • the temperature monitoring step comprises non-invasive monitoring or invasive monitoring, preferably, the none innovative monitoring includes infrared image analysis temperature monitoring method, nuclear magnetic resonance temperature detection method, and ultrasonic temperature detection method.
  • the tumor comprises: a malignant solid tumor, a hematological tumor, a benign tumor, a metastatic tumor, or a combination thereof.
  • the cooling and/or heating comprises direct cooling and/or heating of the tumor cells.
  • the tumor is preferably a malignant solid tumor.
  • the malignant solid tumor includes gastric cancer, liver cancer, pancreatic cancer, gallbladder cancer, colon cancer, rectal cancer, renal cancer, adrenal cancer, cutaneous malignancy, chondroma, thyroid cancer, breast cancer.
  • the tumor is derived from a mammal, such as a mouse, rat or human; preferably, a human.
  • the tumor comprises a tumor isolated from a subject of the tumor and/or a tumor located in the body of the subject.
  • the method comprises an in vitro non-therapeutic method and/or an in vivo therapeutic method.
  • an apparatus for promoting tumor tissue and/or tumor cells to release a tumor immune antigen comprises:
  • a heating element for heating the cooled tumor tissue and/or tumor cells to obtain heated tumor tissue and/or tumor cells; wherein said heating comprises obtaining in I)
  • the cooled tumor tissue and/or tumor cells are heated to T2, and 37 ° C ⁇ T2 ⁇ 60 ° C, preferably, 40 ° C ⁇ T2 ⁇ 55 ° C, more preferably, 45 ° C ⁇ T2 ⁇ 52 ° C;
  • a temperature control element that controls the repetition cycle of the temperature-lowering element and the heating element to start and stop, thereby repeating the temperature-lowering step and the heating step one or more times;
  • a time control element for controlling cooling, heating and/or temperature maintenance time as needed;
  • a temperature monitoring element for temperature monitoring of tumor tissue and/or tumor cells.
  • a temperature feedback component is further disposed between the temperature monitoring component and the temperature control component, and the temperature feedback component is configured to send the temperature control component to the temperature control component after the temperature monitoring component detects that the temperature reaches the set temperature. Instructions to start and/or stop the cooling and/or heating steps.
  • the temperature monitoring component comprises a non-invasive monitor or an invasive monitor, preferably the non-invasive monitor comprises an infrared image analysis temperature monitor, a nuclear magnetic resonance temperature detector, an ultrasonic temperature detector.
  • the device further comprises a tumor immunogenic assay element for quantitative and/or qualitative detection of tumor immune antigens.
  • the temperature reducing element and/or the heating element are each provided with a temperature transmitting element that is in direct and/or indirect contact with tumor tissue and/or tumor cells,
  • the indirect contact comprises contacting a subject having a tumor close to a site of the tumor in the body, such as near the epidermis of the tumor site.
  • HSP70 heat shock protein 70
  • the cooled tumor tissue and/or tumor cells are heated to T2, and 37 ° C ⁇ T2 ⁇ 60 ° C, preferably, 40 ° C ⁇ T2 ⁇ 55 ° C, more preferably, 45 ° C ⁇ T2 ⁇ 52 ° C;
  • HSP70 Heat shock protein 70 isolated and purified from the culture obtained in B).
  • a tumor immune antigen population comprising a tumor immune antigen against a specific tumor, and the tumor immune antigen comprises heat shock protein 70 (HSP70).
  • HSP70 heat shock protein 70
  • the heat shock protein 70 (HSP70) is prepared by the method of the third aspect of the invention.
  • HSP70 heat shock protein 70
  • the pharmaceutical composition is a prophylactic and/or therapeutic vaccine composition.
  • the stimulating organism to produce tumor immunity comprises promoting the conversion of immunosuppressive cells into dendritic cells.
  • a pharmaceutical composition comprising the tumor immune antigen population of the fourth aspect of the invention, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises a tumor therapeutic agent such as a monoclonal antibody, a polyclonal antibody, a chemotherapeutic agent and the like.
  • a tumor therapeutic agent such as a monoclonal antibody, a polyclonal antibody, a chemotherapeutic agent and the like.
  • a method for stimulating the production of a tumor immune antigen, promoting tumor immunity, and/or treating a tumor is provided, and the tumor affected subject of the subject is treated according to any one of the first aspects of the present invention, thereby Stimulation produces tumor immune antigens, promotes tumor immunity, and/or treats tumors.
  • a method of preventing and/or treating a tumor comprising the steps of: administering to a subject in need thereof the tumor immune antigen population of the fourth aspect of the invention and/or the sixth aspect of the invention A pharmaceutical composition to prevent and/or treat a tumor.
  • a tumor treatment system comprising the device according to the second aspect of the present invention, a heat shock protein 70 collection device, a mature dendritic cell culture device, and a mature dendritic cell Transmission device.
  • Figure 1 shows that the survival rate of mice is significantly increased after the cold-hot alternating treatment of the present invention.
  • Figure 2 shows that a large amount of HSP70 is released from tumor tissue after the cold-hot alternating treatment of the present invention.
  • FIG. 3 Western blot analysis of tumor interstitial fluid showed that a large amount of HSP70 protein was released in the cold-heat treatment group.
  • Figure 4 shows a significant increase in HSP70 in peripheral blood of mice in the cold-hot treatment group.
  • Figure 5 shows that the transformation of immunosuppressive cells MDSC into mature dendritic cells is promoted after cold-heat treatment.
  • FIG. 6 shows that HSP70 in serum after cold-heat treatment promotes the transformation of immunosuppressive cells MDSC into mature dendritic cells.
  • tumor immune antigen heat shock protein 70 HSP70
  • the obtained tumor immune antigen can effectively activate the tumor immune system in vivo, such as effectively transforming immunosuppressive cells into mature dendritic cells, thereby enhancing the immune antigen.
  • Present and activate tumor immunity are widely and intensively studied, and for the first time, unexpectedly discovered that tumor tissue and/or cells undergo local sequential cooling-heat treatment (or cold-heat treatment), both in vivo and in vitro, tumor tissue and / or cells will release a large amount of tumor immune antigen heat shock protein 70 (HSP70), and the amount of this release is significantly higher than the amount produced only after simple thermal stimulation of tumor tissue and / or cells.
  • the obtained tumor immune antigen can effectively activate the tumor immune system in vivo, such as effectively transforming immunosuppressive cells into mature dendritic cells, thereby enhancing the immune antigen.
  • Present and activate tumor immunity are widely and intensively studied, and for the first time, unexpectedly discovered that tumor tissue and/or cells undergo local sequential cooling-heat treatment (
  • the method of the present invention is used for preparing a tumor immune antigen and using it as a tumor
  • the seedlings are applied to the desired subject, and in addition, the method of the present invention can also be directly applied to tumors in the body, thereby directly obtaining the therapeutic effect of the tumor.
  • the present invention has been completed.
  • the terms "method of the invention”, “cold-heat treatment (treatment)” and “sequential cooling-heat treatment (treatment)” are used interchangeably and refer to the steps according to the first aspect of the invention, The method of cooling and heat treatment of tumor tissues and/or cells in vivo and/or in vitro to obtain a large amount of tumor immune antigens, particularly HSP70.
