CN110604814A - Purified multi-subtype heat shock protein/peptide complex multiple tumor vaccine and its preparation method - Google Patents

Abstract

The invention relates to a purified multi-subtype heat shock protein/peptide complex multiple tumor vaccine and a preparation method thereof. Specifically, the present invention provides a method for preparing a purified multi-subtype heat shock protein/peptide complex concatenated tumor vaccine, and the purified multi-subtype heat shock protein/peptide complex concatenated tumor vaccine obtained by said method. Tumor vaccine. The vaccine has the advantages of good safety and high immune activity.

Description

Purified multisubtype heat shock protein/peptide complex multiple tumor vaccine and its preparation method

technical field

The present invention relates to a vaccine, especially a purified multi-subtype heat shock protein/peptide complex multi-tumor vaccine aimed at treating tumor tissues by extracting and purifying different subtype heat shock protein/peptide complexes and matching them in proportion. Belongs to the field of protein engineering. The present invention also relates to the preparation method of said vaccine.

Background technique

Tumor immunity is mainly based on cellular immunity, so one of the main purposes of tumor immunology research is to seek tumor-specific antigens, activate specific T lymphocytes (CTL) that kill tumor cells, and enhance the body's anti-tumor rejection. At present, tumor immunotherapy research mainly uses monoclonal antibodies, liposomes, and low-density lipoproteins as carriers, and chemotherapeutic drugs, biotin or radioisotope conjugates as warheads; cytokine genes, co-stimulatory molecules B7 Introduce genes into tumor cell lines, lymphocytes or fibroblasts, and bone marrow hematopoietic stem cells, thereby inhibiting the growth and metastasis of tumor cells and stimulating the body's immune response; directly immunizing animals with nucleic acids to express target antigens in host cells, DNA vaccines that trigger specific humoral immunity and cellular immunity; and peptide vaccines that use tumor cells or virus antigen regions as peptide carriers or adjuvants for immunization. In recent years, breakthroughs have been made in the research of tumor antigen molecules and major histocompatibility complex (MHC) antigen presentation, especially the discovery that heat shock protein (HSP) plays an important role in antigen presentation and activation of CD4 ⁺ T cells and CD8 ⁺ T cells It plays an important role and opens up a new way for the immunotherapy of tumors.

HSP is a kind of highly conserved and widely distributed protein in biological evolution. According to its molecular weight, it can be divided into different subtypes such as HSP110, HSP90, HSP70, HSP60 and HSP28. As a "molecular chaperone", HSP can bind tumor antigens and play the role of presenting antigens, and can bind to various denatured proteins and short peptides in cells, especially in the process of antigen presentation, it will bind a large number of antigenic peptides, thereby Make it have the unique antigen library of the cell. Therefore, heat shock proteins isolated from tumors carry a broad spectrum of tumor antigens. Animals carrying tumors can stimulate the proliferation of T lymphocytes in their own bodies after being vaccinated with heat shock protein/peptide complex vaccines, and activate the body's own immune system in a specific antigen-dependent manner to kill tumors. In this process of anti-tumor, the immunogenicity produced by heat shock protein tumor vaccines is attributed to the fact that these molecular chaperones bind to specific antigenic peptides in tumor cells and present them to antigen-presenting cells (APCs), The tumor-killing effects of CD4 ⁺ T cells and CD8 ⁺ T cells are then stimulated.

Molecular chaperones/peptide complexes extracted from tumors (i.e. HSP/peptide complex vaccines) have been intensively studied in the past two decades and have been proven to be safe and effective in the treatment of many malignant diseases. Personalized HSP/peptide complex tumor vaccines were prepared from specific antigens. However, the personalized HSP/peptide complex vaccines in the current technology use a single HSP as the main component, especially a single HSP vaccine based on HSP70 or Gp96. Phase III clinical trials of Gp96 vaccine have shown that a single Gp96 tumor vaccine has some anti-tumor effects during extensive treatment, but the ability to induce immune responses against tumors still needs to be further improved.

In order to solve this problem, some researchers have tried to use mixed heat shock protein/peptide complex vaccines to treat tumors in recent years, but the components of these mixed heat shock protein/peptide complex vaccines are not clear and have potential risks.

