In-situ tumor vaccine and application thereof

Abstract

The invention provides an in-situ tumor vaccine and application thereof, and belongs to the technical field of vaccines. The in-situ tumor vaccine provided by the invention comprises at least one of Ag85B protein and TTC protein, the amino acid sequence of the Ag85B protein is as shown in SEQ ID NO.1, and the amino acid sequence of the TTC protein is as shown in SEQ ID NO.2. The in-situ tumor vaccine provided by the invention has the advantages that the in-situ tumor vaccine can be used for in-situ tumor treatment; the in-situ tumor vaccine provided by the invention has a remarkable anti-lung cancer effect, when the Ag85B protein and the TTC protein are jointly applied, the lung cancer tumor is obviously reduced by naked eyes, and the volume of the tumor of the combined application group is statistically different from that of a control group.

Description

Technical Field

[0001] The invention belongs to the technical field of vaccines, and in particular relates to an in situ tumor vaccine and application thereof. Background Art

[0002] Lung cancer, also known as primary bronchial lung cancer, is the most common lung malignancy originating from the trachea, bronchial mucosa or glands. According to the histopathological characteristics, there are mainly non-small cell carcinoma (including adenocarcinoma and squamous cell carcinoma) and small cell carcinoma, among which the incidence of non-small cell lung cancer accounts for a relatively high proportion. Conventional cancer vaccines have not been successful in clinical research on non-small cell lung cancer tumor vaccines. Traditional cancer treatments such as surgery, radiotherapy and drug therapy have certain limitations. Due to poor targeting, radiotherapy and drug therapy are prone to damage normal cells and produce adverse reactions. Malignant tumors have biological characteristics of easy invasion and recurrence, so better targeting and less toxic treatment plans are needed. Tumor vaccine is one of the hot topics in recent years. Its principle is to introduce tumor antigens into the patient in various forms such as tumor cells, tumor-related proteins or peptides, genes expressing tumor antigens, etc., to overcome the immunosuppressive state caused by the tumor, enhance immunogenicity, activate the patient's own immune system, and induce the body's cellular and humoral immune responses, thereby achieving the purpose of controlling or eliminating the tumor.

[0003] Tetanus toxin is a strong neurotoxin produced by Clostridium tetani in an anaerobic environment. According to the role of tetanus toxin in the body, it is divided into three parts: A, B, and C. Ag85B is a protein secreted by Mycobacterium tuberculosis (MTB). At present, there is no research in the field on the use of Ag85B protein and tetanus toxin C fragment (TetC, TTC) as in situ tumor vaccines. Summary of the invention

[0004] In view of this, the object of the present invention is to provide an in situ tumor vaccine with significant anti-lung cancer effect.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides an in situ tumor vaccine, which comprises at least one of an Ag85B protein and a TTC protein, wherein the amino acid sequence of the Ag85B protein is shown in SEQ ID NO.1, and the amino acid sequence of the TTC protein is shown in SEQ ID NO.2.

[0007] Preferably, the concentration of the in situ tumor vaccine is 0.2-0.4 μg / μL.

[0008] Preferably, when the in situ tumor vaccine comprises Ag85B protein and TTC protein, the mass ratio of the Ag85B protein to the TTC protein is 1:1.

[0009] The present invention also provides the use of the in situ tumor vaccine in the preparation of a product for treating cancer.

[0010] Preferably, the cancer comprises lung cancer.

[0011] Preferably, the product comprises a medicine.

[0012] The present invention also provides the use of Ag85B protein combined with TTC protein in the preparation of in situ tumor vaccines, wherein the amino acid sequence of the Ag85B protein is shown in SEQ ID NO.1, and the amino acid sequence of the TTC protein is shown in SEQ ID NO.2.

[0013] Preferably, the tumor comprises lung cancer.

[0014] Beneficial effects of the present invention:

[0015] The in situ tumor vaccine provided by the present invention has the following advantages over conventional vaccines: simple and low cost; small systemic side effects; can utilize all tumor antigens in the tumor, thereby avoiding the need to identify antigens; because all antigens in the tumor are utilized, immune escape is minimized; and can be combined with other therapies to exert a synergistic effect.