  • a method that can be used in the present invention includes the following steps:
  • cooling the tumor tissue and/or tumor cells to obtain cooled tumor tissue and/or tumor cells; wherein the cooling comprises cooling the tumor tissue and/or tumor cells to T1, and -50 ° C ⁇ T1 ⁇ 0 ° C;
  • the method of the invention may further comprise the steps of:
  • Steps I) and II) which can be used in the process of the invention may be a stepwise cooling and heating step, respectively, or a one-stage cooling and heating step.
  • step I) is a one-step cooling step, and in step I), the time required for cooling is S1 ⁇ 15min, preferably, S1 ⁇ 10min, more preferably S1 is 5-8min, and after the temperature reaches T1 , for 5-30 min at T1; preferably, for 10-25 min; more preferably for 15-20 min;
  • the time required for heating is S2 ⁇ 15 min, preferably S2 ⁇ 10 min, more preferably S2 is 5-8 min; after the temperature reaches T2, it lasts for 5-30 min at T2; preferably, lasts for 10 -25 min; more preferably, lasting 15-20 min.
  • step II) is preferably a segment heating step, which may comprise a sub-step:
  • step i) reaches the temperature T2a, it is maintained at 0 to 5 min under T2a; and/or in step ii) After reaching T2, it is maintained at T2 for 5-30 min; 10-25 min; more preferably, for 15-20 min; in step ii), after the temperature reaches T2, it is maintained at T2 for 5-30 min; 10-25 min; better Ground, lasting 15-20min.
  • step II) is a one-step heating step
  • the time required for heating is S2 ⁇ 15 min, preferably, S2 ⁇ 10 min, more preferably S2 is 5-8 min, and after reaching T2, Maintain 5-30 min at T2; 10-25 min; more preferably, last 15-20 min.
  • direct or indirect temperature adjustment may be employed for the tumor tissue and/or tumor cells, such as by cooling or heating the tumor tissue with a cooling or heating element.
  • the cooling or heating element can be contacted with tumor tissue (e.g., intraoperatively) or placed adjacent to a local epidermis adjacent to the tumor tissue.
  • the temperature of the tumor tissue and/or cells and/or the generated tumor immune antigen can also be monitored in each step, thereby adjusting the temperature and the maintenance time in each step in time.
  • the temperature monitoring step comprises non-invasive monitoring or invasive monitoring, preferably, the non-invasive monitoring comprises infrared image analysis temperature monitoring method, nuclear magnetic resonance temperature detecting method, ultrasonic temperature detecting method; and other temperatures well known to those skilled in the art. Monitoring methods can also be used in the temperature monitoring of the present invention.
  • Monitoring of tumor immune antigens can be performed quantitatively and/or qualitatively using non-invasive and/or invasive methods, such as quantitative or qualitative monitoring of tumor immune antigens in tumor interstitial fluid or in body samples (eg, serum), Immunohistochemistry, ELISA, for example, are well known to those skilled in the art.
  • non-invasive and/or invasive methods such as quantitative or qualitative monitoring of tumor immune antigens in tumor interstitial fluid or in body samples (eg, serum), Immunohistochemistry, ELISA, for example, are well known to those skilled in the art.
  • the type of tumor which can be used in the method of the present invention is not particularly limited as long as it can produce a tumor immune antigen and can be used in the method of the present invention.
  • Preferred tumor types include malignant solid tumors, hematological tumors, metastases, or a combination thereof, and in addition, benign tumors can also employ the methods of the invention.
  • HSP70 Tumor immune antigen heat shock protein 70
  • Heat shock protein 70 is an important family protein in heat shock proteins and plays an important role in congenital and acquired anti-tumor immune responses. Studies have shown that the body can detect the released HSP70 protein 30 minutes after hyperthermia. Natural killer cells bind to the HSP70 protein on the surface of the tumor cell membrane, which in turn dissolves the tumor cells. The HSP70 protein in the tumor cells binds to the antigen of the tumor to form an immune complex, which is recognized by the antigen-presenting cells (dendritic cells or macrophages) of the body to recognize phagocytosis; at the same time, the dendritic cells are activated to promote the maturation of the dendritic cells and obtain the immune response of the body.
  • the antigen-presenting cells dendritic cells or macrophages
  • a pharmaceutical composition for treating or preventing tumors such as a vaccine composition
  • the antigen-presenting cells of the body are activated after administration (for example, intravenous infusion) to a subject in need thereof to activate the body.
  • Tumor immunological activity for example, intravenous infusion
  • the invention also provides an apparatus for carrying out the method of the invention.
  • the device comprises
  • a heating element for heating the cooled tumor tissue and/or tumor cells to obtain heated tumor tissue and/or tumor cells; wherein said heating comprises obtaining in I)
  • the cooled tumor tissue and/or tumor cells are heated to T2, and 37 ° C ⁇ T2 ⁇ 60 ° C, preferably, 40 ° C ⁇ T2 ⁇ 55 ° C, more preferably, 45 ° C ⁇ T2 ⁇ 52 ° C;
  • a temperature control element that controls the repeating cycle of the temperature-lowering element and the heating element to start and stop, thereby repeating the temperature-lowering step and the heating step one or more times;
  • a temperature monitoring element for temperature monitoring of tumor tissue and/or tumor cells.
  • the temperature reducing element and/or the heating element and/or the temperature control element and/or the temperature monitoring element which can be used in the present invention are not particularly limited, and may be any element in the art which can be used for medical treatment or temperature adjustment of cells.
  • a temperature feedback component is further disposed between the temperature monitoring component and the temperature control component, and the temperature feedback component is configured to issue a startup and/or to the temperature control component after the temperature monitoring component detects that the temperature reaches the set temperature. Stop the instructions for cooling and/or heating steps.
  • the temperature monitoring component comprises a non-invasive monitor or an invasive monitor, preferably the non-invasive monitor comprises an infrared image analysis temperature monitor, a nuclear magnetic resonance temperature detector, an ultrasonic temperature detector.
  • the device further comprises a tumor immunogenic assay element for quantitative and/or qualitative detection of tumor immune antigens.
  • the temperature reducing element and/or the heating element are each provided with a temperature transmitting element that is in direct and/or indirect contact with tumor tissue and/or tumor cells,
  • the invention also provides a method for tumor immune antigen (especially HSP70), which is based on the sequential cooling-heat treatment of the invention, and the tumor cell culture medium is separated and purified after cold-heat treatment of the tumor cells cultured in vitro.
  • tumor immune antigens especially HSP70
  • the preparation method of the present invention comprises:
  • the cooled tumor tissue and/or tumor cells are heated to T2, and 37 ° C ⁇ T2 ⁇ 60 ° C, preferably, 40 ° C ⁇ T2 ⁇ 55 ° C, more preferably, 45 ° C ⁇ T2 ⁇ 52 ° C;
  • tumor immunogen which may be conventionally used in the art to isolate or purify one or more tumor immune antigens.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a safe and effective amount of a tumor immune antigen population and a pharmaceutically acceptable carrier or excipient.
  • Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • Pharmaceutical compositions such as injections, solutions, lyophilized preparations are preferably manufactured under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram body weight to about 5 milligrams per kilogram body weight per day.
