Method for extracting calreticulin from tissues and application of calreticulin

Abstract

The invention discloses a method for extracting calreticulin from tissues and application. The tissue cell extract extracted by the method contains calreticulin and can be used for preparing drugs or vaccines for preventing cancers. Experimental results show that after the tissue cell extract is immunized for several weeks, specific immune response of an organism can be triggered, cancerous cells in the organism can be removed, and therefore the aim of preventing cancers is achieved. The tissue cell extract provided by the invention can be used as a tumor preventive vaccine for preventing autologous or allogenic tumors and reducing the incidence rate of cancers in healthy people.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a method for extracting calreticulin from tissue and its application. Background Art

[0002] Calreticulin (CALR) is a multifunctional protein of the endoplasmic reticulum (ER) that binds to calcium and facilitates the folding and processing of secretory and membrane proteins. It plays a key role in a variety of cellular processes, including calcium homeostasis, protein folding, and quality control within the ER. In addition, calreticulin has been implicated in immune responses and is thought to be involved in cellular processes such as apoptosis (programmed cell death) and the clearance of apoptotic cells. When unfolded proteins accumulate within the ER, calreticulin can maintain intracellular homeostasis. Furthermore, calreticulin mechanistically promotes the initiation of adaptive anti-cancer immunity in the context of immunogenic cell death (ICD). ICD is a functional variant of controlled cell death (RCD) that triggers antigen-specific immune responses in immunocompetent allogeneic hosts. CALR exposed on the surface of tumor cells undergoing ICD mediates a powerful pro-phagocytic effect, thereby promoting antigen uptake by antigen-presenting cells (APCs), particularly immature dendritic cells (DCs), which migrate to lymph nodes to cross-present tumor-specific antigens to naive CD8+ T cells.

[0003] Calreticulin is a component of the peptide loading complex (PLC). The peptide loading complex is a transient multicomponent complex that aggregates at the membrane of the endoplasmic reticulum and ensures the correct loading of cellular antigens onto MHC class I molecules. In addition to calreticulin, the PLC also involves PDIA3, TAP binding protein (TAPBP, also known as tapasin), transporter 1, ATP-binding cassette subfamily B member 1 (TAP1), and transporter 2, ATP-binding cassette subfamily B member 2 (TAP2). The peptide loading complex collectively mediates: (1) assembly of MHC class I heavy chains with β2 microglobulin (B2M); (2) ATP-dependent transport of cytosolic peptides into the endoplasmic reticulum compartment; (3) loading of these peptides onto the antigen-binding pocket of MHC class I molecules; and (4) release of loaded MHC class I for anterograde endoplasmic reticulum-to-Golgi transport and exposure on the plasma membrane. Calreticulin also participates in peptide loading by binding to MHC class I heavy chains prior to their stable interaction with B2M. Specifically, CALR influences PLC function through at least two distinct mechanisms. First, by interacting with PDIA3 in a glycosylation-dependent manner, CALR maintains steady-state levels of TAPBP and MHC class I heavy chains. Second, CALR can retrieve suboptimally assembled MHC class I molecules from the post-ER compartment. Consequently, both mice and human cells lacking CALR exhibit a severe reduction (50%–80%) in peptide loading and exposure on MHC class I molecules, a defect that cannot be rescued by reexpression of CALR variants lacking the C-terminus. Cancer-associated CALR mutants are unable to support PLC activity and are associated with reduced antigen presentation on MHC class I molecules, thereby favoring immune evasion. Even in the absence of oncogenic mutations, CALR levels are reduced in a variety of advanced solid tumors, often associated with reduced MHC class I on the cell surface and poor disease outcome.

