DNA NANOVACCINE, PREPARATION METHOD THEREFOR AND USE THEREOF
20230355766 · 2023-11-09
Inventors
Cpc classification
B82Y5/00
PERFORMING OPERATIONS; TRANSPORTING
A61K39/464499
HUMAN NECESSITIES
A61K2039/55561
HUMAN NECESSITIES
International classification
Abstract
Provided are a DNA nanovaccine, a preparation method therefor and the use thereof. The DNA nanovaccine comprises a DNA nanostructure, a tumor antigen polypeptide-DNA complex and an immunologic adjuvant, and the immunologic adjuvant comprises a double-stranded RNA immunologic adjuvant and/or a CpG immunologic adjuvant. In the present invention, a nanostructure is constructed, wherein the nanostructure is assembled from a DNA template, a DNA chain for assisting in folding and a capture DNA chain. By hybridizing the capture DNA chain with a functional component, the precise positioning and assembling of a tumor antigen molecule and an immunologic adjuvant molecule on the surface of the DNA self-assembled nanostructure is realized; in addition, a controllable DNA molecule “switch” is designed on one side of the tubular DNA nanostructure, which switch can respond to the acid environment of an endosome after entering an antigen-presenting cell, and open the tubular structure responsively to release the tumor antigen and the immunologic adjuvant molecule. The nanostructure has a tumor antigen-specific immunostimulatory effect and is a tumor vaccine used for the immunotherapy and prevention of various types of malignant tumors.
Claims
1. A DNA nanovaccine comprising a DNA nanostructure, a tumor antigen polypeptide-DNA complex, and an immunologic adjuvant; wherein the immunologic adjuvant comprises a double-stranded RNA immunologic adjuvant and/or a CpG immunologic adjuvant.
2. The DNA nanovaccine according to claim 1, wherein the DNA nanostructure is assembled by a DNA template strand, an assisted folding DNA strand, and a capture DNA strand.
3. The DNA nanovaccine according to claim 1, wherein the DNA template strand comprises M13mp18 phage genomic DNA and/or λ phage genomic DNA.
4. The DNA nanovaccine according to claim 1, wherein the capture DNA strand comprises a capture DNA strand I, a capture DNA strand II and a capture DNA strand III.
5. The DNA nanovaccine according to claim 1, wherein the tumor antigen polypeptide-DNA complex, the double-stranded RNA immunologic adjuvant, and the CpG immunologic adjuvant are bound to the DNA nanostructure by a capture DNA strand.
6. The DNA nanovaccine according to claim 1, wherein the shape of the DNA nanovaccine comprises a rectangular two-dimensional structure and/or a tubular three-dimensional structure.
7. A method for preparing the DNA nanovaccine according to claim 1 comprising the following steps: (1) mixing the DNA template strand, the assisted folding DNA strand, and the capture DNA strand in a buffer in proportion, and annealing to obtain a rectangular DNA nanostructure; (2) purifying the annealed product obtained in step (1) by centrifugation, mixing with the tumor antigen polypeptide-DNA complex, the double-stranded RNA immunologic adjuvants and the CpG immunologic adjuvants in proportion, and then annealing; (3) mixing the annealed product obtained in step (2) with the DNA switches in proportion and then annealing; and (4) purifying the annealed product obtained in step (3) by centrifugation to obtain a tubular DNA nanovaccine.
8. The method according to claim 7, wherein the method comprises the following steps: (1) mixing the DNA template strand, the assisted folding DNA strands, and the capture DNA strand in a 1×TAE/Mg.sup.2+ buffer with a molar ratio of 1:(5-20):(5-20) for annealing, and the annealing conditions are: from 95° C. to 65° C., each 1° C. is a gradient, the residence time of each gradient is 5 min; from 65° C. to 25° C., each 1° C. is a gradient, the residence time of each temperature gradient is 10 min, the whole annealing process is 7-9 h, to obtain a rectangular DNA nanostructure; (2) mixing the annealed product obtained in step (1) with a 1×TAE/Mg.sup.2+ buffer and adding to a 100 kDa spin column, centrifuging, and then mixing with the tumor antigen polypeptide-DNA complex, the double-stranded RNA immunologic adjuvants and the CpG immunologic adjuvants with a molar ratio of 1:(2-10):(2-10):(2-10) and annealed, the annealed conditions are: from 45° C. to 25° C., each 1° C. is a gradient, and each gradient stays for 3 to 5 minutes, carries out 6 cycles; (3) mixing and annealing the annealed product obtained in step (2) with the DNA switches in a molar ratio of 1:(1-2), the annealing conditions are: from 45° C. to 25° C., each 1° C. is a gradient, each gradient stays for 3-5 min, and carries out 6 cycles; (4) mixing the annealed product obtained in step (3) with 1×TAE/Mg.sup.2+ buffer, adding to a 100 kDa spin column, and centrifuging to obtain a tubular DNA nanovaccine.