  • the tumor immune antigen population of the present invention can also be used with other therapeutic agents such as tumor suppressors such as monoclonal antibodies, polyclonal antibodies, chemotherapeutic agents and the like.
  • a safe and effective amount of a tumor immune antigen population of the invention is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 8 milligrams per kilogram.
  • the body weight preferably the dose is from about 10 micrograms per kilogram of body weight to about 1 milligram per kilogram of body weight.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the present invention discloses for the first time a method for promoting the secretion of a tumor immune antigen HSP70 by a large amount of tumor cells, which can be effectively applied to the preparation of a tumor vaccine, and can also directly perform immunotherapy on a tumor-affected individual;
  • the antigen obtained by the method of the present invention can effectively promote immunosuppression of cell maturation and activate an anti-tumor immune response.
  • the experimental results show that the method of the present invention can be used to treat tumor-bearing mice, and can promote immunosuppressive cells in mice to the tree. The cells are mature and differentiated.
  • Treatment of tumor-bearing mice by the method of the present invention can significantly improve the survival rate of tumor-bearing mice.
  • mice 6-8 weeks old SPF Balb/c female mice (Shanghai Slack Animal Center) were housed in independent ventilation cages and manually controlled for 12 hours to change light. The mice were free to ingest 60Co radiation-sterilized feed and autoclaved water.
  • Mouse 4T 1 breast cancer cells obtained from Shanghai First People's Hospital, in other embodiments, other breast cancer cells such as T47D cell line can be used), cultured with 10% newborn fetal bovine serum and double Anti-(100 U/mL penicillin, 100 g/mL streptomycin) (Shanghai Shenggong Bioengineering Technology Service Co., Ltd.) in DMEM medium (Hyclone, USA).
  • Magnetic beads for immunocyte sorting were purchased from Miltnyi Biotec, Germany.
  • flow cytometry AF488 anti-mouse CD11c, APC anti-mouse F4/80, Percp/cy5.5 anti-mouse CD86, PE anti-mouse MHC II was purchased from Biolegend.
  • the 4T1 mouse breast cancer cells used in this experiment can be transferred to the lung, liver, bone marrow and brain through a blood-borne pathway, and are highly metastatic breast cancer models.
  • a 1 x 1 0 6 U/0.1 mL 4T1 cell suspension was prepared and placed on ice for use.
  • the animals were anesthetized by intraperitoneal injection of 0.016 g/mL sodium pentobarbital at 0.5 mL/100 g mouse body, and 0.1 mL of cell suspension was subcutaneously injected into the back of the mice. After 21 days of tumor inoculation, the vernier caliper measures the tumor volume and is calculated as follows:
  • the survival status of the mice was comprehensively observed. This part mainly includes: observation of the growth of orthotopic tumors in the control group, ablation and recurrence of orthotopic tumors in the treatment group (daily), and recording changes in body weight before and after treatment (twice a week) for long-term survival of mice. Statistics of rate and survival time. These indicators can effectively reflect the survival of mice, and the statistics of survival rate and survival time are therapeutic effects. The most important indicator of the assessment.
  • Mouse tumors were taken 1 day after cold-hot treatment, and tumors of control mice were taken 22 days after inoculation.
  • the membrane wrapped tumor was placed in a 15 ml centrifuge tube, centrifuged at 2000 rpm for 15 minutes, and the interstitial fluid at the bottom of the centrifuge tube was aspirated.
  • the total protein amount was determined by Bradford method, and 30 ⁇ g of protein was extracted and added to a 4-20% gradient glycine precast gel electrophoresis, and transferred to a PVDF membrane.
  • HSP70 primary antibody (Cell signaling technology) and fluorescent secondary antibody were incubated and blotted strips were recorded.
  • Mouse tumors were taken 1 day after cold-heat treatment and single heat treatment; tumors of control mice were taken 22 days after inoculation. After quick freezing with isopentane, put it into tin foil and store it in a refrigerator at -80 °C. The tumor was removed and fixed on the sample holder with OCT embedding glue. The tumor was cut into 10 ⁇ m slices at -20 ° C ambient temperature using a cryostat, and the sections were adhered to a cover glass and stored in a -80 ° C refrigerator. After the tumor sections were naturally air-dried, they were fixed in acetone for 10 minutes at 4 ° C and rinsed with PBS.
  • mice 2 days and 10 days after cold-heat treatment, the spleen of the mice was taken, and the spleen cell suspension was obtained by GentleMACS gentle tissue processor. The supernatant was discarded by centrifugation at 1500 rpm for 5 minutes, 2 ml of red blood cell lysate was added and blown evenly, and lysed at room temperature. 5 minutes. After adding PEB as much as possible, the supernatant was centrifuged to obtain spleen cells. Immunosuppressive cells with a purity greater than 90% were sorted using the immunosuppressive cell Kit of Miltnyi Biotec, Germany for experiments.
  • mice and control mice were taken, and immunosuppressive cells were sorted from spleen cells by magnetic bead sorting.
  • CD11c and F4/80 as dendritic cells and macrophage surfaces, respectively Characterized antigen; CD86 and MHC II act as surface marker antigens for both cell maturation.
  • 1 ⁇ l of the corresponding fluorescent antibody was added and incubated at 4 ° C for 30 minutes in the dark. After washing with PBS, it was resuspended and detected by flow cytometry.
  • the spleens of the control mice were selected for immunosuppressive cells by magnetic beads, and the serum of the DMEM medium plus the treatment group, the serum of the control group, the serum of the normal mice, and the serum of the treatment group. +HSP70 antibody was incubated for four hours in four media. Flow cytometry was used to detect the expression of cell surface marker antigens CD11c, F4/80, CD86 and MHCII.
  • the survival status of the cold-hot treatment group and the blank control mice was observed for one year (Fig. 1).
  • the survival time of the 16 cold-hot treated mice was greater than 66 days after the inoculation, and the final 11 mice survived to one year.
  • the survival time of the 16 control mice did not exceed 64 days after inoculation.
  • Fig. 2 Brown (light) is the specific staining of the HSP70 protein by the antibody, and blue (dark) is the staining of the nuclei by hematoxylin.
  • HSP70 mainly plays a role in promoting the survival of tumor cells.
  • the ELISA test showed that the HSP70 in the serum of the surgical group was slightly lower than that of the control group after 2 days of treatment.
