CN110604814A - Purified multi-subtype heat shock protein/peptide compound multi-tumor vaccine and preparation method thereof - Google Patents
Purified multi-subtype heat shock protein/peptide compound multi-tumor vaccine and preparation method thereof Download PDFInfo
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- A61P37/04—Immunostimulants
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- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
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- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
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- 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
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Abstract
The invention relates to a purified multi-subtype heat shock protein/peptide compound multi-tumor vaccine and a preparation method thereof. Specifically, the 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 the method. The vaccine has the advantages of good safety and high immunocompetence.
Description
Technical Field
The invention relates to a vaccine, in particular to a purified multi-subtype heat shock protein/peptide compound multi-tumor vaccine aiming at treating tumor tissues, which is prepared by extracting and purifying different subtype heat shock protein/peptide compounds and matching the different subtype heat shock protein/peptide compounds according to a proportion, and belongs to the field of protein engineering. The invention also relates to a preparation method of the vaccine.
Background
Tumor immunity is mainly cellular immunity, so one of the main purposes of tumor immunology research is to search for tumor specific antigen, activate specific T lymphocyte (CTL) killing tumor cells, and enhance the anti-tumor rejection reaction of the organism. At present, monoclonal antibodies, liposomes and low-density lipoprotein are mainly used as carriers in the research of tumor immunotherapy, and chemotherapeutic drugs, biotin or radioactive isotope conjugates are used as the warhead guide therapy; introducing cytokine gene, co-stimulatory molecule B7 gene, etc. into tumor cell strain, lymphocyte or fibroblast, and bone marrow hematopoietic stem cell, thereby inhibiting tumor cell growth and metastasis, and stimulating organism immunoreaction; directly immunizing animals with nucleic acid to express target antigen in host cells and trigger specific DNA vaccine of humoral immunity and cellular immunity; and peptide vaccine and other methods of utilizing tumor cell or virus antigen area polypeptide plus carrier or adjuvant to immunize. In recent years, there has been a breakthrough in the research on the presentation of tumor antigen molecules and Major Histocompatibility Complex (MHC) antigens, especially the discovery that Heat Shock Proteins (HSP) are presenting antigens and activating CD4+T cells and CD8+Has important function in the aspect of T cells, and opens up a new way for the immunotherapy of tumors.
HSPs are proteins that are highly conserved and widely distributed in biological evolution, and can be classified into different subtypes, such as HSP110, HSP90, HSP70, HSP60, HSP28, and the like, according to their molecular weights. HSP as a molecular chaperone (moleculechaliperone) can bind to tumor antigens and play a role in presenting the antigensBinding to various denatured proteins and short peptides in a cell, especially in the course of antigen presentation, binds a large number of antigenic polypeptides, thereby providing a pool of antigens unique to the cell. Thus, heat shock proteins isolated from tumors carry a broad spectrum of tumor antigens. After inoculated with the heat shock protein/peptide complex vaccine, the tumor-carrying animal can stimulate the proliferation of T lymphocytes in the body and activate the body's own immune system in a specific antigen-dependent manner to kill the tumor. In this process of anti-tumor, the immunogenicity conferred by heat shock protein tumor vaccines is attributed to the binding of these chaperones to specific antigenic peptides within the tumor cells, their presentation to Antigen Presenting Cells (APCs), and their subsequent stimulation of CD4+T cells and CD8+Tumor killing by T cells.
Molecular chaperone/peptide complexes extracted from tumors (i.e., HSP/peptide complex vaccines) have been extensively studied over the last two decades and have proven safe and effective in the treatment of many malignant diseases, enabling the preparation of personalized HSP/peptide complex tumor vaccines against specific antigens expressed in patients' tumors. However, the personalized HSP/peptide complex vaccines in the current art have a single HSP as a main component, especially HSP70 or Gp96 based single HSP vaccines. Clinical trials of Gp96 vaccine phase III show that a single Gp96 tumor vaccine exerts a certain anti-tumor effect over a broad range of therapeutic procedures, but the ability to induce immune responses against tumors still needs to be further improved.
To solve this problem, some researchers have tried to treat tumors with mixed heat shock protein/peptide complex vaccines in recent years, but these mixed heat shock protein/peptide complex vaccines have undefined components and are potentially at risk.