Therefore, the technical problem to be solved by the present invention is to overcome the insufficiency of the previous single heat shock protein/peptide complex vaccine in inducing an immune response against tumors and the unclear composition of the mixed heat shock protein and great potential safety hazards, and to provide a vaccine with high therapeutic activity A purified multi-subtype heat shock protein/peptide complex multiple tumor vaccine with good safety.

Contents of the invention

The inventors of the present invention have carried out a large number of theoretical and experimental studies on the mixed heat shock protein/peptide complex vaccines known in the prior art, and found that these mixed heat shock protein/peptide complex vaccines are mostly obtained by ion chromatography and The one obtained by molecular chromatography, therefore contains, in addition to the heat shock protein/peptide complex, other protein impurities of similar molecular weight. The inventors found that it is because of the presence of these impurities that the mixed heat shock protein/peptide complex vaccine has low immune activity and poor safety. The present inventors have creatively conceived a novel method to obtain purified multi-subtype heat shock protein/peptide complex concatenated tumor vaccines by additionally using immunoaffinity chromatography or using immunoaffinity chromatography instead of molecular chromatography To remove various impurities, obtain purified single heat shock protein/peptide complexes of different subtypes, and then mix them to obtain a purified multi-subtype heat shock protein/peptide complex multiple tumor vaccine. By using this method, the safety and immunological activity of the obtained vaccines are significantly improved.

Therefore, the technical problem to be solved by the present invention is solved by the above-mentioned purified multi-subtype heat shock protein/peptide complex multiple tumor vaccine (mpHSP/P).

In a first aspect, the present invention provides a method for preparing a purified multi-subtype heat shock protein/peptide complex multiple tumor vaccine, the method comprising:

(1) Provide tumor cell lysate;

(2) centrifuging the tumor cell lysate and collecting the supernatant;

(3) filtering the supernatant, then passing through an ion chromatography column to collect the protein effluent;

(4) Optionally, passing the protein effluent through a molecular chromatography column;

(5) Purify the product obtained through step (3) or optional step (4) through affinity immunochromatographic columns 1, 2, and 3, which are respectively specific The single HSP of different subtypes is adsorbed, and the purified single HSP/peptide complex 1, the purified single HSP/peptide complex 2 and the purified single HSP/peptide complex 3 are respectively obtained;

(6) mixing said purified single HSP/peptide complex 1, said purified single HSP/peptide complex 2 and said purified single HSP/peptide complex 3 in an appropriate ratio, In order to obtain the purified multi-subtype heat shock protein/peptide complex multiple tumor vaccine.

In a second aspect, the present invention provides a purified multi-subtype heat shock protein/peptide complex multiple tumor vaccine, which is prepared by a method comprising the following steps:

(1) Provide tumor cell lysate;

(2) centrifuging the tumor cell lysate and collecting the supernatant;

(3) filtering the supernatant, then passing through an ion chromatography column to collect the protein effluent;

(4) Optionally, passing the protein effluent through a molecular chromatography column;

(5) Purify the product obtained through step (3) or optional step (4) through affinity immunochromatographic columns 1, 2, and 3, which are respectively specific The single HSP of different subtypes is adsorbed, and the purified single HSP/peptide complex 1, the purified single HSP/peptide complex 2 and the purified single HSP/peptide complex 3 are respectively obtained;

(6) mixing said purified single HSP/peptide complex 1, said purified single HSP/peptide complex 2 and said purified single HSP/peptide complex 3 in an appropriate ratio, In order to obtain the purified multi-subtype heat shock protein/peptide complex multiple tumor vaccine.

The vaccine of the present invention can be used by adding an adjuvant. Unexpectedly, the inventors of the present invention have found that the vaccine of the present invention can achieve excellent immunological activity even when it is used without adding an adjuvant.

In step (1), the tumor cell lysate can be obtained by the following method: fresh tumor tissue is mechanically disrupted (such as shredded), and then lysed. Of course, other techniques known to those skilled in the art can also be used to obtain tumor cell lysates.

