CN116716299A - A macrophage activator based on controllable edge-length RNA nanostructures and its application in the treatment of solid tumors - Google Patents

Abstract

The invention discloses a macrophage activator based on a controllable side length RNA nano structure and application thereof in solid tumor treatment, wherein the RNA nano structure can realize the regulation and control of the solid tumor immune microenvironment, realize the innate immune activation effect stronger than polyIC and enhance the treatment effect of aPD-L1 immunotherapy.

Description

A macrophage activator based on controllable side length RNA nanostructure and its practical application Applications in body tumor treatment

Technical areas:

The invention belongs to the field of materials and biomedicine technology, and in particular relates to a macrophage activation strategy based on a controllable side length RNA nanostructure and its use in immunotherapy or combined therapy of solid tumors.

Background technique:

The innate immune system includes cells such as granulocytes, monocytes, macrophages, dendritic cells (DC cells), and natural killer cells (NK cells). In recent years, macrophage-based immunotherapy has shown good prospects in tumor treatment. Macrophages infiltrate the tumor microenvironment in large numbers and can inhibit the anti-tumor phenotype (M1 type) function of macrophages in the tumor microenvironment by activating or polarizing them or removing the pro-tumor phenotype (M2 type) macrophages. Tumor growth.

Exogenous single-stranded or double-stranded RNA can be recognized by the innate immune system through nucleic acid sensors such as Pattern Recognition Receptors (PRRs), and activate the anti-tumor or anti-viral function of the innate immune system. Synthetic RNA analogs, such as double-stranded polyIC, are used clinically as immune activation adjuvants and have achieved many applications such as anti-upper respiratory tract infection and anti-tumor. However, these RNA analogs are easily degraded by nucleases, which limits their biomedical applications in vivo.

The regulatory mechanism of innate immunity is very precise and complex, posing challenges to the design of immune adjuvants for specific targets. In terms of space, immune recognition-related receptors are only controllably activated in specific compartments (at the organelle level), which puts forward higher requirements for the precise delivery of immune adjuvants; in terms of time, complex ligand processing mechanisms and cellular feedback mechanisms , limiting the activation time of a single adjuvant; on the cellular pathway, a single adjuvant (such as polyIC) activates multiple cellular pathways at the same time, which will bring uncontrollable drug administration risks.

Using RNA nanotechnology, RNA adjuvants of specific shapes and side lengths can be synthesized according to the activation requirements of pattern recognition receptors; by controlling the internal structure of RNA adjuvants, their biological stability can be greatly improved. This strategy of designing adjuvants starting from needs provides new ideas for the design of effective and safe immune adjuvants.

Contents of the invention

The primary purpose of the present invention is to provide a strategy to achieve controllable activation of macrophages by designing RNA nano-adjuvants with different side lengths, and to apply it to immunotherapy and combination therapy of solid tumors, especially pancreatic cancer. The RNA adjuvant of the present invention has strong biological stability, can enhance the uptake of the adjuvant by macrophages, can retain and activate macrophages for a long time, and can be produced on a large scale at low cost. Compared with products such as polyIC, adjuvants based on RNA nanostructures have better immune stimulation effects and can activate the anti-tumor ability of macrophages more in solid tumors.

Specific aspects of the invention include:

A first aspect of the present invention provides a single-stranded two-dimensional RNA nanostructure of a specific shape and size, the shape of the nanostructure is a rectangle or a rhombus, and the side length of the nanostructure is greater than 80 bp, preferably greater than 100 bp;

Preferably, the side length is greater than 110bp, 120bp, 130bp, 140bp, 150bp, 160bp, 170bp, 180bp, 190bp or 200bp.

Preferably, the nanostructure is rectangular, with side lengths of 143bp and 209bp;

Preferably, the nanostructure is rhombus-shaped with a side length of 143 bp.

A second aspect of the present invention provides the use of the RNA nanostructure to prepare macrophage activators.

A third aspect of the present invention provides the use of the RNA nanostructure to prepare immune activators.

The fourth aspect of the present invention provides the use of the RNA nanostructure to prepare cancer therapeutic agents.

The fifth aspect of the present invention provides a pharmaceutical composition or pharmaceutical combination, which contains the above-mentioned RNA nanostructure and an immune checkpoint inhibitor.