[0004] According to current molecular biological and immunological theories, tumorigenesis involves two major and highly interrelated components: (1) the accumulation of genetic and / or epigenetic defects that confer upon (originally) normal cells enhanced proliferative potential, resistance to cell death, the ability to induce neoangiogenesis, and the ability to disseminate and form metastases, and (2) escape from local and systemic control of the host immune system (immune evasion). Thus, malignant transformation (the process by which a healthy cell becomes a tumor cell precursor) and tumor progression (the proliferation of such precursors accompanied by the acquisition of increasingly malignant characteristics), as well as the response of established tumors to therapy, all occur in the context of a bidirectional interaction between (pre-)malignant cells and their host, ultimately determining clinical outcome. In this context, alterations that simultaneously confer intrinsic proliferative advantages on cancer cells and limit the ability of the immune system to recognize and eliminate them are powerful drivers of tumorigenesis and targets for tumor prevention / therapy. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for extracting calreticulin from tissue.

[0006] The method for extracting calreticulin from tissue provided by the present invention comprises the following steps:

[0007] (1) Centrifuge the tissue homogenate or cell suspension and collect the supernatant;

[0008] (2) adding saturated ammonium sulfate to the supernatant obtained in step (1) to 10% saturation, stirring at 0-8°C for 2-4 hours, then centrifuging at 4°C, 12,000 rpm for 20 minutes, discarding the precipitate, and collecting the supernatant;

[0009] (3) Saturated ammonium sulfate was added to the supernatant until the saturation reached 40%, and the mixture was stirred at 4°C overnight. The mixture was then centrifuged at 12,000 rpm for 20 min at 4°C, and the supernatant was discarded to collect the precipitate.

[0010] (4) Resuspend the precipitate obtained in step (3) with 20 mM Tris-HCl buffer (pH 7.0), centrifuge at 12,000 rpm for 20 min at 4°C to obtain the supernatant;

[0011] (5) The supernatant obtained in step (4) was subjected to Capto Q or POROS XS anion exchange column chromatography, with gradient elution using 30% B solution for 5 column volumes, followed by gradient elution using 60% B solution for 5 column volumes, and the eluate was collected;

[0012] (6) The eluate obtained in step (5) was concentrated to a molecular weight cut-off of 50 kD and exchanged into 20 mM PB buffer;

[0013] (7) The solution obtained in step (6) was subjected to GE Sepharose SP or POROS 50HS (cation exchange column chromatography) chromatography to harvest the flow-through sample;

[0014] (8) The flow-through sample obtained in step (7) was concentrated with a molecular weight cut-off of 50 kD and exchanged into 50 mM PB buffer;

[0015] (9) The concentrated solution obtained in step (8) was loaded onto a Superdex 20010 / 300GL molecular sieve chromatography column (flow rate of 0.25 ml / min), and then washed with pH 7.5, 150 mM PBS buffer (flow rate of 0.25 ml / min). The flowthrough at 10 to 13 ml was collected and further concentrated by ultrafiltration using an ultrafiltration tube with a molecular weight cutoff of 50 kD to obtain a solution whose main component was calreticulin.

[0016] The above method further comprises the following steps after step (9): determining the protein concentration in the obtained solution, packaging, and storing at -80°C. Specifically, the protein concentration can be determined by the BCA method;

[0017] The obtained solution (tissue cell extract) was identified by electrophoresis and mass spectrometry to contain calreticulin.

[0018] In step (1) of the above method, the tissue may be placenta, liver, etc., and the cell source may be embryonic stem cells, umbilical cord blood, etc.;

[0019] The centrifugation operation is as follows: centrifuging the tissue homogenate or cell suspension at 17000 g for 1 h, and collecting the supernatant;

[0020] In step (5), the Capto Q is a Cytiva product;

[0021] In step (5), solution B is 20 mM Tris / 1 M NaCl, pH 7.0, wherein the 30% solution B contains 300 mM NaCl; the 60% solution B contains 600 mM NaCl;

[0022] In step (6), the 20 mM PB buffer is 20 mM Na2HPO4·12H2O / NaH2PO4·2H2O, pH 5.5;

[0023] In step (8), the 50 mM PB buffer is 50 mM Na2HPO4·12H2O / NaH2PO4·2H2O, pH 5.5;

[0024] In step (9), the Superdex 20010 / 300GL molecular sieve chromatography column is a product of GE, with a product catalog number of 17517501.