  • the HSP70 in the serum of the monotherapy group was significantly different from that of the control group, while the mice in the cold-heat treatment group were in the peripheral serum. A more pronounced increase in HSP70 can be seen. ( Figure 4)

Abstract

本发明提供了一种肿瘤免疫抗原及其制备方法和应用，通过对肿瘤组织和/或细胞进行局部的序贯性降温-加热处理后，肿瘤组织和/或细胞会放出大量的肿瘤免疫抗原热休克蛋白70，所获得的肿瘤免疫抗原能够激活体内的肿瘤免疫系统，将免疫抑制细胞转化为成熟的树突状细胞，从而提高免疫抗原的提呈并激活肿瘤免疫。

Description

一种肿瘤免疫抗原制备方法及其产物和应用 技术领域
本发明属于医学工程领域，具体地说，本发明涉及一种热物理方法激发肿瘤免疫抗原，尤其是肿瘤组织体内热休克蛋白70(HSP70)水平的方法。
背景技术
恶性肿瘤细胞通过降低自身的免疫原性、下调细胞表面协同刺激分子的表达、释放大量免疫抑制因子、召集多种免疫抑制细胞，形成稳定的肿瘤免疫耐受格局，保护肿瘤细胞逃脱机体的免疫监视并促进肿瘤的发生发展。也正是这种由肿瘤诱导的免疫耐受导致肿瘤治疗的效果不佳。同时，大多数恶性肿瘤都会引起不同程度的免疫抑制，以利于肿瘤逃避机体免疫监视和攻击,促进肿瘤进展。大量研究表明免疫治疗恶性肿瘤不仅能消除原位的肿瘤而且对于远处转移的肿瘤细胞亦有抑制杀伤作用。因而一种新的肿瘤治疗策略越来越受到关注：通过解除免疫抑制,激发机体抗肿瘤作用，从而获得更好的治疗效果。
运用冷治疗和热治疗相结合的方法，增强局部肿瘤细胞坏死，导致细胞结构崩解，释放大量的热休克蛋白70(HSP70)。目前认为热休克蛋白能与肿瘤抗原结合，诱导机体产生抗肿瘤免疫。热休克蛋白70(HSP70)是热休克蛋白中重要的家族蛋白，在先天性和获得性抗肿瘤免疫反应中起着重要的作用。研究表明在热疗后30分钟，机体就能够检测出释放的HSP70蛋白。自然杀伤细胞结合肿瘤细胞膜表面HSP70蛋白，继而溶解肿瘤细胞。肿瘤细胞内HSP70蛋白结合肿瘤的抗原形成免疫复合体，被机体的抗原递呈细胞(树突细胞或巨噬细胞)识别吞噬；同时激活树突细胞，促使树突细胞成熟，获得机体免疫响应。
因此本领域技术人员一直致力于开发一种能够通过促进肿瘤组织大量HSP70蛋白激活肿瘤患者体内免疫响应，改善免疫抑制的方法。
发明内容
本发明提供了一种刺激肿瘤产生热休克蛋白从而激活人体免疫力、抵抗肿瘤的方法。
本发明第一方面，提供了一种促进肿瘤组织和/或肿瘤细胞释放肿瘤免疫抗原热休克蛋白70(HSP70)的方法，包括步骤：
I)将所述的肿瘤组织和/或肿瘤细胞进行降温从而获得经降温的肿瘤组织和/或 肿瘤细胞；其中，所述的降温包括将所述肿瘤组织和/或肿瘤细胞降温至T1，且-50℃≤T1≤0℃，较佳地，-30℃≤T1≤-10℃，更佳地，-25℃≤T1≤-15℃；最佳地，-20℃≤T1≤-18℃；和
II)将I)中获得的经降温的肿瘤组织和/或肿瘤细胞进行加热从而获得经加热的肿瘤组织和/或肿瘤细胞；其中，所述的加热包括将I)获得的经降温的肿瘤组织和/或肿瘤细胞加热至T2，且37℃＜T2≤60℃，较佳地，40℃≤T2≤55℃，更佳地，45℃≤T2≤52℃。
在另一优选例中，步骤I)为分段式降温步骤；和/或
步骤II)为分段式加热步骤。
在另一优选例中，步骤I)中，降温所需的时间S1≤15min，较佳地，S1≤10min，更佳地S1为5-8min；和/或
步骤I)中，温度达到T1后，在T1下持续5-30min；较佳地，持续10-25min；更佳地，持续15-20min。
在另一优选例中，步骤II)中，加热所需的时间S2≤15min，较佳地，S2≤10min，更佳地S2为5-8min；和/或
步骤II)中温度达到T2后，在T2下持续5-30min；较佳地，持续10-25min；更佳地，持续15-20min。
在另一优选例中，所述的降温和/或加热为线性或非线性降温和/或加热。
在另一优选例中，步骤II)还包括以下分段式加热的子步骤：
i)将I)中获得的经降温的肿瘤组织和/或肿瘤细胞加热至T2a，且-10℃＜T2a≤10℃；较佳地-5℃＜T2a≤0℃；和
ii)将i)中获得的T2a温度下的肿瘤组织和/或肿瘤细胞加热至T2。
在另一优选例中，当步骤i)温度达到T2a后，在T2a下维持0-5min；和/或在步骤ii)中，到达T2后，在T2下维持5-30min；10-25min；更佳地，持续15-20min。
在另一优选例中，所述方法还包括任选的步骤：
III)对I)中的降温步骤和II)中的加热步骤进行一次或多次的重复。
在另一优选例中，所述方法中，步骤I)和/或II)还各自包括对肿瘤组织和/或肿瘤细胞的温度监测步骤；和/或
所述方法中，步骤I)和/或II)还各自包括对肿瘤免疫抗原的定量和/或定性检测步骤。
在另一优选例中，所述温度监测步骤包括无创监测或有创监测，优选地，所述无 创监测包括红外图像分析温度监测法、核磁共振温度检测法、超声温度检测法。
在另一优选例中，所述的肿瘤包括：恶性实体肿瘤、血液肿瘤、良性肿瘤、转移瘤或其组合。
在另一优选例中，所述的降温和/或加热包括对所述肿瘤细胞直接接触降温和/或加热。
在另一优选例中，所述的肿瘤优选为恶性实体肿瘤。
在另一优选例中，所述恶性实体肿瘤包括胃癌、肝癌、胰腺癌、胆囊癌、结肠癌、直肠癌、肾癌、肾上腺癌、皮肤恶性肿瘤、软骨瘤、甲状腺癌、乳腺癌。
在另一优选例中，所述的肿瘤来源于哺乳动物，如小鼠、大鼠或人；较佳地，为人。
在另一优选例中，所述肿瘤包括分离自患肿瘤对象的肿瘤和/或位于患肿瘤对象体内的肿瘤。
在另一优选例中，所述方法包括体外非治疗性的方法和/或体内治疗性方法。
本发明第二方面，提供了一种用于促进肿瘤组织和/或肿瘤细胞释放肿瘤免疫抗原的装置，包括：
a)降温元件，所述的降温元件用于将所述的肿瘤组织和/或肿瘤细胞进行降温从而获得经降温的肿瘤组织和/或肿瘤细胞；其中，所述的降温包括将所述肿瘤组织和/或肿瘤细胞降温至T1，且-50℃≤T1≤0℃，较佳地，-30℃≤T1≤-10℃，更佳地，-25℃≤T1≤-15℃；最佳地，-20℃≤T1≤-18℃；