In step (3), the supernatant may be filtered through a strainer and/or a filter or other means and/or devices known to those skilled in the art may be used to filter the supernatant. When carrying out ion chromatography, buffer A can be 20mmol/l Tris solution of pH=8.5; Buffer B can be the mixed solution of 20mmol/l Tris and 1mol/l NaCl of pH=8.5. Of course, other buffers known in the art can also be used as buffer A and/or buffer B, and such technical solutions also fall within the protection scope of the present invention. Through the ion chromatography column, protein samples can be screened and non-protein substances can be filtered.

In step (5), the affinity immunochromatographic columns 1, 2, and 3 respectively and specifically adsorb any one of the HSPs. Preferably, the affinity immunochromatographic columns 1, 2, and 3 respectively and specifically adsorb any one of HSP70, HSP90, Gp96, HSP110, HSP60, and HSP28. Those skilled in the art can easily understand that a chromatographic column that specifically adsorbs HSPs other than HSP70, HSP90, Gp96, HSP110, HSP60, and HSP28 can also be used, and such a technical solution also falls within the protection scope of the present invention. More preferably, the affinity immunochromatographic columns 1, 2, and 3 respectively and specifically adsorb any one of HSP70, HSP90, and Gp96.

In step (5), the purified single HSP/peptide complex 1 is any purified single HSP/peptide complex; the purified single HSP/peptide complex 2 is different Any one of the purified single HSP/peptide complexes than the purified single HSP/peptide complex 1; the purified single HSP/peptide complex 3 is different from the purified single HSP/peptide complex 3 Either of the purified single HSP/peptide complex of peptide complex 1 and the purified single HSP/peptide complex 2. Preferably, the purified single HSP/peptide complex 1, the purified single HSP/peptide complex 2 and the purified single HSP/peptide complex 3 are different in that they are purified Single HSP70/peptide complex, Purified single HSP90/peptide complex, Purified single Gp96/peptide complex, Purified single HSP110/peptide complex, Purified single HSP60/peptide complex any of the purified HSP28/peptide complexes. Those skilled in the art can easily understand that, as mentioned above, a chromatographic column that specifically adsorbs other single HSPs except HSP70, HSP90, Gp96, HSP110, HSP60, and HSP28 can be used to obtain purified single other HSP/peptide complexes , which is a purified single HSP/peptide complex 1, a purified single HSP/peptide complex 2 or a purified single HSP/peptide complex 3, such a technical scheme also falls within the protection of the present invention within range. More preferably, the purified single HSP/peptide complex 1 is any one of HSP70/peptide complex, HSP90/peptide complex, and Gp96/peptide complex; the purified single HSP/peptide complex Peptide complex 2 is any of the HSP70/peptide complex, HSP90/peptide complex, Gp96/peptide complex different from the purified single HSP/peptide complex 1; the purified single HSP/peptide complex 3 is different from said purified single HSP/peptide complex 1 and said purified single HSP/peptide complex 2 HSP70/peptide complex, HSP90/peptide complex, Either of the Gp96/peptide complexes.

In step (5), when performing affinity immunochromatography, buffer A may be 20 mmol/l Tris solution with pH=7.5, and buffer B may be 20 mmol/l sodium citrate buffer with pH=3. Finally, add 50mmol/l Tris with pH=9, mix according to the volume ratio of 1:2 and store in -80°C refrigerator. Of course, other buffers known in the art can also be used as buffer A and/or buffer B, and the volume ratio can be adjusted appropriately according to needs, and such a technical solution also falls within the protection scope of the present invention.

In step (6), the purified single HSP/peptide complex 1, the purified single HSP/peptide complex 2, and the purified single HSP/peptide complex 3 can be derived from a single The ratios of HSP/peptide complex 1, single HSP/peptide complex 2, and single HSP/peptide complex 3 were mixed in ratios determined by Elisa experiments as described below. Of course, those skilled in the art can also adjust the ratio according to the actual situation, and such a technical solution also falls within the protection scope of the present invention.

In an embodiment of the invention, Western blotting was performed on purified single HSP/peptide complex 1, purified single HSP/peptide complex 2, and purified single HSP/peptide complex 3 ( Western blot; WB) identification. Of course, other methods known to those skilled in the art can also be used for identification.

In an embodiment of the present invention, the tumor cell lysate can be derived from any type of tumor known in the art, including but not limited to bone tumor, synovial tumor, fibrous tumor, giant cell tumor, Ewing's tumor, etc., or proposed strains of tumor cells.