Therefore, the technical problem to be solved by the invention is to overcome the defects of insufficient capability of the traditional single heat shock protein/peptide complex vaccine in inducing immunoreaction to resist tumors, undefined mixed heat shock protein components and great potential safety hazard, and provide the purified multi-subtype heat shock protein/peptide complex multi-tumor vaccine with high therapeutic activity and good safety.
Disclosure of Invention
The present inventors have conducted extensive 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 molecular chromatography, and thus contain other protein impurities of similar molecular weight in addition to the heat shock protein/peptide complexes. The present inventors have found that the presence of these impurities makes the mixed heat shock protein/peptide complex vaccine less immunologically active and less safe. The present inventors have creatively conceived a new method for obtaining a purified multi-subtype heat shock protein/peptide complex multi-linked tumor vaccine, wherein various impurities are removed by additionally using immunoaffinity chromatography or using immunoaffinity chromatography instead of molecular chromatography to obtain purified single heat shock protein/peptide complexes of different subtypes, which are then mixed to obtain the purified multi-subtype heat shock protein/peptide complex multi-linked tumor vaccine. By using this method, the safety and immunological activity of the obtained vaccine are significantly improved.
Therefore, the technical problem to be solved by the present invention is solved by the purified multi-subtype heat shock protein/peptide complex concatenated tumor vaccine (mpHSP/P) as described above.
In a first aspect, the present invention provides a method for preparing a purified multi-subtype heat shock protein/peptide complex concatemeric tumor vaccine, comprising:
(1) providing a tumor cell lysate;
(2) centrifuging the tumor cell lysate, and collecting a supernatant;
(3) filtering the supernatant, and then passing through an ion chromatography column to collect a protein effluent;
(4) optionally, passing the protein effluent through a molecular chromatography column;
(5) purifying the product obtained in step (3) or optional step (4) by affinity immunochromatographic columns 1, 2, 3, wherein the affinity immunochromatographic columns 1, 2, 3 specifically adsorb different subtypes of single HSP respectively, and respectively obtain a purified single HSP/peptide complex 1, a purified single HSP/peptide complex 2 and a purified single HSP/peptide complex 3;
(6) mixing the purified single HSP/peptide complex 1, the purified single HSP/peptide complex 2 and the purified single HSP/peptide complex 3 in a proper ratio to obtain the purified multi-subtype heat shock protein/peptide complex multi-combination tumor vaccine.
In a second aspect, the present invention provides a purified multi-subtype heat shock protein/peptide complex concatenated tumor vaccine, which is prepared by a method comprising the following steps:
(1) providing a tumor cell lysate;
(2) centrifuging the tumor cell lysate, and collecting a supernatant;
(3) filtering the supernatant, and then passing through an ion chromatography column to collect a protein effluent;
(4) optionally, passing the protein effluent through a molecular chromatography column;
(5) purifying the product obtained in step (3) or optional step (4) by affinity immunochromatographic columns 1, 2, 3, wherein the affinity immunochromatographic columns 1, 2, 3 specifically adsorb different subtypes of single HSP respectively, and respectively obtain a purified single HSP/peptide complex 1, a purified single HSP/peptide complex 2 and a purified single HSP/peptide complex 3;
(6) mixing the purified single HSP/peptide complex 1, the purified single HSP/peptide complex 2 and the purified single HSP/peptide complex 3 in a proper ratio to obtain the purified multi-subtype heat shock protein/peptide complex multi-combination tumor vaccine.
The vaccine of the present invention may be used with an adjuvant. Unexpectedly, the inventors of the present invention have found that the vaccine of the present invention achieves excellent immune activity when used without the addition of an adjuvant.
In step (1), the tumor cell lysate may be obtained by: fresh tumor tissue is mechanically disrupted (e.g., sheared) and then lysed. Of course, other techniques known to those skilled in the art may also be used to obtain tumor cell lysates.
In step (3), the supernatant may be filtered through a strainer and/or filter or filtered using other means and/or devices known to those skilled in the art. When ion chromatography is performed, the buffer a may be a20 mmol/l Tris solution having a PH of 8.5; the buffer B may be a mixed solution of 20mmol/l Tris and 1mol/l NaCl at PH 8.5. Of course, other buffers known in the art may be used as buffer a and/or buffer B, and such solutions are also within the scope of the present invention. The protein sample can be screened and non-protein substances can be filtered through the ion chromatography column.