The sixth aspect of the present invention provides the use of the pharmaceutical composition for preparing cancer therapeutic agents.

The uses described in the fourth and sixth aspects of the present invention, wherein the cancer is a solid tumor, preferably pancreatic cancer.

The pharmaceutical composition or pharmaceutical combination according to the fifth aspect of the present invention, wherein the immune checkpoint inhibitor is an aPD-L1 antibody.

beneficial effects

The present invention prepares a new type of RNA immune adjuvant based on single-stranded RNA technology. The preparation process is simple and the structure and properties are uniform. RNA nanostructures are highly designable and their morphological characteristics can be precisely controlled. The yield of RNA nanostructures prepared by the invention is as high as more than 90%.

By precisely folding RNA single strands without using modified bases or liposomes and other delivery tools, the present invention has excellent biological stability and can resist the degradation of RNase A and T2. Compared with commercialized Adjuvants (polyIC) can be more stable and stay in target organelles for a long time.

The present invention is based on the strategy of RNA origami side length control to achieve controllable activation of macrophages. The present invention precisely controls the side length of RNA origami and can realize the regulation of macrophages from immune resting to immune activated state. The nanostructure with a side length of 77 bp does not activate macrophages; increasing the side length to 143 bp can achieve an activation effect slightly higher than that of the commercial adjuvant polyIC; increasing the side length to 209 bp, the RNA nanostructure of the present invention achieves a much higher activation effect than polyIC immune activating effect.

The RNA nanostructure of the present invention can realize the regulation of the immune microenvironment of solid tumors and enhance the therapeutic effect of aPD-L1 immunotherapy. Through intratumoral injection, RNA nanostructures achieve a stronger innate immune activation effect than polyIC. Compared with aPD-L1 monotherapy, the combined treatment of aPD-L1 and RNA nanostructures more effectively inhibited the growth of solid tumors.

Picture description:

Figure 1 is the structural characterization result of the RNA nano-adjuvant synthesized by the present invention: Figure a is a rhombic RNA nanostructure with a side length of 77 base pairs (bp) and a width of 10 RNA double strands; Figure b is a side length of 143 A rhombus-shaped RNA nanostructure with 6 RNA double-stranded bases (bp). Figure c shows a rectangular RNA nanostructure with a side length of 143 base pairs (bp) and a width of 7 RNA double-strands. Figure a shows the side length. It is a rectangular RNA nanostructure with 209 base pairs (bp) and a width of 5 RNA double strands. The design diagram of each structure and its structure synthesis yield are shown in the upper left corner;

Figure 2 is the in vitro verification of the biological stability of the RNA nanostructure of the present invention: Figures a and b are the agarose gel chart results of the 143bp and 209bp RNA nanostructures respectively after incubation with 0.5 RNase A and T2 for 24 hours. Compared with unfolded single-stranded RNA, RNA nanostructures can resist the enzymatic digestion of the above two RNases and demonstrate good biological stability;

Figure 3 is a cell-level verification of the biological stability of the RNA nanostructure of the present invention: Figure a shows single-stranded RNA (ssRNA), polyIC and the RNA nanostructure (ssRNA origami) prepared by the present invention in mouse macrophage RAW 264.7 Different residence times. The retention time of RNA nanostructures exceeds 24 hours, indicating their long-term stability in cells. Picture b shows the RNA retention area in picture a through quantitative processing by ImageJ software;

Figure 4 shows the characterization of the activation effect of RNA nanostructures of the present invention on macrophages: using flow cytometry to verify the activation effect of RNA nanostructures with different side lengths and shapes on macrophages (expression of CD40); increasing the sides of the RNA nanostructures Long length can achieve controllable activation effect; 143bp and 209bp rectangular RNA nanostructures show higher activation effect than commercial reagent polyIC;

Figure 5 is a diagram showing the effect of the RNA nanostructure of the present invention on inducing the anti-cancer phenotype of tumor macrophages in mice. Single-cell sequencing technology was used to sequence and analyze macrophages in mouse solid tumors after intratumoral injection of RNA nanostructures. The results showed that RNA nanostructures can activate more anti-cancer macrophages than commercial polyIC reagents. And it has good synergy with PDL1 antibody to enhance the anti-cancer phenotype of macrophages;