[0016] The in situ tumor vaccine provided by the present invention has a significant anti-lung cancer effect. When Ag85B protein and TTC protein are used in combination, the lung cancer tumor is significantly reduced as can be seen with the naked eye, and there is a statistical difference in the volume of the tumor in the combined application group compared with that in the control group. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The results of SDS-PAGE test, the left picture shows the result of Ag85B recombinant protein, and the right picture shows the result of TTC recombinant protein;

[0018] Figure 2 Results of in situ vaccine immunization tumor-bearing mouse models in different groups. The left picture shows the tumor size, and the right picture shows the statistical results of tumor weight, where * indicates p < 0.05, and ** indicates p < 0.01;

[0019] Figure 3 The results of Example 3 are shown in the upper row. The results of CTL cell flow cytometry and CD8 + T cell statistical results, the next row NK cell flow cytometry results and NK cell statistical results of different groups, where ns means no significant difference, * means p < 0.05, ** means p < 0.01, *** means p < 0.001;

[0020] Figure 4 The test results of Example 4, from left to right are the flow cytometry test results of the Ag85B+TTC group, Ag85B group, TTC group, and PBS group, as well as the statistical results of Treg cells in different groups, where ns means no significant difference, and **** means p<0.0001. DETAILED DESCRIPTION

[0021] The present invention provides an in situ tumor vaccine, which comprises at least one of an Ag85B protein and a TTC protein, wherein the amino acid sequence of the Ag85B protein is shown in SEQ ID NO.1, and the amino acid sequence of the TTC protein is shown in SEQ ID NO.2.

[0022] In the present invention, Ag85B protein and TTC protein can be artificially synthesized by a biological company, or prepared and expressed by a prokaryotic expression system. The specific method is: according to the amino acid sequence of Ag85B protein and TTC protein, they are transferred into Escherichia coli through a plasmid vector for culture and induction, the bacteria are broken to collect the protein and highly purified to obtain recombinant Ag85B protein and recombinant TTC protein.

[0023] In the present invention, the concentration of the in situ tumor vaccine is preferably 0.2-0.4 μg / μL, more preferably 0.25-0.35 μg / μL. When the in situ tumor vaccine comprises Ag85B protein and TTC protein, the mass ratio of Ag85B protein to TTC protein is preferably 1:1.

[0024] The present invention also provides the use of the above-mentioned in situ tumor vaccine in the preparation of a product for treating cancer. In the present invention, the cancer preferably includes lung cancer; the product preferably includes a drug, and the present invention has no particular limitation on the specific dosage form of the drug.

[0025] The present invention also provides the use of Ag85B protein combined with TTC protein in preparing an in situ tumor vaccine, wherein the amino acid sequence of the Ag85B protein is shown in SEQ ID NO. 1, and the amino acid sequence of the TTC protein is shown in SEQ ID NO. 2. In the present invention, the type of tumor preferably includes lung cancer.

[0026] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0027] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0028] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0029] Example 1

[0030] Synthesis and preparation of Ag85B recombinant protein and TTC recombinant protein

[0031] 1. Clone the gene sequence encoding Ag85B or TTC protein into pET-30a plasmid to obtain the respective recombinant vectors;

[0032] 2. Protein expression:

[0033] Transformation: The pET-30a recombinant vector was transformed into BL21 (DE3) competent cells, and then coated on LB solid plates containing kanamycin at a final concentration of 30 μg / mL and cultured;

[0034] Activation: Pick a single clone and culture it in liquid culture medium containing kanamycin at a final concentration of 30 μg / mL;

[0035] Induction: When the OD value reached 0.6, add the inducer 0.5mM IPTG and continue to culture, culture overnight at 20℃ and culture for 6h at 37℃, respectively. The negative control was without the addition of inducer.

[0036] Collect bacteria: centrifuge, discard the supernatant, and collect the bacteria;

[0037] Expression detection: Buffer A (PBS, pH 7.4) was added to the collected bacteria for suspension, and the cells were fully dissolved using an ultrasonic disruptor and centrifuged. The precipitate after centrifugation was dissolved using buffer B (8 M Urea, 50 mM Tris-HCl, 300 mM NaCl, pH 8.0), and the supernatant and precipitate were treated separately, and samples were prepared for SDS-PAGE detection;

[0038] Large-scale expression: The bacterial liquid was cultured in a medium containing kanamycin at a final concentration of 30 μg / mL. When the OD value reached 0.6, 0.5 mM inducer IPTG was added. The culture was carried out overnight at 37°C for large-scale expression, and the bacterial cells were collected by centrifugation.