9. A pharmaceutical composition comprising the DNA nanovaccine according to claim 1.
10. (canceled)
11. (canceled)
12. A method for treating a subject via immunotherapy of a tumor comprising administering the DNA nanovaccine according to claim 1 to a subject in need thereof, thereby treating a subject via immunotherapy of the tumor.
13. A method for preventing tumor growth in a subject comprising administering the DNA nanovaccine according to claim 1 to the subject in need thereof, thereby preventing tumor growth in the subject.
14. (canceled)
15. (canceled)
16. The DNA nanovaccine according to claim 3, wherein the nucleotide sequence of the M13mp18 phage genomic DNA is as shown in SEQ ID NO: 1.
17. The DNA nanovaccine according to claim 4, wherein, the capture DNA strand I is formed by adding a capture sequence I complementary to the DNA sequence of the tumor antigen polypeptide-DNA complex at the 5′ end of the assisted folding DNA strand, the nucleotide sequence of the capture sequence I is as shown in SEQ ID NO: 16-24; the capture DNA strand II is formed by adding a capture sequence II complementary to the cohesive end sequence of the double-stranded RNA immunologic adjuvant at the 5′ end of the assisted folding DNA strand, the nucleotide sequence of the capture sequence II is as shown in SEQ ID NO: 25-33; and/or the capture DNA strand III is formed by adding a capture sequence III complementary to the 5′ end extension sequence of the CpG immunologic adjuvant at the 5′ end of the assisted folding DNA strand, the nucleotide sequence of the capture sequence III is as shown in SEQ ID NO: 34-42.
18. The DNA nanovaccine according to claim 5, wherein, the number of the tumor antigen polypeptide-DNA complex is 10-30; the number of the double-stranded RNA immunologic adjuvant is 10-30; the number of the CpG immunologic adjuvant is 10-30; the amino acid sequence of the tumor antigen polypeptide is as shown in SEQ ID NO: 11; the nucleotide sequence of the DNA template of the double-stranded RNA immunologic adjuvant is as shown in SEQ ID NO: 13-14; and/or the nucleotide sequence of the CpG immunologic adjuvant is as shown in SEQ ID NO: 15.
19. The DNA nanovaccine according to claim 18, wherein, the number of the tumor antigen polypeptide-DNA complex is 15-20; the number of the double-stranded RNA immunologic adjuvant is 15-20; and/or the number of the CpG immunologic adjuvant is 15-20.
20. The DNA nanovaccine according to claim 6, wherein, the length of the rectangular two-dimensional structure is 80-100 nm; the width of the rectangular two-dimensional structure is 50-70 nm; the bottom diameter of the tubular three-dimensional structure is 10-25 nm; and/or the height of the tubular three-dimensional structure is 80-100 nm.
21. The DNA nanovaccine according to claim 20, wherein, the length of the rectangular two-dimensional structure is 90-100 nm; the width of the rectangular two-dimensional structure is 50-60 nm; the bottom diameter of the tubular three-dimensional structure is 19-20 nm; and/or the height of the tubular three-dimensional structure is 90-100 nm.
22. The DNA nanovaccine according to claim 20, wherein the DNA nanovaccine with the tubular three-dimensional structure has DNA switches.
23. The DNA nanovaccine according to claim 22, wherein the number of the DNA switches is 5-10; and/or the nucleotide sequence of the DNA switches is as shown in SEQ ID NO: 43-58.