Figure 6 is a diagram showing the anti-cancer effect of the RNA nanostructure of the present invention on the mouse pancreatic cancer subcutaneous tumor model. Figure a shows the mouse treatment dosing plan, i.t. is intratumoral injection, and i.p. is intraperitoneal injection. A total of three injections were given. The mouse model is the Panc02 pancreatic cancer subcutaneous tumor model. Pictures b and c are tumor growth charts and mouse survival curves of different treatment methods respectively;

Figure 7 Flow chart of the design and preparation of RNA nanoadjuvants with different side lengths.

Detailed ways

The present invention will be described in further detail below with reference to the examples and drawings, but the implementation of the present invention is not limited thereto. The drugs and reagents used in the examples can be purchased from chemical companies, or can be prepared by methods disclosed in the art.

Reagents, equipment and methods

Reagents

BamHI restriction endonuclease was purchased from New England Biolabs, fluorescent molecule Cy5-UTP and commercial PolyIC were purchased from APExBio Company, RNase A was purchased from Thermo Fisher Scientific, RNase T2 was purchased from Shanghai Sangon Biotechnology Company, and flow cytometry was used The antibody anti-CD40-FITC was purchased from Thermo Fisher Scientific, and the therapeutic anti-PDL1 was purchased from Bio X Cell in the United States. The DNA plasmid containing the single-stranded RNA nanoadjuvant sequence was synthesized by Suzhou Genewiz Company. The restriction endonuclease BamHI required for DNA plasmid linearization and the kit required for DNA transcription into RNA HiScribe T7 High Yield RNA Synthesis Kit were purchased from New England Biolab.

The sequence (5’-3’) of a possible RNA nanoadjuvant (rectangle with a side length of 143 bp) integrated on the DNA plasmid:

TGGCGTTGGGCTACTCTCCGTTGCGAGCAAGAATGAAGGAGCGCCCTGCGACCCTTCTCGGCAGCCGACCCTTTCCTCGATGGACCTGGGCCGAGGAGCCTAACGAGGACCCAGCAGCTTTGCGGGACCGCTCGGACGGGCTGTCCGAGCCACCGGTGTCGGAAATAGTGGGTCTCCCTCGAATCCCTTACCTTCATCCTGTCTCGAGAGTCGTAAGAACCGATGCGCGGCGAGTTCGCGGACCTAACGAGCAAA GACACGGAGAGCATCGGGGTAGCCGCGCCCGGTACGCGGCTAGCTGCAGGCCTAGCCCAGACGCCGCCACATCCGTTACAGTTAGCTATGGTGGGTGTCCGCGTTCACTTGAGTAGACCTCAACTGACTACTAGGGAGTATCTCTCCTCTATTTCCGGTGCCGAAGAGGTGCGCTGACCGCGCACCTGTCAAATGGTCGCCGCCGCCCCTAGGGGGAGAGTAATGGGGTATAGGGACAGCGCCCGGTCGTGCCAT CCGTGGCTAATGACCCAGTATTGCTTCATTCTCCTTCGAAGGGCTAGGACCGTCGATGTAAAGGTGGGAGGCCTTTACAGCCGGTCTGCGCTCGCCGGCGTCGCAACGATTCGAACTGCCTCGCCATGGAACTGTTTTGCGCGAACCGAGTGTCAGCGCGGCCCGTCCTTCGAGGGTTGCGGCTGGCGGCGACACCCGAGCGACTATGCCTTCGACATAGTCGCGTCCGCGCCTGACCTATTAGCTGGCCGGCA GCACGGGATAGGCCTTGCTACGGAAACAGAGATGACAGGCTGGGGGTCGCATCCAGCCCCGGATGTGACTCGCGCAGCGAGCGCTCTCAACTCTATGAGTTCAGGCGACTCATAGTGAAGCCGACCATAGTTTTCTCAGCAGGCCAGCCCTACCCTCTGCCACGTACTTCGCAGAGCAGGACGTCTGGGAGGATGAAGGTTCCCCGCTCTCCCCTGGTGGGCAGGTCAGGACAGGTTATCGTGACCGATAACCTGTTCCTG CGTGCCCACCAGCTTGGAACGGGGAACCACACACAACCCAGACGGGGTCGGCTGCGAAGTACCATAGCTAGGGTAGGGAATCGTTGCTGAGAAAACTATGGTCGGCTCACCCGTTAGAGGGATTAACGGGTAGTTGAGACCTTGGCCTGCGCGAGTTCACGGACGGGCTGGACCTCCTCACCAGCCTGTTTACGACTTTTCCGTAGTCCCTATTATCCCGTGCTGCCGGCCAGCTAATACGCAGGACGCGGACGCG GCATTATGCCAACTAATGCCGCTCGGGTGACACGGTGAGCCGCAAGGTTGACCGGACGGGCCAGGTCCATACTCGGTTGTGAACGCCAGGTGTTCCATGGCGAGGCAGTTCGCTGGCCTGCGACGCCGGGCCAAGGAGACCGGCATAGCTCACTGAAGATGAGCTATTCGACGGTACCCTGACTTCGAAGGAGCGTATACCAATACTGTCGCCGCGGCCACGGATGGCACGACCGGGCGCTGCAAGGCCACCCCATTATTC CAAGCCTAGGGGCCACCGTGTCCATTTGACCACCCTCCAGCGCCGAGGGTGCTTCGGCACCCGGCGGAGAGGAGGTTAGGCTTAGTAGTCAGTTGAGGTCTACTCAACGCGCAAAGGACACCCACGTGGCAGAACTGTAAGTCCGTGAGCGGCGTCTGTCAGGGTCCTGCAGCGTGGCCCGCTCTCTGCGGGCCACCCCCGATGACTTCCGGGTCTTTGCTCGTTAGGTCCGCGAACGGTCATTACATCGGTTCCATCT CTGCTCGAGACTTGTGTGGTGGTAAGGGAGGTCAACCAGACCCACTCCGCCGGACACCGGTGGAGAGAGGCCATCTCTCTCTCGGTCCCGCAAAGCTGCTGGGTCCTCATACTCCCCCTCGGCCCGCGCTGACCGAGGAAATCCTGCTCTGCCGAGAATGAGGAGGGGGCGCTCGTATACGCTTGCTCGCACCGGAAGTTAGCCCAACGCCA (SEQ ID NO: 1)