[0039] 3. Protein purification:

[0040] Collect crude protein: The bacterial cells were dissolved with buffer C (50 mM Tris, 300 mM NaCl, 0.2 mM PMSF, 0.1% Triton X-100, pH 8.0), ultrasonically disrupted, and the crude protein in the supernatant was collected by centrifugation;

[0041] Balance: Take 5 ml Ni-NTA and wash the column with 5 times the column volume of Binding buffer at a flow rate of 5 ml / min;

[0042] Incubation: Incubate the crude protein and the equilibrated column packing for 1 h;

[0043] Loading onto column: Load the incubated product onto the column and collect the outflow;

[0044] Equilibration: Wash and equilibrate the column with Binding buffer D (50 mM Tris, 300 mM NaCl, pH 8.0);

[0045] Washing: Wash the column with Washing buffer E (50 mM Tris, 300 mM NaCl, 20 / 50 mM Imidazole, pH 8.0) and collect the flow-through;

[0046] Elution: Elution with Elutionbuffer F (50 mM Tris, 300 mM NaCl, 500 mM Imidazole, pH 8.0) and collect the effluent;

[0047] Purification detection: The crude protein, washing effluent and elution effluent were processed separately, and samples were prepared for SDS-PAGE detection.

[0048] 4. Osmotic concentration:

[0049] For Ag85B recombinant protein: the purest fraction was dialyzed into 50 mM Tris, 300 mM NaCl, pH 8.0. After the infiltration, it was concentrated with PEG20000, filtered through a 0.45 μm filter membrane, and then packaged and stored at -80°C.

[0050] For TTC recombinant protein: the purest fraction was dialyzed into 50 mM Tris, 300 mM NaCl, 0.1% sarkosyl, 2 mM DTT, pH 8.0. After the infiltration, it was concentrated with PEG20000, filtered through a 0.45 μm filter membrane, and packaged and stored at -80°C.

[0051] The Ag85B recombinant protein and TTC recombinant protein obtained after osmotic concentration were subjected to SDS-PAGE detection. The results are as follows. Figure 1 As shown, it shows that the present invention successfully expressed the Ag85B recombinant protein and the TTC recombinant protein using the prokaryotic system. The amino acid sequence of the Ag85B recombinant protein with a His tag was sequenced as shown in SEQ ID NO.3, and the amino acid sequence of the TTC recombinant protein with a His tag was shown in SEQ ID NO.4.

[0052] Example 2

[0053] Example 1 Experiment on preparing Ag85B protein and TTC protein as in situ tumor vaccine to immunize tumor-bearing mouse model

[0054] One week before mice were immunized with tumors, 10 μg Ag85B and 10 μg TTC recombinant proteins were diluted to 25 μL with double distilled water, and then mixed with AddaVax at a volume ratio of 1:1. 25 μL was injected into the left and right upper limbs of the mice for pre-immunization. One week after pre-immunization, 1×10 6 Lewis lung cancer cells (LLC) were implanted in C57BL / 6J mice at 1-2 cm below the right armpit. After the tumor grew to 3-5 mm (about 3-5 days), the mice were randomly divided into PBS group, Ag85B group, TTC group and Ag85B+TTC group, with 6 mice in each group. The vaccine injection process of each group was as follows:

[0055] PBS group: 50 μL PBS was injected into the tumor (single injection dose), for a total of 3 injections, one injection every 4 days.

[0056] Ag85B group: 50 μL PBS containing 10 μg Ag85B was injected into the tumor (single injection dose), for a total of 3 injections, one injection every 4 days.

[0057] TTC group: 50 μL PBS containing 10 μg TTC was injected into the tumor (single injection dose), for a total of 3 injections, one injection every 4 days.

[0058] Ag85B+TTC group: 10 μg Ag85B and 10 μg TTC recombinant proteins were diluted to 50 μL with double distilled water and then injected intratumorally (in situ tumor vaccine injection). The dose for one injection was 50 μL, and a total of 3 injections were given, with an interval of 4 days between each injection.