24. The method according to claim 12, wherein, the tumor is selected from one or more of the following: melanoma, breast cancer, colon cancer.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0096] Hereinafter, the embodiments of the present invention will be described in detail with reference to the drawings, in which:
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BEST MODE FOR CARRYING OUT THE INVENTION
[0104] In order to further illustrate the technical means adopted by the present invention and its effects, the present invention will be further described below with reference to the embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention, but not to limit the present invention.
[0105] If the specific technology or condition is not indicated in the embodiment, it shall be performed according to the technology or condition described in the literature in the art or according to the product specification. The reagents or instruments that do not indicate the manufacturer are all conventional products that are commercially available from regular channels.
EXAMPLE 1
Preparation of Rectangular Lamellar DNA Nanostructure
[0106] Mixed M13mp18 template strand, assisted folding DNA strand (“staple” strand) and capture DNA strand at final concentrations of 20 nM and 100 nM for the template strand and the assisted folding DNA strand respectively; the mixture was slowly annealed by a gradient PCR instrument. The annealed conditions were as follows: from 95° C. to 65° C., each 1° C. is a gradient, and each gradient stays for 5 min; from 65° C. to 25° C., each 1° C. is a gradient, each temperature gradient stays for 10 min; the whole annealed process was 8 h, and the rectangular lamellar DNA nanostructure was obtained.
[0107] After the annealed procedure, the rectangular DNA nanostructure samples were taken out and centrifuged with a 100 kDa spin column (MWCO) to remove excess staple strands and capture DNA strands. The centrifugation conditions were as follows: added 350 μL 1×TAE-Mg buffer to 100 μL sample, centrifuge at 4800 rpm/min for 3 min, the volume of the remaining solution in the spin column is about 100 μL, and repeat the centrifugation 4 times. The final collected samples were analyzed by 1% agarose gel electrophoresis and the morphology of the lamellar structure was observed under an atomic force microscope.
[0108] The results were shown in
EXAMPLE 2
Preparation of Tubular DNA Nanovaccine Loaded with Tumor Antigen and Immunologic Adjuvant
[0109] The purified rectangular lamellar DNA nanostructure solution, tumor antigen polypeptide-DNA complex, double-stranded RNA immunologic adjuvant, and CpG immunologic adjuvant were mixed uniformly according to the molar ratio of 1:5:5:5, and put into the gradient PCR instrument, slowly decreased from 45° C. to 25° C., each 1° C. is a gradient, and the residence time of each gradient was 5 min; 6 cycles were performed.
[0110] After the annealed procedure, the samples connected with the tumor antigen polypeptide and the two immunologic adjuvants were mixed with the DNA molecular “switch” in a molar ratio of 1:1 and annealed. The annealed conditions were as follows: from 45° C. to 25° C., each 1° C. is a gradient, and the residence time of each gradient was 5 min, and 6 cycles were carried out; and then the PCR products were separated by centrifugation with a 100 kDa spin column, purified and recovered by agarose gel electrophoresis, and the purified tubular DNA nanovaccine composite structure loaded with antigen and adjuvant was obtained.
[0111] The results were shown in
EXAMPLE 3
Evaluation of Lymph Node Targeting Effect of Tubular DNA Nanovaccine
[0112] A certain dose of the Cy5 fluorescently labeled tubular DNA nanovaccine of example 2 (the tumor antigen polypeptide is SEQ ID NO: 11: SIINFEKLRRG) was inoculated at the base of the tail of C57BL/6 mice, and the mice were anesthetized and killed 24 hours later, fluorescence imaging was performed on the inguinal lymph nodes of mice to evaluate the lymph node targeting effect of the tubular DNA nanovaccine.
[0113] The results were shown in
EXAMPLE 4
Evaluation of Anti-Tumor Metastasis Effect of Tubular DNA Nanovaccine
[0114] 2.0×10.sup.5 mouse B16-OVA melanoma cells were injected into C57BL/6 mice through the tail vein, and this time was counted as day 0; a certain dose of the tubular DNA nanovaccine of example 2 (tumor antigen polypeptide is SEQ ID NO: 11: SIINFEKLRRG) was inoculated into the tail base of the melanoma model mice on the 1st and 7th days, and the mice were killed on the 16th day, surgical removal of mouse lung tissue to observe the formation of metastasis in mouse lung tissue.