In step (5), the affinity immunochromatographic columns 1, 2, 3 respectively adsorb any one of HSP differently and specifically. Preferably, the affinity immunochromatographic columns 1, 2, 3 specifically adsorb any one of HSP70, HSP90, Gp96, HSP110, HSP60, HSP28 differently, respectively. It is easily understood by those skilled in the art that a chromatography column specifically adsorbing other HSPs than HSP70, HSP90, Gp96, HSP110, HSP60, HSP28 may also be used, and such a technical solution also falls within the scope of the present invention. More preferably, the affinity immunochromatographic columns 1, 2, 3 specifically adsorb any one of HSP70, HSP90, Gp96 differently, respectively.
In step (5), the purified single HSP/peptide complex 1 is any one of the purified single HSP/peptide complexes; the purified single HSP/peptide complex 2 is any one purified single HSP/peptide complex other than the purified single HSP/peptide complex 1; the purified single HSP/peptide complex 3 is any one purified single HSP/peptide complex other than the purified single HSP/peptide complex 1 and the purified single HSP/peptide complex 2. Preferably, the purified single HSP/peptide complex 1, purified single HSP/peptide complex 2 and purified single HSP/peptide complex 3 are different and are any one of a purified single HSP 70/peptide complex, a purified single HSP 90/peptide complex, a purified single Gp 96/peptide complex, a purified single HSP 110/peptide complex, a purified single HSP 60/peptide complex, a purified single HSP 28/peptide complex, respectively. It is easily understood by those skilled in the art that, as described above, purified single other HSP/peptide complexes, which are purified single HSP/peptide complex 1, purified single HSP/peptide complex 2 or purified single HSP/peptide complex 3, may be obtained using a chromatography column that specifically adsorbs other single HSPs than HSP70, HSP90, Gp96, HSP110, HSP60, HSP28, and such a technical solution also falls within the scope of the present invention. More preferably, the purified single HSP/peptide complex 1 is any one of HSP 70/peptide complex, HSP 90/peptide complex, Gp 96/peptide complex; the purified single HSP/peptide complex 2 is any one of HSP 70/peptide complex, HSP 90/peptide complex, Gp 96/peptide complex different from the purified single HSP/peptide complex 1; the purified single HSP/peptide complex 3 is any one of HSP 70/peptide complex, HSP 90/peptide complex, Gp 96/peptide complex different from the purified single HSP/peptide complex 1 and the purified single HSP/peptide complex 2.
In step (5), in the case of performing affinity immunochromatography, buffer a may be a20 mmol/l Tris solution at PH 7.5, and buffer B may be a20 mmol/l sodium citrate buffer at PH 3. And finally adding 50mmol/l Tris with pH being 9, mixing according to the volume ratio of 1:2, and storing in a refrigerator at-80 ℃. Of course, other buffers known in the art may be used as buffer a and/or buffer B, and the volume ratio may be appropriately adjusted as needed, and such a technical solution also falls within the scope of the present invention.
In step (6), purified single HSP/peptide complex 1, purified single HSP/peptide complex 2 and purified single HSP/peptide complex 3 may be mixed in the ratio of single HSP/peptide complex 1, single HSP/peptide complex 2 and single HSP/peptide complex 3 in the originating tumor tissue, as determined by the Elisa experiment, for example, as described below. Of course, the person skilled in the art can adjust the ratio according to the actual situation, and such a technical solution also falls within the scope of the present invention.
In an embodiment of the present invention, the purified single HSP/peptide complexes 1, 2 and 3 are identified by Western blotting (Western blot; WB). Of course, other methods known to those skilled in the art may also be used for identification.
In embodiments of the invention, the tumor cell lysate can be derived from any type of tumor known in the art, including but not limited to bone tumors, synovial tumors, fibrous tumors, giant cell tumors, ewing's tumors, and the like, or established tumor cells.
In embodiments of the invention, the tumor cell lysate may be derived from fresh autologous tumor tissue surgically removed from a tumor patient, including but not limited to bone tumor, synovial tumor, fibrous tumor, giant cell tumor, ewing's tumor, etc., or established tumor cells.