[0059] On the third day after the last injection of the orthotopic tumor vaccine, the mice were anesthetized, their eyeballs were removed and blood was collected. The mice were then killed by cervical dislocation, and the spleens were removed under sterile conditions and the subcutaneous tumors of the mice were removed.

[0060] Tumor growth results are shown in Figure 2 .Depend on Figure 2 The results showed that the in situ tumor vaccines Ag85B and TTC had good tumor treatment effects. In the subcutaneous tumor model of C57 mice inoculated with LLC, the tumor was significantly reduced in size and the tumor growth was significantly inhibited.

[0061] Example 3

[0062] Experimental study on cytotoxic T lymphocytes (CTL cells) and NK cell responses in tumor infiltrating lymphocytes (TIL)

[0063] The mouse tumor infiltrating tissue lymphocyte separation kit (Solebo, China, catalog number: P9000) was used to separate the lymphocytes from the tumor removed in Example 2, and to detect CTL cells and NK cells. The specific method is as follows:

[0064] (1) Preparation of single-cell suspension of tumor tissue

[0065] The tumor tissue was peeled off under sterile conditions and minced with scissors. The minced tumor tissue was digested in complete culture medium containing 1 mg / mL type IV collagenase (Solerbo, China, catalog number: C8160) and 0.1 mg / mL DNaseⅠ (Solerbo, China, catalog number: D8071) and incubated at 37°C in a shaker for 2 hours. The digestion solution was collected, filtered through a 200-mesh nylon mesh, centrifuged at 300g for 10 minutes, and the supernatant was discarded. The cell pellet was washed twice with PBS, and the supernatant was discarded after centrifugation. Finally, the pellet was resuspended with whole blood and tissue diluent to obtain a single cell suspension of tumor-infiltrating tissue.

[0066] (2) Lymphocyte separation method

[0067] Take a 15mL centrifuge tube and add an equal amount of separation solution to the single cell suspension of tumor infiltrating tissue (the separation solution should not be less than 3mL, and the total volume should not exceed two-thirds of the centrifuge tube). Carefully pipette the single cell suspension and add it to the surface of the separation solution, and pay attention to keep the interface between the two liquid surfaces clear. Centrifuge at room temperature with a horizontal rotor at 850g for 25min. After centrifugation, the cells in the centrifuge tube are divided into four layers from top to bottom. The first layer is the diluent layer; the second layer is the ring-shaped milky white lymphocyte layer; the third layer is the transparent separation liquid layer; and the fourth layer is the red blood cell layer. Use a pipette to carefully pipette the second layer of ring-shaped milky white lymphocytes into another clean 15mL centrifuge tube, add 10mL of cell washing solution to the centrifuge tube to wash the white film layer cells, and centrifuge at 300g for 10min. Discard the supernatant, resuspend the cells with 5mL of cell washing solution, and centrifuge at 300g for 10min. Discard the supernatant, resuspend the cells with 5mL of cell washing solution, and centrifuge at 300g for 10min. Discard the supernatant, resuspend the cells with 5mL of cell washing solution, and centrifuge at 300g for 10min. Discard the supernatant, resuspend the cells for later use.

[0068] (3) Detection of CTL cells and NK cells in mouse tumor lymphocytes

[0069] The concentration of the cell suspension obtained in step (2) was adjusted to 1×10 7 / mL, take 100μL in a 1.5mL EP tube. Cell surface target staining: before staining, add 1μg CD16 / 32 antibody to each tube for blocking, block for 10min, no need to wash, directly place the antibody mixture in the test tube. Then place the cells in a 4℃ refrigerator and stain for 30min away from light. anti-CD3-APC (100235, Biolegend, USA) + anti-CD8-PE (100707, Biolegend, USA); Anti-CD45-APC (103111, Biolegend, USA) + Anti-CD3-PE-Cy7 (100320, Biolegend, USA) + anti-NK1.1-PE (156503, Biolegend, USA). Wash the cells twice with sterile PBS, centrifuge at 4℃, 300g for 5min, and discard the supernatant. The cells were resuspended in 500 μL PBS, filtered through a 200-mesh nylon mesh, and then the cell samples were detected by flow cytometry.

[0070] Results Figure 3 The results showed that the in situ tumor vaccine Ag85B+TTC recombinant protein treatment could significantly enhance cellular immunity, and the proportion of CTL cells and (or) NK cells in tumor tissues was significantly increased compared with the group injected with Ag85B or TTC recombinant protein and PBS alone.