The sequence (5’-3’) of a possible RNA nanoadjuvant (rectangle with a side length of 209 bp) integrated on the DNA plasmid:

CGTAGCGTACAGGGATTCGAATGTGGGACGAAGGCGCCGTACGTTGGGAGAAACCTTGTCGAGGCACGGTGGTATGCCGGAGAGATCTAAATTACAGTCAGCAGGGCTAACTTACTATCGTATATGGGAGATATACCGTTCGCGAGTTTGAAAACAGCGGGTTACCGGAAGCCCGAATAGACAGCCGCGTCAGATTCGCAGACGTATCTGCGACCAACGGGCGACTGCTTGCTTCCCGGGTACGGTTTGAT CCTGAGCGCCGTTGGGCCGCGTTTCCTCGTGCGTAGGGCGATAGTAGCCCTTTCGTTTTTCAAATAAGCGGCGCCTTGGGAGATGAGCTAGACAGTTGAGGGTTCGACTCGGAGAAGGCTGGCGCCTTACGTTTCCTGGAAGGTGCGTATTTGGCATGCCGGGGGCGGCATGCTCTATGGATGCCACTGGACTAAGACTTCACTCCCTAGCAGATCACCGCTTGAGGTATCACCAACATGACCGTTGCCAAGGCGAA GACGCTGCCGGTAAGTTGGTCCCGTACGAAAGTGGGACGTCGGAGCGAGCTCCGATGAGCCCTAGTTCCGATCAACCGTACTCAGTGCGAATGCCTACATGCGGAGCCGGGCCTCGGGGCCCGGGAGTACCTGCGCTTCTCCACATCCCTCCACGTCGATGGGCCCTTTCAAACCAGACCCTTTCAGCTCGCCGGTCTCTTTCGTACCCGACAGGGGCCAGAGGCTTACCTTTATCTATCTGATATTCACCGTGCCACC CGCGCCCCCCGTGAACGCGATGGAGTGAAGCTTTAGTCTTTTGGCTTCTCCATCACATTCGTGCCCGAATGTGATATTGCGAGATGTCGGGACGTTATGAGGCTGGTGAGCTGCCCGAGTCGACAATCGATGCGTGCAACAGACAATGATAGATACGGTCTCTCAATGTAAGTGATGGCGTCTCGTACTCCAGTTGTCGAGGAAAGAAAAATGCTTTTGAACCGAGATCCGACGTGGAAGCCGGATGCTAGGGG AACGCGACCCAATCGGGTCGCGTTGGCTGTCTATCCGGCTGCGGAGTGGGATCTCGGTAGGGCTCCATTTTTCGTGGCCCGACAACTGGAGTACGAGACGCCATCACTGCTGACGAGAGACCGAGACGCCTATTGTCTGATGTTGGTATCGATTGTTCATATCGGCAGCTCACCAGCCTCATAACGTCCTTCGAATCCGCAATATAGCCCTAAACCATTAGGGCTATGGAAGACACCTTCCGACTAAAGCCTCAGTTG ATCGCGTTCACGGGGGGCGGGTGATGCCCGCAATATCAAAGGCGTCTAAGGTAAGCTGAAAAACCCTGTCGGTTTGGGACGAGACCGGCGAGCTGAAAGGGTCTGAGTGACGGGGGCCCATCCACTCCGCGGGATGTGCAGCAGTCCAGGTACTCGACTGCTTCCGAGCAGTCCTCCGCATGTAGGCATTCGCACTGAAGGTCCGAGATCGGAACTTCAAAAGATCGGAGCGGTATATCACGTCCCACCGTCCAAA GGGACCAACTTACCGGCAGCGTCTTTTGGTTGACAACGGTCTTGCACGCGATACCTCAGTTTCTCCTCTGCTAGCAACTGAGGTCTTAGTCCAGTGGCATCCATAGATACGTGACCGGGGTCACGTACAAATACGAGCCAAAAAGGAAACGTGCGCGAAGAGCCTTCTCGATATGAAACCCTCAACTGTCTAGCTCATCTCTCAACCAACCGCTTATTCTCTGGCCGAAAGGGCGTTAACAACCCCTACGCACGGGCCACC GCGGCCCAACGGCGCTCAGGATCATCGGACCTCCGGGAAGGAGAAGCGGCCCGTTGGACTGAGCACCGGCTCAGTATCTGACGCCCCCTAGCATTCGGGCTTCCGGTAACCCGCTGTCCGTCACTTCGCGAACTCGCTCCGTCCCATATAGTGTTAACAGTTAGCCCTTACATTTGTAATTTAGATCTCTCCGGCATACGCGGGCATCTCGACAAGAGCGGTGACAACGTACCTTCGCGCCGTCCCACACGACATCTCCTGTACGC TACG (SEQ ID NO: 2)

The sequence (5’-3’) of a possible RNA nanoadjuvant (a rhombus with a side length of 77 bp) integrated on a DNA plasmid:

AATGTGCCAGCGAAGGGGGTCCGGATCGTATCAGTGGGCCGCAACTCACATCAGCTCCGTGACCGGGGGAATCCAACGGTCTACTTCCTGTCGACCCCCGTCGCTGCAAATTGATGCGGGTTCGACACGGAAGGAAAGCCGCCCGGCTTTCCCTGTGCATACCACGTACTCGTTTCGGTGGCTAGGCGTGCATCTCATTGTGCCTATTAAAGTTTGGGGTGTCCGAACTCGGAATATATCGAGCTATGTAGTCGGCTACAT AGACGAAGTTACTTCCCTGCGTAGCTCGTCAGCCTTAGAGCAAACGTGGCACACCTGTCGGTGATCGGGTGAGTAGGTATTCCCCCGTTGCCAATCCCGCTATCTACCTAGTCTGAGTCGGAGCTGAGTTCCGGAGCGCCGGAACTCGGGGGCGTCCGCCCGATCACCGACGGTAGGAACAGCACTGCGAGCTCCGCGCCGGGCACTGTGAGGGTCGCACACGAAGAGACCTCCCCCGAGTGGGACGCATCCCACTCC GGCCGATCCCGCGAGGACACCCCCGTCGTGCGGTTCAGCCGCTATGAGCTGGAGCAAATGGGGGATTGGTCATTCGAGGCCAAGTGGATATGCTGGCGCGGAAGCCGCAAATAAGTGGCGTCCCAATGGAGGACCCTCCATTGCGAGCCACTTCCAGAGTTCCCCGCCCGCCAGCAGCTCCGGCTTTTTTGATGTTCCCATAGCGGGATTCGGCACGGAACGAGCCAAAGGGGGGCAGCGACGGCACACTTAACGAGTGT GGTGCGCGACGGATATTCCTATCCGTCCGGTCAGCGCACGCTGCATCGACCGCTAGCCACCTTAGAGAAGGGGCAAAGCTCCGCATTCCTACCGTCGGATCCTCAACCTTCCCGTGTGGGTGCATCTGCGGATGCACCCTGGTTCGCTCGTGCCTCTGCCTTCTCCCCCATTGCCCAGTGAAGCAGGAGGCAGAACCCGATCACTGGTGTGCCTTGATGAGCCGGCCGAGGATTCCCTACGCTCGGTAAGATCCCAGTT ACCCCTCACAGGCATCTGTGAGGTGGGAGGGCCCATGCCGGCAGTGCCCCCAAACTTGCGGCCTTCTTCGCCCAATCACAACTCACAGTCGCTACTTCTTATCCAAGTTCCCTATGACACCGTAAGCGGTGTCACTCCCCTCGGCGCAACATTGGCCTCCGCACGACGGAGGCTTGTCTCGGCTTAGCCAGGAGGCTGACGCCCCCCGCAGTATGCGTCGCCCCTCGGCCCACAGGTGCCGGACAATCCCCTCGAGTGGCCGG CACGTGCCGGCCACTCCCGGACCCATTGTCCGGCACCTTGCGGCATAGGGGCGACTTCCGAGTCGGGGGGCCGACAACGCCTGGCTAACTCTTCGTAAGCCTCCCGAGGGTCGAGGCCAATGTTGCGCCGAGGGGAGCTACCCAGCAATCTGGGTAGTAGGGAACGCTCATAGGAAGTAGCGTCCCCGGGTTGTGATTGTGCCACGAAGGCCGCATGCTCCCCGGCACTGCGGAGCTGAGCCCTCCCACCCTGTTTCACAAAAAAA CAGGGGGTAACTGGGATCTTACCGAGCGTAGCCGGCCTTCGGCCGGCACAATGAGGCACACCAACAGTCGCGTTCTGCCTGGAGCTCGACTGGGCAGCTTGAGTGAAGGCAGAGGCACGAGCGAACCAGCTCGGGTTCCCGAACCCGAGCACACGGGTGGCCTCGGGATCCGACAAGGCGTATGCGGAGCGGGGAATATTCTCTAAGGTAGGTTACGGTCGATACAGGAAGCGCTGACCGTGTCACAAGATGTT GTGACAGCGCACCACACTCGTTAAGTGTGCCGTAGGCCGCCCCCTTTGTACGTGGTCGTGCCGAAACAGCTAATGGGAACACGGGCGGAGCCGGAGCGGTTTGACGGCGGGGAACTCTGGAAGTGGCTCGCTTCTCGGTGATCCGAGAAGGGACGCCACTTATTTGCGGCTTCCGGTCAAACCTATCCACTAAGGTTGAAATGACCAAAACTCAAGCTTGCTCCATTGGATAACGGCTGAACGACCCTCGGGGGGTGTC CTCGCGGGATCGGCGGCGTGCGCTGTCGCACGCCGGGGGAGGTGCCGAGACGTGCGACCCCCGGGGAGCCCGGCGCCCTGCTTCCAGTGCTGTACGCCTTGTCGGTGATTCAAAAAACGCCCCCGTCGATACCTGGCGGTATCGACAGCTCCGACTCAGACTAGGTAGATTAGCTGTTTGGCAACGTTTGCCCCCCTACTCACGCGACTGTCGACAGGTGGGCGAAGTTTGCTCTACGTTGTCGGAGCTACGCAGGGA AGTAACTTCGTTAGCTTCCGTCAGGAAGCTACTCGATATAGCATACTGTCGGACACGGGGAGCATTAATAGGCTCATCAAGATGCACGCTAACCTACCGAAACGAGGCTCGTTCATGCACAGGGCCCCCACCGCTGGGGGCCTTCCGTGTCGAACCCGCATCAATTTCGGCCTACGGGGGTCGGCAGCGTGTAGACCGTTAGGCCGGCCCGGTCACCGGCATGGTGTGAGTTGATGCCGCATGATACGATCCGGAGGGT CCGGGCTGGCACATT (SEQ ID NO: 3)