In an embodiment of the present invention, the tumor cell lysate can be derived from fresh self-tumor tissues surgically removed from tumor patients, including but not limited to bone tumors, synovial tumors, fibrous tumors, giant cell tumors, Ewing tumors, etc., or proposed strains of tumor cells.

The present invention also relates to the use of the multi-subtype heat shock protein/peptide complex multiple tumor vaccine prepared by the method of the present invention or purified according to the present invention in the preparation of drugs for inhibiting tumor growth.

The present invention also relates to the preparation of the multi-subtype heat shock protein/peptide complex multiple tumor vaccine prepared by the method of the present invention or the purification of the present invention for activating cytokines IL-2, TNF-α, IFN- Uses of gamma in medicine.

The present invention also relates to the use of the multi-subtype heat shock protein/peptide complex multiple tumor vaccine prepared by the method of the present invention or purified according to the present invention in the preparation of drugs for increasing CD4 ⁺ /CD8 ⁺ T cells .

The present invention also relates to the use of the multi-subtype heat shock protein/peptide complex multiple tumor vaccine prepared by the method of the present invention or purified according to the present invention for inhibiting tumor growth.

The present invention also relates to the use of the multi-subtype heat shock protein/peptide complex multiple tumor vaccine prepared by the method of the present invention or purified according to the present invention for activating cytokines IL-2, TNF-α, IFN-γ use.

The present invention also relates to the use of the multi-subtype heat shock protein/peptide complex multi-tumor vaccine prepared by the method of the present invention or purified according to the present invention for increasing CD4 ⁺ /CD8 ⁺ T cells.

Through the above preparation method, several heat shock protein/peptide complexes with immune activation function in tumor tissue can be extracted and purified in a single way. The purified single HSP/peptide complexes obtained by the above method include HSP70/peptide complexes, HSP90/peptide complexes, Gp96/peptide complexes, etc., and then they are reasonably compounded at the ratio obtained by component quantification (such as Elisa experiment) apply. The purified multi-subtype heat shock protein/peptide complex multi-tumor vaccine of the present invention has unique advantages compared with the vaccines known in the prior art. First, HSP70, Gp96, and HSP90 carry a large number of various tumor antigens, which are similar to the role of an antigen library to form a multivalent vaccine, which not only effectively activates specific immunity, but also stimulates the body to produce an innate immune response. Through the activation of the immune system, anti-tumor cytokines such as IL-12, IL-2, TNF-α, and IFN-γ are produced, and the corresponding anti-tumor CD4 ⁺ T cells and CD8 ⁺ T cells are activated to achieve excellent Antitumor effect. Second, the preparation process of the vaccine of the present invention includes purification by affinity immunochromatography, which effectively eliminates the immunosuppressive factors (such as TGF-β, etc.) present in the tumor tissue, thereby improving the immune effect of the vaccine to the greatest extent, and avoid possible side effects. Thirdly, the vaccine of the present invention does not need additional adjuvants, is easy to prepare, and eliminates possible side effects of adjuvants. Fourth, the vaccine of the present invention can be used in combination with existing antitumor drugs to further enhance the antitumor effect of tumor vaccines.

Brief introduction to the drawings

Figure 1: Identification and quantification of HSP70/peptide complexes, HSP90/peptide complexes, Gp96/peptide complexes in MCA207 sarcoma cells. A: The stable expression of three heat shock protein/peptide complexes was identified by immunohistochemical fluorescent staining. The blue fluorescence indicates the nucleus, and the red fluorescence in the three groups indicates the HSP70/peptide complex, HSP90/peptide complex, and Gp96/peptide complex respectively Complex. B: Elisa assay quantifies the content of each of the three heat shock protein/peptide complexes in sarcoma cells, and provides the mixing ratio of the three heat shock protein/peptide complexes for the preparation of mpHSP/P.

Figure 2: Protein efflux recording curves. A: The protein elution record curve obtained after passing through the ion chromatography column, where the peak appears to indicate protein elution. B: The protein efflux record curve and WB identification chart obtained after passing through the affinity immunochromatographic column; by collecting each fragment with protein efflux and using WB for identification, it shows that the HSP70/peptide complex is collected from the A47 fragment , the HSP90/peptide complex was collected from the A46 fragment, and the Gp96/peptide complex was collected from the A4 fragment.