[0025] The obtained solution is subjected to electrophoresis and Coomassie Brilliant Blue staining for identification: the electrophoresis can be performed using SDS-PAGE, such as conventional 15% concentration protein gel electrophoresis.

[0026] The tissue cell extract prepared by the above method also falls within the scope of protection of the present invention. The tissue cell extract contains calreticulin.

[0027] The use of the tissue cell extract prepared by the above method in the preparation of drugs (or vaccines) for preventing cancer also falls within the scope of protection of the present invention.

[0028] The cancer includes colon cancer (such as colon cancer caused by MC38 mouse colon cancer cells) and melanoma (such as melanoma caused by B16 melanoma cells).

[0029] The drug (or vaccine) can specifically inhibit the occurrence and / or growth of colon cancer and melanoma.

[0030] The present invention also protects a drug or vaccine for preventing cancer, the active ingredient of which is the tissue cell extract (containing calreticulin).

[0031] The cancer includes colon cancer (such as colon cancer caused by MC38 mouse colon cancer cells) and melanoma (such as melanoma caused by B16 melanoma cells).

[0032] The drug (or vaccine) can specifically inhibit the occurrence and / or growth of colon cancer and melanoma.

[0033] When the tissue cell extract of the present invention is used as a vaccine or medicine, HCD, HCP, endotoxin detection, sterility detection, and concentration determination can be performed when the tissue cell extract is prepared, and then the extract can be packaged and directly frozen or freeze-dried for storage.

[0034] The tissue cell extract of the present invention is used as a vaccine or medicine. For the immunized object, the immunization dose is not less than 100 μg each time, and not less than 3 times in total, and the immunization is performed by intramuscular injection or subcutaneous injection.

[0035] After several weeks of immunization, the tissue cell extract of the present invention can trigger a specific immune response in the body, eliminating cancerous cells in the body, thereby achieving the purpose of cancer prevention. The tissue cell extract of the present invention can be used as a tumor preventive vaccine for the prevention of autologous or allogeneic tumors, reducing the incidence of cancer in healthy people. The tissue cell extract of the present invention can also be used for the immune prevention and treatment of allogeneic tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The results of SDS-PAGE analysis of tissue extracts are shown. Lanes: from left to right: Page-ruler protein marker, mouse placenta extract 0.1 μg, 1 μg, 2 μg, 10 μg. DETAILED DESCRIPTION

[0037] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0038] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0039] The quantitative tests in the following examples were all repeated three times, and the results were averaged. PMSF is phenylmethylsulfonyl fluoride, with a molecular formula of C7H7FO2S.

[0040] C57BL / 6 mice: 6-8 weeks old, purchased from Beijing Weitonglihua Company, strain C57BL / 6J.

[0041] MC38 mouse colon cancer cells were purchased from the National Laboratory Cell Resource Sharing Platform with the product number 1101MOU-PUMC000523.

[0042] B16 mouse melanoma cells were purchased from the National Laboratory Cell Resource Sharing Platform with the product number 1101MOU-PUMC000215.

[0043] The PBS solution used in the examples has a pH of 7.4 and has the following formula: 8 g of NaCl, 0.2 g of KCl, 3.63 g of Na2HPO4·12H2O, and 0.24 g of KH2PO4 were weighed and dissolved in 900 ml of double-distilled water. The pH was adjusted to 7.4 with hydrochloric acid, and the volume was made up to 1 L with water. The solution was stored at room temperature until use.