[0071] Example 4

[0072] Experiment on reducing Treg cell response in spleen lymphocytes

[0073] (1) Preparation of single-cell suspension of spleen lymphocytes

[0074] The spleen of the mouse in Example 2 was obtained under sterile conditions, ground, and then filtered through a 200-mesh nylon mesh to obtain a single cell suspension, and centrifuged at 4°C and 300g for 5 min, and the supernatant was discarded. 700 μL of red blood cell lysis solution (Solebo, China, Cat. No.: R1010) was added to the cell pellet and mixed evenly. After lysing for 2 min, RPMI1640 containing 10% FBS was used to terminate the lysis, and the cell pellet was centrifuged at 4°C and 450g for 5 min, the supernatant was discarded, and the cell pellet was resuspended with PBS to obtain a single cell suspension.

[0075] (2) Detection of Treg cells in mouse spleen lymphocytes

[0076] Using eBioscience TM Foxp3 / transcription factor flow fixation permeabilization buffer (Thermo, Germany, Cat. No.: 00-5523-00) was used for intracellular flow cytometry staining:

[0077] The concentration of single cell suspension was adjusted to 1×107 / mL, take 100μL into a 1.5mL EP tube.

[0078] For cell surface target staining: directly add an appropriate amount of antibody mixture to a test tube. Then place the cells in a 4°C refrigerator and stain for 30 minutes away from light: anti-CD3-PE-Cy7 (100320, Biolegend, USA) + anti-CD4-PE-Cy5 (100409, Biolegend, USA) + Anti-CD25-APC (102011, Biolegend, USA).

[0079] Wash the cells twice with sterile PBS, centrifuge at 4°C, 300g for 5min, and discard the supernatant; add 1mL of Foxp3 fixation / permeabilization working solution to each tube and pulse vortex, incubate at 2°C in the dark for 60min; add 1.5mL of 1× permeabilization buffer to each tube, centrifuge at 500g for 5min at room temperature, and discard the supernatant; resuspend the pellet in the remaining volume of 1× permeabilization buffer (usually 100μL). Without washing, add the recommended amount of anti-Foxp3-PE (320008, Biolegend, USA), and incubate at room temperature in the dark for 60min; add 1.5mL of 1× permeabilization buffer to each tube, centrifuge at 500g for 5min at room temperature, and discard the supernatant; add 1.5mL of 1× permeabilization buffer to each tube, centrifuge at 500g for 5min at room temperature, and discard the supernatant; resuspend the cells with 500μL of PBS, filter through a 200-mesh nylon mesh, and detect the cell samples by flow cytometry.

[0080] Results Figure 4 The results showed that the in situ tumor vaccine recombinant Ag85B protein + recombinant TTC protein could significantly weaken the Treg cell immune response after treatment, and the proportion of Treg cells in tumor tissue was significantly lower than that in the PBS group.

[0081] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An in situ tumor vaccine, It is characterized in that The in situ tumor vaccine comprises at least one of Ag85B protein and TTC protein, the amino acid sequence of the Ag85B protein is shown in SEQ ID NO.1, and the amino acid sequence of the TTC protein is shown in SEQ ID NO.

2.

2. The in situ tumor vaccine according to claim 1, It is characterized in that The concentration of the in situ tumor vaccine is 0.2-0.4 μg / μL.

3. The in situ tumor vaccine according to claim 1, It is characterized in that When the in situ tumor vaccine includes Ag85B protein and TTC protein, the mass ratio of the Ag85B protein to the TTC protein is 1:

1.

4. Use of the in situ tumor vaccine according to any one of claims 1 to 3 in the preparation of a product for treating cancer.

5. The use according to claim 4, It is characterized in that The cancer includes lung cancer.

6. The use according to claim 4, It is characterized in that The products include pharmaceuticals.

7. Application of Ag85B protein combined with TTC protein in the preparation of in situ tumor vaccines, It is characterized in that The amino acid sequence of the Ag85B protein is shown in SEQ ID NO.1, and the amino acid sequence of the TTC protein is shown in SEQ ID NO.

2.

8. The use according to claim 7, It is characterized in that The tumor includes lung cancer.

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