Figure 3: Cytotoxicity assay of mpHSP/P. A: The cytotoxicity of mpHSP/P to cells at different concentrations was determined by flow cytometry. B: Cell viability at different vaccine concentrations. Physiological saline was used as a negative control (con).

Figure 4: Comparison of mpHSP/P with a single Gp96/peptide complex vaccine and a comparative mixed heat shock protein/peptide complex vaccine (mHSP/P, as detailed in the comparative example below). A: Tumor growth curve. B: Mouse survival curve. Physiological saline was used as a negative control (con).

Figure 5: Concentrations of anti-tumor cytokines IL-2, TNF-α, IFN-γ in tumor tissues measured at day 14 after injection of mpHSP/P, single Gp96/peptide complex vaccine or mHSP/P. Physiological saline was used as a negative control (con).

Figure 6: Infiltration of CD4 ⁺ T cells and CD8 ⁺ T cells in tumor tissues observed at day 14 after injection of mpHSP/P, single Gp96/peptide complex vaccine or mHSP/P. Blue fluorescence indicates nuclei, red fluorescence indicates CD4 ⁺ T cells, and green fluorescence indicates CD8 ⁺ T cells. Physiological saline was used as a negative control (con).

Example

The beneficial effects of the present invention are further described through the following examples. It should be understood that these examples are only for the purpose of illustration and are not intended to limit the protection scope of the present invention. The protection scope of the present invention is limited only by the claims.

Example 1: Preparation of the purified multi-subtype heat shock protein/peptide complex multiple tumor vaccine of the present invention

(1) Materials and methods

(1) Source of material: the tumor tissue taken out after injection of mouse MCA207 tumor cell 5e5 into the subcutaneous culture of mice for 3-4 weeks.

(2) Preparation steps of the vaccine:

1) Shred the tumor tissue, add 2-3 times the cell lysate (RIPA:cocktail=100:1) and put it in a homogenizer for 4 hours.

2) The cell lysate obtained above was ultracentrifuged at 25000 rpm (75000 g) at 4 degrees for 2.5 h, and the supernatant was collected.

3) Filter the collected supernatant before and after through the filter and the filter, and then collect the filtered protein suspension.

4) The above-treated sample supernatant is passed through an ion chromatography column (buffer A: Tris solution of 20mmol/l and PH=8.5, buffer B: under PH=8.5, Tris of 20mmol/l and 1mol/l mixed solution of NaCl). After all the samples passed through the ion chromatography column, the section where the protein flowed out was collected (Fig. 2A).

5) Concentrate the sample obtained in the previous step through the concentration test tube.

6) Pass the concentrated sample through the affinity immunochromatographic columns 1, 2, and 3 that specifically adsorb HSP70, HSP90, and Gp96, respectively, and collect the fragments with protein effluent. The buffer of the affinity immunochromatography column is divided into two buffers, A and B. Buffer A: Tris solution with 20mmol/l and pH=7.5, buffer B: 20mmol/l sodium citrate buffer with pH=3. Finally, 2 volumes of 50 mmol/l Tris at pH=9 were added.

8) After identification by WB, the prepared purified single HSP70/peptide complex (A47 truncated), the purified single HSP90/peptide complex (A46 truncated), the purified single Gp96/ The peptide complex (A4 truncation) is mixed in a certain ratio to obtain the purified multi-subtype heat shock protein/peptide complex multiple tumor vaccine (mpHSP/P) of the present invention, and the mixing ratio is the tumor tissue determined by the Elisa experiment. The ratios of these 3 heat shock protein/peptide complexes in , as described below.

9) Aliquot and freeze-dry for later use.

(3) Determination and identification:

1) Using the materials obtained after the above step 3), the stable expression of the three heat shock protein/peptide complexes was identified by immunohistochemical fluorescent staining (Figure 1A). The blue fluorescence indicates the nucleus, and the red in the three groups of figures indicates HSP70 respectively /peptide complexes, HSP90/peptide complexes, and Gp96/peptide complexes; the contents of the three heat shock protein/peptide complexes in sarcoma cells were quantitatively determined by Elisa assay (Fig. Mixing ratio of heat shock protein/peptide complexes.