The invention also relates to the application of the purified multi-subtype heat shock protein/peptide complex concatenated tumor vaccine prepared by the method or the purified multi-subtype heat shock protein/peptide complex concatenated tumor vaccine in the preparation of a medicament for inhibiting tumor growth.
The invention also relates to application of the multi-subtype heat shock protein/peptide complex concatenated tumor vaccine prepared by the method or purified by the method in preparation of a medicament for activating cytokines IL-2, TNF-alpha and IFN-gamma.
The invention also relates to the application of the purified multi-subtype heat shock protein/peptide complex concatenated tumor vaccine prepared by the method or the purified multi-subtype heat shock protein/peptide complex of the invention in preparing a vaccine for increasing CD4+/CD8+Use in the manufacture of a medicament for T cells.
The invention also relates to the application of the purified multi-subtype heat shock protein/peptide complex concatenated tumor vaccine prepared by the method or the purified multi-subtype heat shock protein/peptide complex concatenated tumor vaccine for inhibiting tumor growth.
The invention also relates to application of the purified multi-subtype heat shock protein/peptide complex multi-tumor vaccine prepared by the method or the purified multi-subtype heat shock protein/peptide complex multi-tumor vaccine for activating cytokines IL-2, TNF-alpha and IFN-gamma.
The invention also relates to the use of the purified multi-subtype heat shock protein/peptide complex concatenated tumor vaccine prepared by the method or the purified multi-subtype heat shock protein/peptide complex of the invention for increasing CD4+/CD8+Use of T cells.
By the preparation method, several heat shock protein/peptide complexes with immune activation in tumor tissues can be extracted and purified in a single mode. The purified single HSP/peptide complexes obtained by the above method include HSP 70/peptide complex, HSP 90/peptide complex, Gp 96/peptide complex, etc., and then administered in a rational ratio by component quantification (e.g., Elisa experiment). The purified multi-subtype heat shock protein/peptide complex concatenated tumor vaccine of the invention has unique advantages compared with the vaccines known in the prior art. Firstly, a large number of tumor antigens are carried by HSP70, Gp96, HSP90 and the like, and a multivalent vaccine is formed by the action similar to an antigen library, so that specific immunity is effectively activated, and the body is stimulated to generate an innate immune response. By activating immune system, anti-tumor cytokines such as IL-12, IL-2, TNF-alpha, IFN-gamma and the like are generated, and corresponding anti-tumor CD4 is activated+T cells and CD8+T cells, achieving excellent antitumor effects. Secondly, the preparation process of the vaccine comprises purification by affinity immunochromatography, and immunosuppressive factors (such as TGF-beta and the like) existing in tumor tissues are effectively eliminated, so that the immune effect of the vaccine is improved to the maximum extent, and possible side effects are avoided. Thirdly, the vaccine of the invention does not need to add an adjuvant, is simple and convenient to prepare, and eliminates the possible side effect brought by the adjuvant. Fourthly, the vaccine of the invention can be used in combination with the existing antitumor drugs to further enhance the antitumor effect of the tumor vaccine.
Brief description of the drawings
FIG. 1: identification and quantitative determination of HSP 70/peptide complex, HSP 90/peptide complex, Gp 96/peptide complex in MCA207 sarcoma cells. A: immunohistochemical fluorescent staining identified stable expression of 3 heat shock protein/peptide complexes, blue fluorescence indicated cell nucleus, and red fluorescence in the three panels indicated HSP 70/peptide complex, HSP 90/peptide complex, and Gp 96/peptide complex, respectively. B: the Elisa experiment quantitatively determines the content of each of 3 heat shock protein/peptide complexes in sarcoma cells, and provides a mixing ratio of the three heat shock protein/peptide complexes for mpHSP/P preparation.
FIG. 2: protein efflux was recorded. A: the protein efflux was recorded after passing through the ion chromatography column, where the peak was present indicating protein efflux. B: protein outflow recording curve and WB identification chart obtained after passing through the affinity immunochromatographic column; by collecting each fragment from which protein was eluted and identifying it with WB, it was revealed that HSP 70/peptide complex was collected from the A47 fragment, HSP 90/peptide complex was collected from the A46 fragment, and Gp 96/peptide complex was collected from the A4 fragment.