[0044] Example 1. Preparation of tissue cell extracts

[0045] Tissue and cell extracts were prepared from the placenta, liver, mouse umbilical cord blood, and embryonic stem cells of female C57BL / 6 mice, respectively. The preparation methods are as follows:

[0046] (1) For placenta and liver tissues: collect the tissues, mince them, grind them in 25 mM NaHCO3 solution with a pH of 7.4 at a mass (g) to volume (ml) ratio of 1:6, and then prepare the tissue homogenate using a tissue homogenizer.

[0047] For cord blood and embryonic stem cells: Prepare a cell suspension by adding 25 mM NaHCO3 solution, pH 7.4, at a volume (ml)-volume (ml) ratio of 1:5.

[0048] The tissue homogenate or cell suspension was centrifuged (17000g for 1 h) and the supernatant was collected;

[0049] (2) Saturated ammonium sulfate was slowly added to the supernatant obtained in step (1) until the saturation reached 10%, and the mixture was slowly stirred at 4°C for 3 h, and then centrifuged at 4°C and 12,000 rpm for 20 min, the precipitate was discarded, and the supernatant was collected;

[0050] (3) Slowly add saturated ammonium sulfate to the supernatant until the saturation reaches 40%, slowly stir at 4°C overnight, then centrifuge at 4°C, 12,000 rpm for 20 min, discard the supernatant, and collect the precipitate;

[0051] (4) Resuspend the precipitate obtained in step (3) with 50 ml of 20 mM Tris-HCl buffer, pH 7.0, and centrifuge at 12,000 rpm for 20 min at 4°C to obtain the supernatant;

[0052] (5) The supernatant obtained in step (4) was subjected to CaptoQ anion exchange column chromatography, and gradient elution was performed with 30% B solution (ion exchange column chromatography, the buffer system of the sample was pH 7.0, 20 mM Tris-HCl, B solution was 20 mM Tris / 1 M NaCl, pH 7.0, wherein the 30% B solution contained 300 mM NaCl), eluted for 5 column volumes, and then gradient eluted with 60% B solution (B solution was 20 mM Tris / 1 M NaCl, pH 7.0, wherein the 60% B solution contained 600 mM NaCl), eluted for 5 column volumes, and the eluate was collected;

[0053] (6) The eluate obtained in step (5) was concentrated to a molecular weight cut-off of 50 kD and exchanged into 20 mM PB buffer (i.e., 20 mM Na2HPO4·12H2O / NaH2PO4·2H2O, pH 5.5);

[0054] (7) subjecting the solution obtained in step (6) to GE Sepharose SP chromatography to harvest a flow-through sample;

[0055] (8) The flow-through sample obtained in step (7) was concentrated with a molecular weight cut-off of 50 kD and exchanged into 50 mM PB buffer (i.e., 50 mM Na2HPO4·12H2O / NaH2PO4·2H2O, pH 5.5);

[0056] (9) The concentrated solution obtained in step (8) was loaded onto a Superdex 20010 / 300GL molecular sieve chromatography column (flow rate of 0.25 ml / min), and then washed with pH 7.5, 150 mM PBS buffer (flow rate of 0.25 ml / min). The flowthrough at 10 to 13 ml was collected and further concentrated by ultrafiltration using an ultrafiltration tube with a molecular weight cutoff of 50 kD to obtain a solution whose main component was calreticulin.

[0057] The protein concentration in the solution was determined by BCA method, and the solution was finally packaged and stored at -80°C.

[0058] The tissue extracts (placenta, liver, mouse umbilical cord blood, embryonic stem cell extracts) were subjected to conventional 15% SDS-PAGE protein gel electrophoresis and mass spectrometry identification. Figure 1The extract was identified by mass spectrometry as the main component of calreticulin, with 258 effective peptides. The results showed that the main component was calreticulin.

[0059] The purified extracts of placenta, liver, mouse umbilical cord blood, and embryonic stem cells were diluted to 1 mg / ml with PBS solution, and the experiments of Example 2 and Example 3 were carried out.