2) WB identification was performed on the protein samples obtained after the above two chromatography, and the identification results proved that a single HSP70/peptide complex (A47 truncation) and a single HSP-90/peptide complex (A46 truncation) were purified respectively. segment), a single Gp96/peptide complex (A4 segment) (Fig. 2B).

Comparative Example: Preparation of Mixed Heat Shock Protein/Peptide Complex Vaccine

(1) Materials and methods

(1) Source of material: the tumor tissue taken out after injection of mouse MCA207 tumor cell 5e5 into the subcutaneous culture of mice for 3-4 weeks.

(2) Preparation steps of the vaccine:

1) Shred the tumor tissue, add 2-3 times the cell lysate (RIPA:cocktail=100:1) and put it in a homogenizer for 4 hours.

2) The cell lysate obtained above was ultracentrifuged at 25000 rpm (75000 g) at 4 degrees for 2.5 h, and the supernatant was collected.

3) Filter the collected supernatant before and after through the filter and the filter, and then collect the filtered protein suspension.

4) The above-treated sample supernatant is passed through an ion chromatography column (buffer A: Tris solution of 20mmol/l and PH=8.5, buffer B: under PH=8.5, Tris of 20mmol/l and 1mol/l mixed solution of NaCl). After all the samples pass through the ion chromatography column, the section with the protein flowing out is collected.

5) Concentrate the sample obtained in the previous step through the concentration test tube.

6) Purify the concentrated sample a second time through a molecular chromatography column (buffer Tris 20mmol/L PH=8.5), collect the eluted proteins of different fragments for SDS-PAGE experiments to identify the properties of the proteins, and then retain the samples containing Fractional mixing of HSP70/peptide complexes, HSP90/peptide complexes, and Gp96/peptide complexes resulted in a comparative mixed heat shock protein/peptide complex vaccine (mHSP/P).

6) Aliquot and freeze-dry for later use.

Embodiment 2: biological safety test

(1) Test materials

(1) Experimental group samples: mpHSP/P.

(2) Negative control group: normal saline.

(3) Experimental cells and related equipment: L929 cell line, flow cytometer.

(2) Experimental methods and results

Add mpHSP/P in different amounts of 10ug, 20ug, and 30ug into the culture dish of L929 cells, and use flow cytometry to measure the cell viability.

Experimental results: mpHSP/P at different concentrations did not exhibit cytotoxicity, thus proving its biological safety (Figure 3).

Embodiment 3: Therapeutic immune effect test

(1) Test materials

(1) Experimental group: mpHSP/P.

(2) Control group: physiological saline (negative control), purified single Gp96/peptide complex vaccine, mHSP/P.

The purified single Gp96/peptide complex vaccine, mHSP/P and mpHSP/P were all extracted from the same tumor by the inventors.

(3) Experimental animals: C57 mice, weighing 18-20 g, female, purchased from Speiford Company. MCA207 tumor cells, donated by Professor Zong Kangla of Stanford University.

(2) Test methods and results

MCA207 tumor cells 5e5 were planted in mice for 3 days and then injected with purified single Gp96/peptide complex vaccine, mHSP/P, mpHSP/P or saline. The doses of normal saline, purified single Gp96/peptide complex vaccine, mHSP/P and mpHSP/P were all 30ug/time/mouse, injected subcutaneously, and immunized 3 times, with an interval of 7 days between each immunization and the last immunization. Each group contained 15 mice.

Experimental results: the mice in the negative control group (physiological saline injection group) all died after 30-40 days. Mice injected with mpHSP/P did not show any adverse effects. Compared with injection of saline, mHSP/P or purified single Gp96/peptide complex vaccine, injection of mpHSP/P significantly inhibited tumor growth and prolonged the survival time of mice. Therefore, the therapeutic immune effect of the vaccine of the present invention is obviously better than that of the control group, and has stronger anti-tumor ability (Figure 4).

Example 4: Molecular and cytological experiments of stimulation and anti-tumor

(1) Test materials

(1) Experimental group: mpHSP/P.

(2) Control group: physiological saline (negative control), purified single Gp96/peptide complex vaccine, mHSP/P.

The purified single Gp96/peptide complex vaccine, mHSP/P and mpHSP/P were all extracted from the same tumor by the inventors.