[0115] The results were shown in
EXAMPLE 5
Evaluation of Antitumor Effect of Tubular DNA Nanovaccine
[0116] 2.0×10.sup.5 mouse B16-OVA melanoma cells were inoculated on the back of C57BL/6 mice, and this time was counted as day 0; on the 4th day after inoculation, the melanoma was basically formed. On the 4th day and the 11th day, a certain dose of the tubular DNA nanovaccine of example 2 (the tumor antigen polypeptide is SEQ ID NO: 11: SIINFEKLRRG) was inoculated at the tail base of the mice respectively, the tumor volume was measured every 2 days, and the changes of tumor volume were statistically analyzed. The tumor volume was calculated according to the following formula, wherein d is the smallest diameter of the tumor, D is the largest diameter of the tumor, and the mice in the control group were injected with normal saline.
Volume=(d.sup.2×D)/2
[0117] The results were shown in
EXAMPLE 6
Preparation of Tubular DNA Nanovaccine Loaded with Tumor Antigen gp100 and Immunologic Adjuvant
[0118] For melanoma B16F10, the antigenic polypeptide gp10025-33 (KVPRNQDWL) was selected. Mixed the purified rectangular lamellar DNA nanostructure solution with tumor antigen polypeptide-DNA complex, double-stranded RNA immunologic adjuvant, and CpG immunologic adjuvant in a molar ratio of 1:5:5:5 uniformly, and put into the gradient PCR instrument, slowly decreased from 45° C. to 25° C., each 1° C. is a gradient, and the residence time of each gradient was 5 min; 6 cycles were performed.
[0119] After the annealed procedure, the samples connected with the tumor antigen polypeptide and the two immunologic adjuvants were mixed with the DNA molecular “switch” in a molar ratio of 1:1 and annealed. The annealed conditions were as follows: from 45° C. to 25° C., each 1° C. is a gradient, the residence time of each gradient was 5 min, and 6 cycles were performed; then the PCR products were separated by centrifugation with a 100 kDa spin column, purified and recovered by agarose gel electrophoresis, the purified tubular DNA nanovaccine composite structure loaded with antigen and adjuvant was obtained.
[0120] The results were shown in
EXAMPLE 7
Preparation of Tubular DNA Nanovaccine Loaded with Tumor Antigen Adpgk and Immunologic Adjuvant
[0121] For colorectal tumors, the antigen Adpgk polypeptide (ASMTNMELM) was selected. Mixed the purified rectangular lamellar DNA nanostructure solution with tumor antigen polypeptide-DNA complex, double-stranded RNA immunologic adjuvant, and CpG immunologic adjuvant in a molar ratio of 1:5:5:5 uniformly, and put into the gradient PCR instrument, slowly decreased from 45° C. to 25° C., each 1° C. is a gradient, and the residence time of each gradient was 5 min; 6 cycles were performed.
[0122] After the annealed procedure, the samples connected with the tumor antigen polypeptide and the two immunologic adjuvants were mixed with the DNA molecular “switch” in a molar ratio of 1:1 and annealed. The annealed conditions were as follows: from 45° C. to 25° C., each 1° C. is a gradient, the residence time of each gradient was 5 min, and 6 cycles were performed; then the PCR products were separated by centrifugation with a 100 kDa spin column, purified and recovered by agarose gel electrophoresis, the purified tubular DNA nanovaccine composite structure loaded with antigen and adjuvant was obtained.
[0123] The results were shown in
EXAMPLE 8
Evaluation of Anti-Melanoma Effect of Tubular DNA Nanovaccine
[0124] 2.0×10.sup.5 mouse B16-F10 melanoma cells were inoculated on the back of C57BL/6 mice, and this time was counted as day 0; on the 4th day after inoculation, the melanoma was basically formed; On the 4th day and the 11th day, a certain dose of the tubular DNA nanovaccine of example 6 was respectively inoculated at the tail base of the mice, the tumor volume was measured every 2 days, and the changes of tumor volume were statistically analyzed. The tumor volume was calculated according to the following formula, wherein d is the smallest diameter of the tumor, D is the largest diameter of the tumor, and the mice in the control group were injected with normal saline.