FIG. 3: cytotoxicity assay of mpHSP/P. A: flow cytometry was used to determine the killing toxicity of mpHSP/P on cells at various concentrations. B: viability of cells at different vaccine concentrations. Saline was used as a negative control (con).
FIG. 4: comparison of mpHSP/P with a single Gp 96/peptide complex vaccine and a comparative mixed heat shock protein/peptide complex vaccine (mHSP/P, as detailed in the comparative examples below). A: tumor growth curve. B: mouse survival curves. Saline was used as a negative control (con).
FIG. 5: the concentrations of the antitumor cytokines IL-2, TNF- α, IFN- γ in the tumor tissues measured at day 14 after the injection of mphsHSP/P, a single Gp 96/peptide complex vaccine or mHSP/P. Saline was used as a negative control (con).
FIG. 6: CD4 in tumor tissues observed at day 14 after injection of mpHSP/P, single Gp 96/peptide complex vaccine or mHSP/P+T cells and CD8+Infiltration of T cells. Blue fluorescence for nuclei and red fluorescence for CD4+T cells, green fluorescence CD8+T cells. Saline was used as a negative control (con).
Examples
The benefits of the present invention are further illustrated by the following examples, it being understood that these examples are for illustrative purposes only and are in no way intended to limit the scope of the present invention. The scope of the invention is only limited by the claims.
Example 1: the preparation of the purified multi-subtype heat shock protein/peptide compound multi-linked tumor vaccine
Materials and methods
(1) Sources of materials: mouse MCA207 tumor cell 5e5 was injected into tumor tissue removed from mice after 3-4 weeks of subcutaneous culture.
(2) The preparation steps of the vaccine are as follows:
1) tumor tissues were minced, and 2-3 fold cell lysate (RIPA: cocktail ═ 100:1) was placed in the homogenizer for 4 h.
2) The cell lysate obtained above was ultracentrifuged at 25000rpm (75000g) at 4 ℃ for 2.5 hours, and the supernatant was collected.
3) And filtering the collected supernatant by a filter screen and a filter before and after, and collecting the filtered protein suspension.
4) The sample supernatant subjected to the above treatment was subjected to an ion chromatography column (buffer a: tris solution at 20mmol/l and PH 8.5, buffer B: a mixed solution of 20mmol/l Tris and 1mol/l NaCl at PH 8.5). After all the sample passed through the ion chromatography column, a protein-eluting cut was collected (FIG. 2A).
5) And (4) passing the sample obtained in the last step through a concentration test tube, and concentrating the sample.
6) The concentrated sample is passed through affinity immunochromatographic columns 1, 2 and 3 which specifically adsorb HSP70, HSP90 and Gp96, respectively, and the protein-eluted fragments are collected. The buffer solution of the affinity immunochromatography column is divided into A, B two buffer solutions, namely a buffer solution A: tris solution at 20mmol/l and PH 7.5, buffer B: 20mmol/l sodium citrate buffer at pH 3. Finally, 2 volumes of 50mmol/l Tris pH 9 were added.
8) After WB identification, the purified single HSP 70/peptide complex (A47 segment), the purified single HSP 90/peptide complex (A46 segment) and the purified single Gp 96/peptide complex (A4 segment) are mixed according to a certain proportion to obtain the purified multi-subtype heat shock protein/peptide complex multi-tumor vaccine (mpHSP/P), and the mixing proportion is the proportion of the 3 heat shock protein/peptide complexes in the tumor tissues determined by an Elisa experiment, and is described in the following.
9) Subpackaging and freeze-drying for later use.
(3) And (3) determination and identification:
1) identifying the stable expression of 3 heat shock protein/peptide complexes by immunohistochemical fluorescent staining using the material obtained after the above step 3) (FIG. 1A), blue fluorescence representing the nucleus, red colors in the three panels representing HSP 70/peptide complex, HSP 90/peptide complex, Gp 96/peptide complex, respectively; the respective contents of the 3 heat shock protein/peptide complexes in sarcoma cells were quantitatively determined using the Elisa experiment (FIG. 1B), providing a mixture ratio of the three heat shock protein/peptide complexes for mpHSP/P preparation.
2) WB identification was carried out on the protein samples obtained by the above two chromatographies, and the results of the identification demonstrated that a single HSP 70/peptide complex (A47 fragment), a single HSP-90/peptide complex (A46 fragment), and a single Gp 96/peptide complex (A4 fragment) were purified, respectively (FIG. 2B).