(3) Experimental animals: C57 mice, weighing 18-20 g, female, purchased from Speiford Company. MCA207 tumor cells, donated by Professor Zong Kangla of Stanford University.

(4) Related reagents and equipment for molecular detection: microplate reader, ELISA kit for anti-tumor cytokines (IFN-γ, TNF-α, IL-2), fluorescence microscope, CD4 and CD8 antibodies.

(2) Test methods and results

MCA207 tumor cells 5e5 were planted in mice for 3 days and then injected with purified single Gp96/peptide complex vaccine, mHSP/P, mpHSP/P or saline. By subcutaneous injection, the dosage is as described in Example 4, immunized 3 times, and the interval between each immunization and the last immunization is 7 days. Two weeks after immunization, the mouse tumors were lysed, centrifuged, and filtered, and then ELISA was used to detect the contents of anti-tumor cytokines (IFN-γ, TNF-α, IL-2) in different groups. The mice were killed 3-4 weeks after the immunization, and CD4 ⁺ /CD8 ⁺ T cell staining was done on the tumor tissue of the mice, and the anti-tumor effect of the immune system in the control group and the experimental group was compared after 2 weeks.

Comparing the experimental group with the control group, it was found that the IFN-γ, TNF-α, IL-2 anti-tumor cytokines in the mpHSP/P group were higher than those in the other groups (Figure 5), and the anti-tumor CD4 ⁺ /CD8 ⁺ T cells increased (Figure 6).

Claims (8)

1. A method for preparing a purified multi-subtype heat shock protein/peptide complex multiple tumor vaccine, said method comprising: (1) Provide tumor cell lysate; (2) centrifuging the tumor cell lysate and collecting the supernatant; (3) filtering the supernatant, then passing through an ion chromatography column to collect the protein effluent; (4) Optionally, passing the protein effluent through a molecular chromatography column; (5) Purify the product obtained through step (3) or optional step (4) through affinity immunochromatographic columns 1, 2, and 3, which are respectively specific The single HSP of different subtypes is adsorbed, and the purified single HSP/peptide complex 1, the purified single HSP/peptide complex 2 and the purified single HSP/peptide complex 3 are respectively obtained; (6) mixing said purified single HSP/peptide complex 1, said purified single HSP/peptide complex 2 and said purified single HSP/peptide complex 3 in an appropriate ratio, In order to obtain the purified multi-subtype heat shock protein/peptide complex multiple tumor vaccine. 2. The method according to claim 1, wherein said purified single HSP/peptide complex 1, said purified single HSP/peptide complex 2 and said purified single The HSP/peptide complex 3 was mixed at the ratio of single HSP/peptide complex 1, single HSP/peptide complex 2, and single HSP/peptide complex 3 in the source tumor tissue. 3. The method according to claim 1, wherein the purified single HSP/peptide complex 1 is any one of HSP70/peptide complex, HSP90/peptide complex, Gp96/peptide complex species; the purified single HSP/peptide complex 2 is different from the purified single HSP/peptide complex 1 in the HSP70/peptide complex, HSP90/peptide complex, Gp96/peptide complex any of; said purified single HSP/peptide complex 3 is HSP70 different from said purified single HSP/peptide complex 1 and said purified single HSP/peptide complex 2 /peptide complex, HSP90/peptide complex, Gp96/peptide complex. 4. The method according to any one of claims 1-3, wherein the tumor cell lysate is derived from bone tumors, synovial tumors, fibrous tumors, giant cell tumors, Ewing's tumors, or established strains of tumor cells. 5. A purified multisubtype heat shock protein/peptide complex multiple tumor vaccine prepared by the method of any one of the preceding claims. 6. Use of the purified multi-subtype heat shock protein/peptide complex multiple tumor vaccine according to claim 5 in the preparation of a drug for inhibiting tumor growth. 7. Use of the purified multi-subtype heat shock protein/peptide complex multiple tumor vaccine according to claim 5 in the preparation of drugs for activating cytokines IL-2, TNF-α, IFN-γ. 8. Use of the purified multi-subtype heat shock protein/peptide complex multiple tumor vaccine according to claim 5 in the preparation of a medicament for increasing CD4 ⁺ /CD8 ⁺ T cells.