b)加热元件，所述的加热元件用于将经降温的肿瘤组织和/或肿瘤细胞进行加热从而获得经加热的肿瘤组织和/或肿瘤细胞；其中，所述的加热包括将I)中获得的经降温的肿瘤组织和/或肿瘤细胞加热至T2，且37℃＜T2≤60℃，较佳地，40℃≤T2≤55℃，更佳地，45℃≤T2≤52℃；
c)温度控制元件，所述的温度控制元件于控制所述降温元件和加热元件的重复循环启动和停止，从而对降温步骤和加热步骤进行一次或多次的重复；
d)时间控制元件，所述时间控制元件用于根据需要控制降温、加热和/或温度维持时间；和任选的
e)温度监测元件，所述温度监测元件用于对肿瘤组织和/或肿瘤细胞的温度监测。
在另一优选例中，所述温度监测元件和温度控制元件之间还设有温度反馈元件，所述温度反馈元件用于在温度监测元件监测到温度达到设定温度后，向温度控制元件发出启动和/或停止降温和/或加热步骤的指令。
在另一优选例中，所述温度监测元件包括无创监测器或有创监测器，优选地所述无创监测器包括红外图像分析温度监测器、核磁共振温度检测器、超声温度检测器。
在另一优选例中，所述的装置还包括肿瘤免疫抗原测定元件，用于对肿瘤免疫抗原的定量和/或定性检测。
在另一优选例中，所述降温元件和/或加热元件各自设有一与肿瘤组织和/或肿瘤细胞直接和/或间接接触的温度传递元件，
在另一优选例中，所述的间接接触包括与患有肿瘤的对象接近体内肿瘤的部位接触，如接近肿瘤部位的表皮。
本发明第三方面，提供了一种制备热休克蛋白70(HSP70)的方法，包括步骤：
A)将培养的肿瘤组织和/或肿瘤细胞进行降温，从而获得经降温的肿瘤组织和/或肿瘤细胞；其中，所述的降温包括将所述肿瘤组织和/或肿瘤细胞降温至T1，且-50℃≤T1≤0℃，较佳地，-30℃≤T1≤-10℃，更佳地，-25℃≤T1≤-15℃；最佳地，-20℃≤T1≤-18℃；
B)将A)中获得的经降温的肿瘤组织和/或肿瘤细胞进行加热，从而获得含有肿瘤免疫抗原群的肿瘤组织和/或肿瘤细胞培养物；其中，所述的加热包括将A)获得的经降温的肿瘤组织和/或肿瘤细胞加热至T2，且37℃＜T2≤60℃，较佳地，40℃≤T2≤55℃，更佳地，45℃≤T2≤52℃；
C)从B)中获得的培养物中分离并纯化的热休克蛋白70(HSP70)。
本发明第四方面，提供了一种肿瘤免疫抗原群，所述的肿瘤免疫抗原群中含有针对特定肿瘤的肿瘤免疫抗原，且所述的肿瘤免疫抗原包括热休克蛋白70(HSP70)。
在另一优选例中，所述的热休克蛋白70(HSP70)是由本发明第三方面所述的方法制备获得。
本发明第五方面，提供了本发明第四方面所述的热休克蛋白70(HSP70)的用途，用于预防和/或制备治疗肿瘤和/或刺激机体产生肿瘤免疫的药物组合物。
在另一优选例中，所述药物组合物为预防性和/或治疗性疫苗组合物。
在另一优选例中，所述刺激机体产生肿瘤免疫包括促进免疫抑制细胞转化为树突状细胞。
本发明第六方面，提供了一种药物组合物，所述的药物组合物含有本发明第四方面所述的肿瘤免疫抗原群，和药学上可接受的载体。
在另一优选例中，所述的药物组合物中还含有肿瘤治疗剂，如单克隆抗体、多克隆抗体、化疗剂等。
本发明第七方面，提供了一种刺激产生肿瘤免疫抗原、促进机体肿瘤免疫、和/或治疗肿瘤的方法，将需要的对象的肿瘤患处本发明第一方面任一所述方法进行处理，从而刺激产生肿瘤免疫抗原、促进机体肿瘤免疫、和/或治疗肿瘤。
本发明第八方面，提供了一种预防和/或治疗肿瘤的方法，包括步骤：向需要的对象施用本发明第四方面所述的肿瘤免疫抗原群和/或本发明第六方面所述的药物组合物，从而预防和/或治疗肿瘤。
本发明第九方面，提供了一种肿瘤治疗系统，所述治疗系统包括本发明第二方面所述的装置、热休克蛋白70采集装置、成熟树突状细胞培养装置和成熟树突状细胞回输装置。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了经本发明冷-热交替治疗后，小鼠的生存率显著上升。
图2显示了经本发明冷-热交替处理后，肿瘤组织释放出大量的HSP70。
图3肿瘤间质液蛋白质印迹检测显示冷-热处理组释放大量HSP70蛋白。
图4显示了冷-热治疗组小鼠外周血清中HSP70明显增加。
图5显示了冷-热治疗后促进了免疫抑制细胞MDSC向成熟的树突状细胞转化。
图6显示了冷-热治疗后血清中的HSP70促进了免疫抑制细胞MDSC向成熟的树突状细胞转化。
具体实施方式
本发明人经过广泛而深入地研究，首次意外地发现，对肿瘤组织和/或细胞进行局部的序贯性降温-加热处理(或冷-热治疗)后，无论在体内还是体外，肿瘤组织和/或细胞会放出大量的肿瘤免疫抗原热休克蛋白70(HSP70)，且该释放的量显著高于仅对肿瘤组织和/或细胞进行单纯热刺激后产生的量。利用本发明序贯性降温-加热处理后，所获得的肿瘤免疫抗原能够有效地激活体内的肿瘤免疫系统，如能有效地将免疫抑制细胞转化为成熟的树突状细胞，从而提高免疫抗原的提呈并激活肿瘤免疫。因此，本发明方法用于制备肿瘤免疫抗原，并将其作为肿瘤疫 苗施用于需要的对象，此外，本发明方法也可直接用于体内的肿瘤，从而直接获得肿瘤治疗效果。在此基础上，完成了本发明。
促进肿瘤组织和/或肿瘤细胞释放肿瘤免疫抗原的方法
如本文所用，术语“本发明方法”、“冷-热治疗(处理)”和“序贯性降温-加热治疗(处理)”可互换使用，指的是按本发明第一方面的步骤，对体内和/或体外的肿瘤组织和/或细胞先后进行降温-加热处理，从而获得大量肿瘤免疫抗原，尤其是HSP70的方法。
可用于本发明的方法包括以下步骤：
I)将所述的肿瘤组织和/或肿瘤细胞进行降温从而获得经降温的肿瘤组织和/或肿瘤细胞；其中，所述的降温包括将所述肿瘤组织和/或肿瘤细胞降温至T1，且-50℃≤T1≤0℃；和
II)将I)中获得的经降温的肿瘤组织和/或肿瘤细胞进行加热从而获得经加热的肿瘤组织和/或肿瘤细胞；其中，所述的加热包括将I)获得的经降温的肿瘤组织和/或肿瘤细胞加热至T2，且37℃＜T2≤60℃。
在另一优选例中，-30℃≤T1≤-10℃，更佳地，-25℃≤T1≤-15℃；最佳地，-20℃≤T1≤-18℃；40℃≤T2≤55℃，更佳地，45℃≤T2≤52℃。
优选地，本发明方法还可包括步骤：
III)对I)中的降温步骤和II)中的加热步骤进行一次或多次的重复。
可用于本发明方法的步骤I)和II)分别可以为分段式降温和加热步骤，也可以是一段式降温和加热步骤。