[0060] Example 2: Preventive Effects of Tissue Extracts on Colon Cancer

[0061] C57BL / 6 mice were divided into five groups, 10 mice in each group, and treated as follows:

[0062] Group 1: Placenta-derived calreticulin (diluent) was injected subcutaneously in the abdomen for three immunizations (0.5 ml each time). The single immunization dose of placenta-derived calreticulin was 50 μg / mouse.

[0063] Group 2: Liver-extracted calreticulin (diluent) was injected subcutaneously in the abdomen for three immunizations (0.5 ml each time). The single immunization dose of liver-extracted calreticulin was 50 μg / mouse.

[0064] Group 3: Calreticulin (diluent) extracted from umbilical cord blood of mice was injected subcutaneously in the abdomen for three immunizations (0.5 ml each time). The single immunization dose of calreticulin extracted from umbilical cord blood was 50 μg / mouse.

[0065] Group 4: Calreticulin (diluent) extracted from mouse embryonic stem cells was injected subcutaneously in the abdomen for three immunizations (0.5 ml each time). The single immunization dose of calreticulin extracted from embryonic stem cells was 50 μg / mouse.

[0066] Group 5: 0.5 ml PBS solution was injected subcutaneously into the abdomen for three times.

[0067] In the five groups above, the first immunization was performed on the first day of the experiment; the second immunization was performed on the 7th day; and the third immunization was performed on the 14th day. On the 21st day, each group was subcutaneously inoculated with MC38 colon cancer cells (5×10 5 cells), and the days were counted from the inoculation of colon cancer cells. The mice were killed on the 28th day, the tumors were removed and weighed, and the tumor incidence was calculated.

[0068] The statistical results of tumor incidence (the proportion of mice with tumor weight greater than 0.2 g) and tumor weight 28 days after inoculation of tumor cells are shown in Table 1.

[0069] Table 1 Tumor incidence and tumor weight (g) on ​​day 28 after subcutaneous inoculation

[0070]

[0071]

[0072] The results showed that calreticulin extracted from various tissues had a certain degree of effect in preventing tumor occurrence.

[0073] Example 3: Preventive Effects of Tissue Extracts on Melanoma

[0074] C57BL / 6 mice were divided into six groups, 10 mice in each group, and treated as follows:

[0075] Group 1: Placenta-derived calreticulin (diluent) was injected subcutaneously in the abdomen for three immunizations (0.5 ml each time). The single immunization dose of placenta-derived calreticulin was 50 μg / mouse.

[0076] Group 2: Liver-extracted calreticulin (diluent) was injected subcutaneously in the abdomen for three immunizations (0.5 ml each time). The single immunization dose of liver-extracted calreticulin was 50 μg / mouse.

[0077] Group 3: Calreticulin (diluent) extracted from umbilical cord blood of mice was injected subcutaneously in the abdomen for three immunizations (0.5 ml each time). The single immunization dose of calreticulin extracted from umbilical cord blood was 50 μg / mouse.

[0078] Group 4: Calreticulin (diluent) extracted from mouse embryonic stem cells was injected subcutaneously in the abdomen for three immunizations (0.5 ml each time). The single immunization dose of calreticulin extracted from embryonic stem cells was 50 μg / mouse.

[0079] Group 5: 0.5 ml PBS solution was injected subcutaneously into the abdomen for three times.

[0080] In the six groups above, the first immunization was performed on the first day of the experiment; the second immunization was performed on the 7th day; and the third immunization was performed on the 14th day. On the 21st day, each group was subcutaneously inoculated with B16 melanoma cells (5×10 5 cells), and the number of days was counted from the inoculation of melanoma cells. Mice were killed on the 28th day, the tumors were removed and weighed, and the tumor incidence was calculated.

[0081] The statistical results of tumor incidence (the proportion of mice with tumor weight greater than 0.2 g) and tumor weight 28 days after inoculation of tumor cells are shown in Table 2.