Volume=(d.sup.2×D)/2
[0125] Tumor dimensions were shown in Table 1 below. Compared with the control group, the DNA nanovaccine treatment group can effectively inhibit the proliferation of melanoma in tumor-bearing mice, showing a significant tumor therapeutic effect.
TABLE-US-00010 TABLE 1 Evaluation results of anti-melanoma effect of tubular DNA nanovaccine Days after Control group DNA nanovaccine group inoculation tumor size (mm.sup.3) tumor size (mm.sup.3) 4 30.1 (±6.6) 42.2 (±9.2) 6 46.3 (±14.5) 53.3 (±11.1) 8 121.7 (±30.8) 89.3 (±20.6) 10 233.7 (±72.7) 114.6 (±37.9) 12 469.3 (±174.7) 168.6 (±61.5) 14 735.3 (±240.8) 223.3 (±86.2) 16 1125.2 (±337.6) 363.5 (±144.4)
EXAMPLE 9
Evaluation of Anti-Colorectal Tumor Effect of Tubular DNA Nanovaccine
[0126] 1.0×10.sup.5 mouse MC-38 colorectal cancer cells were inoculated on the back of C57BL/6 mice, and this time was counted as day 0; on the 4th day after inoculation, the colorectal tumors were basically formed; On the 4th day and the 11th day, 100 nM (100 μL) of the tubular DNA nanovaccine of example 7 was inoculated at the tail base of the mice respectively, the tumor volume was measured every 2 days for 20 consecutive days, the changes of tumor volume were statistically analyzed. The tumor volume was calculated according to the following formula, wherein d is the smallest diameter of the tumor, D is the largest diameter of the tumor, and the mice in the control group were injected with normal saline.
Volume=(d.sup.2×D)/2
[0127] Tumor dimensions were shown in Table 2 below. Compared with the control group, the DNA nanovaccine treated group could effectively inhibit the proliferation of colorectal tumors in tumor-bearing mice, showing a significant tumor therapeutic effect.
TABLE-US-00011 TABLE 2 Evaluation results of the anti-colorectal tumor effect of tubular DNA nanovaccine Days after Control group DNA nanovaccine group inoculation tumor size (mm.sup.3) tumor size (mm.sup.3) 4 68.5 (±10.8) 62.6 (±14.7) 6 102.2 (±27.0) 83.3 (±47.1) 8 170.0 (±28.0) 112.0 (±20.6) 10 256.4 (±49.1) 118.0 (±50.3) 12 317.3 (±65.8) 139.1 (±76.7) 14 406.7 (±95.8) 154.6 (±85.7) 16 502.9 (±112.3) 177.6 (±98.3) 18 591.0 (±144.1) 198.7 (±118.6) 20 760.2 (±187.8) 213.9 (±113.1)
[0128] In summary, the present invention uses the circular DNA single strand of the M13mp18 bacteriophage as the main strand, and the excess short-strand DNA as the auxiliary strand, through the hybridization and complementation of the main strand and the programmable auxiliary strand at a specific position, a two-dimensional rectangular lamellar DNA nanostructure is formed by folding. According to according to the principle of base complementary pairing, the tumor-specific antigen polypeptide, double-stranded RNA immunologic adjuvant and CpG immunologic adjuvant were connected to the surface of the self-assembled two-dimensional lamellar DNA nanostructure by using the capture DNA strand, then, acid-responsive DNA “switch” was hybridized on the two long sides of a rectangular lamellar DNA nanostructure, so that the rectangular structure was coiled and closed to form a tubular structure, a tubular DNA nanoparticle vaccine loaded with tumor antigen and immunologic adjuvant and controlled “switch” to respond to the acidic environment of antigen presenting cells in vivo was obtained. The nanoparticle vaccine has a bottom diameter of 19 nm and a height of 90 nm, which can be used as a nanoscale molecular machine for the loading of tumor antigen and immunologic adjuvant, and also can be effectively transported to lymph nodes for controllable release. It is expected to provide a new formulation of nanovaccine for tumor immunotherapy.
[0129] The applicant declares that the present invention illustrates the detailed method of the present invention through the above mentioned embodiments, but the present invention is not limited to the above mentioned detailed method, that is, it does not mean that the present invention must rely on the above mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.