其中优选地，步骤I)为一段式降温步骤，步骤I)中，降温所需的时间S1≤15min，较佳地，S1≤10min，更佳地S1为5-8min，而在温度达到T1后，在T1下持续5-30min；较佳地，持续10-25min；更佳地，持续15-20min；
步骤II)中，加热所需的时间S2≤15min，较佳地，S2≤10min，更佳地S2为5-8min；温度达到T2后，在T2下持续5-30min；较佳地，持续10-25min；更佳地，持续15-20min。
而步骤II)则优选为分段加热步骤，其可以包括子步骤：
i)将I)中获得的经降温的肿瘤组织和/或肿瘤细胞加热至T2a，且-10℃＜T2a≤10℃；较佳地-5℃＜T2a≤0℃；和
ii)将ii)中获得的T2a温度下的肿瘤组织和/或肿瘤细胞加热至T2。
其中，当步骤i)温度达到T2a后，在T2a下维持0-5min；和/或在步骤ii)中， 到达T2后，在T2下维持5-30min；10-25min；更佳地，持续15-20min；在步骤ii)中，温度达到T2后，在T2下维持5-30min；10-25min；更佳地，持续15-20min。
当步骤II)为一段式加温步骤时，步骤II)中，加热所需的时间S2≤15min，较佳地，S2≤10min，更佳地S2为5-8min，且在达到T2后，在T2下维持5-30min；10-25min；更佳地，持续15-20min。
在本发明中，对肿瘤组织和/或肿瘤细胞可采用直接或间接的温度调整，优选为直接温度调整，例如利用降温或加热元件对肿瘤组织进行接触降温或加热。当本发明方法用于体内时，可将降温或加热元件接触肿瘤组织(如术中)或置于接近肿瘤组织的局部表皮附近。
此外，在本发明方法中，还可以在各步骤中对肿瘤组织和/或细胞的温度和/或所产生的肿瘤免疫抗原进行监测，从而及时调整各步骤中的温度以及维持时间。其中，所述温度监测步骤包括无创监测或有创监测，优选地，所述无创监测包括红外图像分析温度监测法、核磁共振温度检测法、超声温度检测法；其它本领域技术人员所熟知的温度监测方法也可用于本发明的温度监测中。而对肿瘤免疫抗原的监测可采用无创和/或有创方法进行定量和/或定性测定，例如对肿瘤组织间质液中或机体样本(如血清)中的肿瘤免疫抗原进行定量或定性监测，例如采用免疫组化、ELISA法，其均为本领域技术人员所熟知。
可采用本发明方法的肿瘤类型没有特殊限制，只要能够产生肿瘤免疫抗原均可用于本发明方法。优选的肿瘤类型包括恶性实体肿瘤、血液肿瘤、转移瘤或其组合，此外，良性肿瘤也可采用本发明方法。
肿瘤免疫抗原热休克蛋白70(HSP70)
热休克蛋白70(HSP70)是热休克蛋白中重要的家族蛋白，在先天性和获得性抗肿瘤免疫反应中起着重要的作用。研究表明在热疗后30分钟，机体就能够检测出释放的HSP70蛋白。自然杀伤细胞结合肿瘤细胞膜表面HSP70蛋白，继而溶解肿瘤细胞。肿瘤细胞内HSP70蛋白结合肿瘤的抗原形成免疫复合体，被机体的抗原递呈细胞(树突细胞或巨噬细胞)识别吞噬；同时激活树突细胞，促使树突细胞成熟，获得机体免疫响应。采用本发明肿瘤免疫抗原热休克蛋白70，可制备治疗或预防肿瘤的药物组合物，例如疫苗组合物，向需要的对象施用(如静脉输注)后激活机体的抗原提呈细胞从而激活机体的肿瘤免疫活性。
本发明装置
本发明还提供了一种用于实施本发明方法的装置。其中，所述的装置包括
a)降温元件，所述的降温元件用于将所述的肿瘤组织和/或肿瘤细胞进行降温从而获得经降温的肿瘤组织和/或肿瘤细胞；其中，所述的降温包括将所述肿瘤组织和/或肿瘤细胞降温至T1，且-50℃≤T1≤0℃，较佳地，-30℃≤T1≤-10℃，更佳地，-25℃≤T1≤-15℃；最佳地，-20℃≤T1≤-18℃；
b)加热元件，所述的加热元件用于将经降温的肿瘤组织和/或肿瘤细胞进行加热从而获得经加热的肿瘤组织和/或肿瘤细胞；其中，所述的加热包括将I)中获得的经降温的肿瘤组织和/或肿瘤细胞加热至T2，且37℃＜T2≤60℃，较佳地，40℃≤T2≤55℃，更佳地，45℃≤T2≤52℃；
c)温度控制元件，所述的温度控制元件于控制所述降温元件和加热元件的重复循环启动和停止，从而对降温步骤和加热步骤进行一次或多次的重复；和任选的
d)温度监测元件，所述温度检测元件用于对肿瘤组织和/或肿瘤细胞的温度监测。
可用于本发明的降温元件和/或加热元件和/或温度控制元件和/或温度监测元件没有特殊限制，可以为本领域任何可以医用或对细胞进行温度调节处理的元件。
优选地，所述温度监测元件和温度控制元件之间还设有温度反馈元件，所述温度反馈元件用于在温度监测元件监测到温度达到设定温度后，向温度控制元件发出启动和/或停止降温和/或加热步骤的指令。所述温度监测元件包括无创监测器或有创监测器，优选地所述无创监测器包括红外图像分析温度监测器、核磁共振温度检测器、超声温度检测器。此外，所述的装置还包括肿瘤免疫抗原测定元件，用于对肿瘤免疫抗原的定量和/或定性检测。
优选地，所述降温元件和/或加热元件各自设有一与肿瘤组织和/或肿瘤细胞直接和/或间接接触的温度传递元件，
肿瘤免疫抗原制备方法
本发明还提供了一种肿瘤免疫抗原(尤其是HSP70)的方法，该方法基于本发明序贯性降温-加热处理，对体外培养的肿瘤细胞进行冷-热处理后，分离并纯化肿瘤细胞培养液中的肿瘤免疫抗原。优选地，本发明制备方法包括：
A)将培养的肿瘤组织和/或肿瘤细胞进行降温，从而获得经降温的肿瘤组织和/或肿瘤细胞；其中，所述的降温包括将所述肿瘤组织和/或肿瘤细胞降温至T1，且-50℃≤T1≤0℃，较佳地，-30℃≤T1≤-10℃，更佳地，-25℃≤T1≤-15℃；最佳地， -20℃≤T1≤-18℃；
B)将A)中获得的经降温的肿瘤组织和/或肿瘤细胞进行加热，从而获得含有肿瘤免疫抗原群的肿瘤组织和/或肿瘤细胞培养物；其中，所述的加热包括将A)获得的经降温的肿瘤组织和/或肿瘤细胞加热至T2，且37℃＜T2≤60℃，较佳地，40℃≤T2≤55℃，更佳地，45℃≤T2≤52℃；
C)从B)中获得的培养物中分离并纯化的肿瘤免疫抗原。
在获得了含有本发明肿瘤免疫抗原的细胞培养液后，优选分离并纯化肿瘤免疫抗原，所采用的方法可以是本领域常规技术，可对一种或多种肿瘤免疫抗原进行分离或纯化。
药物组合物
本发明还提供了一种药物组合物，它含有安全有效量的肿瘤免疫抗原群以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液、冻干制剂宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重-约5毫克/千克体重。此外，本发明的肿瘤免疫抗原群还可与其他治疗剂(例如肿瘤抑制剂，如单克隆抗体、多克隆抗体、化疗剂等)一起使用。