[0082] Table 2 Melanoma incidence and tumor weight (g) on ​​day 28 after subcutaneous inoculation

[0083]

[0084]

[0085] The results showed that calreticulin extracted from various tissues had a certain degree of effect in preventing tumor occurrence.

[0086] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A method for extracting calreticulin from tissue, comprising the following steps: (1) Centrifuge the tissue homogenate or cell suspension and collect the supernatant; (2) adding saturated ammonium sulfate to the supernatant obtained in step (1) to 10% saturation, stirring at 0-8°C for 2-4 hours, then centrifuging at 4°C, 12,000 rpm for 20 minutes, discarding the precipitate, and collecting the supernatant; (3) Saturated ammonium sulfate was added to the supernatant until the saturation reached 40%, and the mixture was stirred at 4°C overnight. The mixture was then centrifuged at 12,000 rpm for 20 min at 4°C, and the supernatant was discarded to collect the precipitate. (4) Resuspend the precipitate obtained in step (3) with 20 mM Tris-HCl buffer (pH 7.0), centrifuge at 12,000 rpm for 20 min at 4°C, and obtain the supernatant; (5) The supernatant obtained in step (4) was subjected to Capto Q or POROS XS anion exchange column chromatography, with gradient elution using 30% B solution for 5 column volumes, followed by gradient elution using 60% B solution in Tris solution for 5 column volumes, and the eluate was collected; (6) The eluate obtained in step (5) was concentrated to a molecular weight cut-off of 50 kD and exchanged into 20 mM PB buffer; (7) subjecting the solution obtained in step (6) to GE Sepharose SP or POROS 50HS chromatography to harvest the flow-through sample; (8) The flow-through sample obtained in step (7) was concentrated with a molecular weight cut-off of 50 kD and exchanged into 50 mM PB buffer; (9) The concentrated solution obtained in step (8) was loaded onto a Superdex 20010 / 300GL molecular sieve chromatography column, and then washed with a pH 7.5, 150 mM PBS buffer solution, and the flowthrough at 10 to 13 ml was collected. The solution was further concentrated by ultrafiltration using an ultrafiltration tube with a molecular weight cutoff of 50 kD to obtain a solution whose main component was calreticulin, i.e., a tissue cell extract.

2. The method according to claim 1, characterized in that In step (1), the tissue is placenta or liver, and the cell source is embryonic stem cells or umbilical cord blood; The centrifugation operation is as follows: centrifuging the tissue homogenate or cell suspension at 17000 g for 1 hour and collecting the supernatant.

3. The method according to claim 1, characterized in that In step (5), solution B is 20 mM Tris / 1 M NaCl, pH 7.0, wherein 30% solution B contains 300 mM NaCl; and 60% solution B contains 600 mM NaCl.

4. The method according to claim 1, wherein In step (6), the 20 mM PB buffer is 20 mM Na2HPO4·12H2O / NaH2PO4·2H2O, pH 5.5; In step (8), the 50 mM PB buffer is 50 mM Na2HPO4·12H2O / NaH2PO4·2H2O, pH 5.

5.

5. The method according to claim 1, wherein The concentrate was loaded onto a Superdex 20010 / 300GL molecular sieve chromatography column at a flow rate of 0.25 ml / min; the flow rate of washing with PBS buffer was 0.25 ml / min.

6. A tissue cell extract prepared by the method according to any one of claims 1 to 5, wherein the tissue cell extract contains calreticulin.

7. Use of the tissue cell extract according to claim 6 in the preparation of a drug or vaccine for preventing cancer.

8. The use according to claim 7, characterized in that The cancers include colon cancer and melanoma.

9. The use according to claim 7 or 8, characterized in that The drug or vaccine inhibits the occurrence and / or growth of colon cancer and melanoma.

10. A drug or vaccine for preventing cancer, comprising the tissue cell extract according to claim 6 and a pharmaceutically acceptable carrier.

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