Comparative example: preparation of mixed heat shock protein/peptide complex vaccine
Materials and methods
(1) Sources of materials: mouse MCA207 tumor cell 5e5 was injected into tumor tissue removed from mice after 3-4 weeks of subcutaneous culture.
(2) The preparation steps of the vaccine are as follows:
1) tumor tissues were minced, and 2-3 fold cell lysate (RIPA: cocktail ═ 100:1) was placed in the homogenizer for 4 h.
2) The cell lysate obtained above was ultracentrifuged at 25000rpm (75000g) at 4 ℃ for 2.5 hours, and the supernatant was collected.
3) And filtering the collected supernatant by a filter screen and a filter before and after, and collecting the filtered protein suspension.
4) The sample supernatant subjected to the above treatment was subjected to an ion chromatography column (buffer a: tris solution at 20mmol/l and PH 8.5, buffer B: a mixed solution of 20mmol/l Tris and 1mol/l NaCl at PH 8.5). After all the samples pass through the ion chromatographic column, the protein outflow sections are collected.
5) And (4) passing the sample obtained in the last step through a concentration test tube, and concentrating the sample.
6) The concentrated sample was purified twice by molecular chromatography column (buffer Tris 20mmol/L PH 8.5), the eluted proteins of different segments were collected and subjected to SDS-PAGE to identify the nature of the protein, and then the segments containing HSP 70/peptide complex, HSP 90/peptide complex, and Gp 96/peptide complex were left to mix to obtain a comparative mixed heat shock protein/peptide complex vaccine (mHSP/P).
6) Subpackaging and freeze-drying for later use.
Example 2: biosafety test
(I) test materials
(1) Experimental group samples: mpHSP/P.
(2) Negative control group: physiological saline.
(3) Experimental cells and related equipment: l929 cell line, flow cytometer.
(II) Experimental methods and results
mpHSP/P was added to a culture dish of L929 cells at different amounts of 10ug, 20ug, 30ug, and the cell survival status was determined by flow cytometry.
The experimental results are as follows: no cytotoxicity was shown at different concentrations of mpHSP/P, thus demonstrating the biosafety (FIG. 3).
Example 3: therapeutic immune efficacy test
(I) test materials
(1) Experimental groups: mpHSP/P.
(2) Control group: physiological saline (negative control), purified Gp 96-only/peptide complex vaccine, mHSP/P.
Purified single Gp 96/peptide complex vaccine, mHSP/P and mpHSP/P were all extracted from the same tumor by the inventors.
(3) Experimental animals: c57 mice, 18-20g in weight, female, were purchased from Spbefu. MCA207 tumor cells, gifted by professor zongka, university of stanford.
(II) test methods and results
MCA207 tumor cells 5e5 were implanted in mice 3 days later and then injected with purified single Gp 96/peptide complex vaccine, mHSP/P, mpHSP/P or normal saline. The dosages of physiological saline, purified single Gp 96/peptide complex vaccine, mHSP/P and mpHSP/P are 30 ug/time/single, subcutaneous injection is carried out, and immunization is carried out for 3 times, wherein each immunization is separated from the last immunization by 7 days. Each group contained 15 mice.
The experimental results are as follows: the negative control group (saline-injected group) mice all died after 30-40 days. The mice injected with mpHSP/P did not show any adverse reactions. Compared with the injection of physiological saline, mHSP/P or purified single Gp 96/peptide complex vaccine, the injection of the mpHSP/P obviously inhibits the growth of tumors and prolongs the survival time of mice. Therefore, the therapeutic immune effect of the vaccine of the invention is obviously better than that of the control group, and the vaccine has stronger anti-tumor capability (figure 4).
Example 4: molecular and cytological assays for stimulating anti-tumor activity
(I) test materials
(1) Experimental groups: mpHSP/P.
(2) Control group: physiological saline (negative control), purified Gp 96-only/peptide complex vaccine, mHSP/P.
Purified single Gp 96/peptide complex vaccine, mHSP/P and mpHSP/P were all extracted from the same tumor by the inventors.
(3) Experimental animals: c57 mice, 18-20g in weight, female, were purchased from Spbefu. MCA207 tumor cells, gifted by professor zongka, university of stanford.