使用药物组合物时，是将安全有效量的本发明肿瘤免疫抗原群施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约8毫克/千克体重，较佳地该剂量是约10微克/千克体重-约1毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明有益效果
(1)本发明首次揭示了一种促进肿瘤细胞大量分泌肿瘤免疫抗原HSP70的方法，其可有效应用于肿瘤疫苗的制备，也可直接对肿瘤罹患个体进行免疫治疗；
(2)本发明方法获得的抗原能够有效促免疫抑制细胞成熟，激活抗肿瘤免疫应答的方法，实验结果表明采用本发明的方法处理荷瘤小鼠，能够促进小鼠体内的免疫抑制细胞向树突细胞成熟分化。
(2)采用本发明的方法治疗荷瘤小鼠，能够显著提高荷瘤小鼠的生存率。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
通用材料和方法
1.动物、细胞系及主要试剂
6-8周龄SPF级Balb/c雌性小鼠(上海斯莱克动物中心)，饲养在独立换气盒笼中，人工控制12小时昼夜变换光照。小鼠自由摄取60Co辐射灭菌的饲料及高温灭菌水。小鼠4T 1乳腺癌细胞(获自上海市第一人民医院，在其它实施方式中，完全可以使用其它的乳腺癌细胞如T47D细胞系)，培养在加有10％的新生胎牛血清及双抗(100U/mL青霉素、100g/mL链霉素)(上海生工生物工程技术服务有限公司)的DMEM培养基中(美国Hyclone公司)。用于免疫细胞分选的磁珠购于德国Miltnyi Biotec公司。用于流式细胞术AF488anti-mouse CD11c,APC anti-mouse F4/80,Percp/cy5.5anti-mouse CD86,PE anti-mouse MHC II购于Biolegend公司。
2. 4T1乳腺癌模型的建立及肿瘤大小的测定
本实验所用的4T1小鼠乳腺癌细胞可通过血源性途径向肺脏、肝脏、骨髓和脑部等转移，是高度转移性的乳腺癌模型。制备1×1 06U/0.1mL 4T1细胞悬液置于冰上待用。按0.5mL/100g小鼠体重腹腔注射0.016g/mL戊巴比妥钠对动物进行麻醉，在小鼠背部皮下注射0.1mL细胞悬液。肿瘤接种21d后，游标卡尺测量肿瘤体积，按如下公式计算：
Figure PCTCN2017105727-appb-000001
Figure PCTCN2017105727-appb-000002
3.冷-热治疗的疗效评估
治疗后，综合性观察小鼠的生存状况。这部分内容主要包括：观察对照组原位肿瘤的生长情况、治疗组原位肿瘤的消融和复发情况情况(每天)，记录小鼠治疗前后的体重变化(一周两次)，作小鼠长期生存率和生存时间的统计。这些指标均能有效地反映小鼠的生存状况，其中生存率和生存时间的统计是治疗效果 评估的最重要指标。
4.肿瘤间质液蛋白质印迹分析
冷-热治疗后1天取小鼠肿瘤，接种22天后取对照组小鼠肿瘤。滤膜包裹肿瘤置入15ml离心管中，2000rpm离心15分钟，吸取离心管底部间质液。Bradford法测定总蛋白量，提取30μg蛋白加入至4-20％梯度甘氨酸预制胶电泳，转至PVDF膜。孵育HSP70一抗(Cell signaling technology)和荧光二抗，印迹条带曝光记录。
5.肿瘤免疫组化染色
冷-热治疗、单热治疗后1天取小鼠肿瘤；接种22天后取对照组小鼠肿瘤。经异戊烷速冻后，包入锡纸放至－80℃冰箱保存。肿瘤取出后用OCT包埋胶固定在样品托上，在－20℃环境温度下，利用冰冻切片机将肿瘤切成10μm薄片，将切片粘附到盖玻片上，放入－80℃冰箱保存。肿瘤切片自然风干后，在4℃条件下，置于丙酮中固定10分钟，PBS冲洗。使用0.3％过氧化物酶(甲醇稀释H2O2溶液)灭活20分钟，PBS冲洗。滴加5％BSA封闭30分钟。加入一抗anti-HSP70(Abcam,按1:1000PBS稀释)，置于37℃孵育1个小时，PBS冲洗。加入生物素标记的二抗，置于37℃孵育1小时，用免疫组化检测试剂盒中的链霉亲和素-HRP和过氧化物酶底物二氨基联苯胺(DAB)显色。用苏木素复染核。在显微镜明场下观察、拍照并记录。
6. ELISA检测外周血清中HSP70
治疗2天后，冷-热治疗组、手术切除肿瘤组、单热治疗组和无任何治疗对照组小鼠眼球静脉采血。收集的静脉血静置1小时，2000rpm离心20分钟，提取上层血清。按ELISA检测样本要求处理血清样本，检测血清中HSP70表达量。
7.磁珠分选免疫抑制细胞
冷-热治疗后2天和10天，取小鼠脾脏，采用GentleMACS温和组织处理器获得脾脏细胞混悬液，1500rpm离心5分钟弃上清液，加入2ml红细胞裂解液并吹打均匀，室温下裂解5分钟。加入PEB尽量稀释后离心去上清，获得脾脏细胞。采用德国Miltnyi Biotec公司的免疫抑制细胞Kit分选出纯度大于90％的免疫抑制细胞用于实验。
8.免疫抑制细胞分化实验
8.1体内实验
冷-热治疗10天后，取小鼠和对照组小鼠的脾脏，采用磁珠分选方法从脾脏细胞中分选出免疫抑制细胞。CD11c和F4/80分别作为树突细胞和巨噬细胞表面的 特征标记抗原；CD86和MHC II作为两种细胞成熟的表面标记抗原。加入1μl相应荧光抗体，在4℃条件下避光孵育30分钟。经PBS清洗后重悬，流式细胞仪检测。
8.2体外实验血清采集
冷-热治疗2天后的治疗组和对照组小鼠，以及正常组的小鼠，通过小鼠眼球静脉采血。收集的静脉血静置1小时，2000rpm离心20分钟，吸取上层血清，零下80摄氏度保存。
8.3体外实验
接种后23天，取对照组小鼠的脾脏采用磁珠分选出免疫抑制细胞，分别与DMEM培养液加治疗组小鼠血清、对照组小鼠血清、正常小鼠血清以及治疗组小鼠血清+HSP70抗体四种培养基孵育24小时。流式细胞仪检测细胞表面标记抗原CD11c、F4/80、CD86和MHCII表达。
9数据统计
本实验中所有实验数据的统计与分析都采用Graph pad Prism软件进行，利用Student’s t test作组间显著性差异分析，结果以平均值±标准差显示。P<0.05认为数据间存在显著差异。
实施例1冷-热治疗提高小鼠生存率