(4) Related reagents and equipment for molecular detection: enzyme-linked immunosorbent assay (ELISA), an ELISA kit for anti-tumor cytokines (IFN-gamma, TNF-alpha and IL-2), a fluorescence microscope, and CD4 and CD8 antibodies.
(II) test methods and results
MCA207 tumor cells 5e5 were implanted in mice 3 days later and then injected with purified single Gp 96/peptide complex vaccine, mHSP/P, mpHSP/P or normal saline. The immunization was performed 3 times, each 7 days apart from the last immunization, using subcutaneous injections in the amounts described in example 4. After 2 weeks of immunization, mice were tumor lysed, centrifuged, filtered and tested for anti-tumor in different groups by ELISAThe content of cytokines (IFN-. gamma., TNF-. alpha., IL-2). Mice were sacrificed 3-4 weeks after immunization and tumor tissue blocks of mice were made CD4+/CD8+T cell staining, and comparing the anti-tumor effect of the immune system after 2 weeks in the control and experimental groups.
Comparing the experimental group with the control group, it was found that: IFN-gamma, TNF-alpha, IL-2 antitumor cytokines were increased in the mpHSP/P group compared to the other groups (FIG. 5), and anti-tumor CD4 invaded in tumor tissues+/CD8+T cells were increased (fig. 6).
Claims (8)
1. A method for preparing a purified multi-subtype heat shock protein/peptide complex concatenated tumor vaccine, which comprises:
(1) providing a tumor cell lysate;
(2) centrifuging the tumor cell lysate, and collecting a supernatant;
(3) filtering the supernatant, and then passing through an ion chromatography column to collect a protein effluent;
(4) optionally, passing the protein effluent through a molecular chromatography column;
(5) purifying the product obtained in step (3) or optional step (4) by affinity immunochromatographic columns 1, 2, 3, wherein the affinity immunochromatographic columns 1, 2, 3 specifically adsorb different subtypes of single HSP respectively, and respectively obtain a purified single HSP/peptide complex 1, a purified single HSP/peptide complex 2 and a purified single HSP/peptide complex 3;
(6) mixing the purified single HSP/peptide complex 1, the purified single HSP/peptide complex 2 and the purified single HSP/peptide complex 3 in a proper ratio to obtain the purified multi-subtype heat shock protein/peptide complex multi-combination tumor vaccine.
2. The method of claim 1, wherein said purified single HSP/peptide complex 1, said purified single HSP/peptide complex 2 and said purified single HSP/peptide complex 3 are mixed in the ratio of single HSP/peptide complex 1, single HSP/peptide complex 2 and single HSP/peptide complex 3 in the originating tumor tissue.
3. The method of claim 1, wherein the purified single HSP/peptide complex 1 is any one of HSP 70/peptide complex, HSP 90/peptide complex, Gp 96/peptide complex; the purified single HSP/peptide complex 2 is any one of HSP 70/peptide complex, HSP 90/peptide complex, Gp 96/peptide complex different from the purified single HSP/peptide complex 1; the purified single HSP/peptide complex 3 is any one of HSP 70/peptide complex, HSP 90/peptide complex, Gp 96/peptide complex different from the purified single HSP/peptide complex 1 and the purified single HSP/peptide complex 2.
4. The method of any one of claims 1-3, wherein the tumor cell lysate is derived from bone tumor, synovial tumor, fibrous tumor, giant cell tumor, Ewing's tumor, or established tumor cells.
5. The purified multi-subtype heat shock protein/peptide complex concatenated 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 concatemeric tumor vaccine of claim 5 in the preparation of a medicament for inhibiting tumor growth.
7. Use of the purified multi-subtype heat shock protein/peptide complex concatemeric tumor vaccine of claim 5 in the preparation of a medicament for activating cytokines IL-2, TNF-a, IFN- γ.
8. The use of the purified multi-subtype heat shock protein/peptide complex concatemeric tumor vaccine of claim 5 in preparing a vaccine for increasing CD4+/CD8+Use in the manufacture of a medicament for T cells.
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| CN112111016A (en) * | 2020-09-24 | 2020-12-22 | 和泓尚医(成都)生物科技有限公司 | Method for extracting and purifying Gp 96-polypeptide compound (CTL-OT) |
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