对冷-热治疗组(n＝16)的小鼠进行了冷-热治疗处理，其中，10分钟内降温至-20℃，降温维持时间5min，10分钟内加热至50℃，维持时间20min。
而空白对照组小鼠(n＝16)未接受治疗。
观察冷-热治疗组和空白对照小鼠的生存状况一年(图1)。16只冷-热治疗小鼠生存时间大于接种后66天，最终11只小鼠存活至一年。而16只对照组小鼠存活时间都未超过接种后64天。
实施例2冷-热治疗促局部肿瘤释放HSP70
将实施例3中冷-热治疗、单热治疗1天后，接种22天后，取原位肿瘤切片免疫组化。(图2)棕色(浅色)为抗体对HSP70蛋白的特异性染色，蓝色(深色)为苏木素对细胞核的染色。
在对照组中，看到一些分离的HSP70的存在，多存在于细胞内，并未释放出来， 此类HSP70主要起到促进肿瘤细胞存活的作用。
在单热治疗组和冷热结合治疗组中，皆可观察到区域性HSP70的释放，但是在单热组中，HSP70释放区域较为局限，有些细胞核完整的区域并未释放HSP70；
而在冷-热治疗组中，细胞坏死性死亡及细胞破碎严重，并伴随着更大面积的HSP70的释放。综上，实验结果证明，冷-热治疗作用于原位肿瘤，在产生更严重的细胞坏死性死亡的同时，诱发了更多HSP70的释放。
肿瘤间质液蛋白质印迹检测显示冷-热治疗组的HSP70蛋白水平明显多于对照组。(图3)
ELISA检测提示，治疗2天后手术组原位肿瘤切除后，血清中HSP70略低于对照组；单热治疗组血清中的HSP70与对照组差异很小，而冷-热治疗组小鼠外周血清中可见更为明显的HSP70的增加。(图4)
实施例3冷-热治疗促进免疫抑制细胞成熟分化
3.1体内实验
冷-热治疗10天后，冷-热治疗组小鼠脾脏免疫抑制细胞表面CD11C的表达水平高于对照组。且冷-热治疗组免疫抑制细胞表面MHC II和CD86的表达水平明显高于对照组。(图5)说明冷-热治疗后促进了免疫抑制细胞MDSC向成熟的树突状细胞转化。
3.2体外实验
免疫抑制细胞与四种血清培养基孵育24小时后，冷-热治疗组血清孵育的免疫抑制细胞表面的CD11C表达明显高于对照组和冷-热治疗组加入HSP70中和抗体；而与正常小鼠血清孵育的免疫抑制细胞无差异。(图6)流式检测发现，冷-热治疗组血清孵育的免疫抑制细胞表面抗原CD86和MHCII表达，明显高于对照组和冷-热治疗组加入HSP70中和抗体；高于正常小鼠血清孵育的免疫抑制细胞的表达。说明冷-热治疗后血清中的HSP70促进了免疫抑制细胞MDSC向成熟的树突状细胞转化。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种促进肿瘤组织和/或肿瘤细胞释放肿瘤免疫抗原热休克蛋白70(HSP70)的方法，其特征在于，包括步骤：
    I)将所述的肿瘤组织和/或肿瘤细胞进行降温从而获得经降温的肿瘤组织和/或肿瘤细胞；其中，所述的降温包括将所述肿瘤组织和/或肿瘤细胞降温至T1，且-50℃≤T1≤0℃；和
    II)将I)中获得的经降温的肿瘤组织和/或肿瘤细胞进行加热从而获得经加热的肿瘤组织和/或肿瘤细胞；其中，所述的加热包括将I)获得的经降温的肿瘤组织和/或肿瘤细胞加热至T2，且37℃＜T2≤60℃。
  2. 如权利要求1所述的方法，其特征在于，
    步骤I)为分段式降温步骤；和/或
    步骤II)为分段式加热步骤。
  3. 如权利要求1所述的方法，其特征在于，步骤I)中，降温所需的时间S1≤15min；和/或
    步骤I)中，温度达到T1后，在T1下持续5-30min。
  4. 如权利要求1所述的方法，其特征在于，步骤II)中，加热所需的时间S2≤15min；和/或
    步骤II)中温度达到T2后，在T2下持续5-30min。
  5. 如权利要求4所述的方法，其特征在于，步骤II)还包括以下分段式加热的子步骤：
    i)将I)中获得的经降温的肿瘤组织和/或肿瘤细胞加热至T2a，且-10℃＜T2a≤10℃；和
    ii)将i)中获得的T2a温度下的肿瘤组织和/或肿瘤细胞加热至T2。
  6. 如权利要求1所述的方法，其特征在于，所述方法还包括任选的步骤：
    III)对I)中的降温步骤和II)中的加热步骤进行一次或多次的重复。
  7. 如权利要求1-6任一所述的方法，其特征在于，所述方法中，步骤I)和/或II)还各自包括对肿瘤组织和/或肿瘤细胞的温度监测步骤；和/或
    所述方法中，步骤I)和/或II)还各自包括对肿瘤免疫抗原的定量和/或定性检测步骤。
  8. 如权利要求1所述的方法，其特征在于，所述的肿瘤包括：恶性实体肿瘤、血液肿瘤、良性肿瘤、转移瘤或其组合。
  9. 一种用于促进肿瘤组织和/或肿瘤细胞释放肿瘤免疫抗原的装置，其特征在于，包括：
    a)降温元件，所述的降温元件用于将所述的肿瘤组织和/或肿瘤细胞进行降温从而获得经降温的肿瘤组织和/或肿瘤细胞；其中，所述的降温包括将所述肿瘤组织和/或肿瘤细胞降温至T1，且-50℃≤T1≤0℃；
    b)加热元件，所述的加热元件用于将经降温的肿瘤组织和/或肿瘤细胞进行加热从而获得经加热的肿瘤组织和/或肿瘤细胞；其中，所述的加热包括将I)中获得的经降温的肿瘤组织和/或肿瘤细胞加热至T2，且37℃＜T2≤60℃；
    c)温度控制元件，所述的温度控制元件于控制所述降温元件和加热元件的重复循环启动和停止，从而对降温步骤和加热步骤进行一次或多次的重复；
    d)时间控制元件，所述时间控制元件用于根据需要控制降温、加热和/或温度维持时间；和任选的
    e)温度监测元件，所述温度监测元件用于对肿瘤组织和/或肿瘤细胞的温度监测。
  10. 一种制备热休克蛋白70(HSP70)的方法，其特征在于，包括步骤：
    A)将培养的肿瘤组织和/或肿瘤细胞进行降温，从而获得经降温的肿瘤组织和/或肿瘤细胞；其中，所述的降温包括将所述肿瘤组织和/或肿瘤细胞降温至T1，且-50℃≤T1≤0℃；
    B)将A)中获得的经降温的肿瘤组织和/或肿瘤细胞进行加热，从而获得含有肿瘤免疫抗原群的肿瘤组织和/或肿瘤细胞培养物；其中，所述的加热包括将A)获得的经降温的肿瘤组织和/或肿瘤细胞加热至T2，且37℃＜T2≤60℃；
    C)从B)中获得的培养物中分离并纯化的热休克蛋白70(HSP70)。
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