The sequence (5’-3’) of a possible RNA nanoadjuvant (a diamond with a side length of 143 bp) integrated on a DNA plasmid:

CGAACACTAGATAGCAGTTCTTCGTTTAGCGAATGAACCATTGGATAGTATGCACGTTGTGACTCCCAGGTGTGGCTTTGAAGTAATGTGTACCTATACTCAAGTGAGCGACTGACCATACTCACGTGGTTAAGAGTATCAAGTTAGGCGACTACGAGACATTCAGATGCATCAAAAGATGCATCGGGCAGTGAGTTGATATGTCCACCTTAGGTGTCAATCCTCTTTAGTGCGTTTCATGTCTCGTGATACCCTTCGC TGATACAGTTATCACCCACACCTGTATATCCCAGTCAGTCGTTCGATCGGACATTACAAAAGTAATGTCGAATTGCGCCATTGAACCATGTATTACAATCTTGAAGATAGCACTTAACCCACGTGGGTCATACGGATAGTGTCGTGTAGACTGCCACTGGGACACCTACGGTTTGATCTAGTTTGCATTGTTCCAGTGTGAAATGCTAGCCTGTTCACCGACAGACACCACCAAAAGGTGGTGTGTACGAGTGAACGT CCACTTATCTGTCACACTGTTGATCTCTACATGCTCGATAGCCTCACAATGTCAATATCCTACGTAGATGTGCATGATCACTTGAAGTTTGTCAGTCATTGTACAGATCCAAGATTGTGTGCGACACTGTTTAGTGCCTTCATGAGTCATCAAAAGATGACTCCACACGCATTGCAGATGCGCCACGATCGTTACTGAATGCATGTAACGCTTACATCAGAATCACGAGCCCGATAGAGAATGAGTAGACTAGTACATTGTGTA GCAACCTGGTGCGATGTTCAAAGGTGACTTCAAAAGAAGTCACCATGGCGCACTAACAGTAAAGAATAGATACTCTAGGCGGCACTGACACAATATGGCCGCCGTATGAGGTGGCTAAGTTGACTAGTCCGTAAAGTAACGACACGTCTTGATTAGCTGTTTGGAGCATGGTTCAACCTGCGACCGCTAAACTGCTAGTCTCCATCTAGAAATAGATGGAGACCTTGATCATAGCGGTCGCAGGTATCGACTCGCTC CAAACCTAAACAGAAGACGTGCTTAGGAATTACGGACTAGTCAACTTAGCCACCGGTTTCATCGGCCATATGCACTATATGCCGCCTTCAAACGTTATTCTTTAAGCATGTAGCGCCATGCTACGTCGAAAACGACGTAGCTTTGAACATCGCACCAGGTTGCTAGTGAGGTAACTAGTCTTGAGTATGTCTATCGGGATGCATCATCTGATGTGGTTAAGTCATGCATTCTTCCTAAGTCGTGGCGCATCTGCAATGCG TGTGGTTTGCCGAAAACGGCAAACATGAAGGCAAGCTAATCTGTCGCACTGTAGTCGGGATCTGTATGTATCAGGACAAACTGTAGCAAGTCATGCACATCTACGTAGGATATTGTACCTCACAGGCTATCGCTGTTAGTGAGATCAAACTATCATCAGATAAGTGGACGTTCACTCGTACTCTCAATGAAAACATTGAGACTGTCGGTGTGAACAGGCTAGCATTATGATAGTGAACAATGTCGTAGTCATCAAACCGAGATACCACCAG TGGCAGTCTACACGACACTATATGAAACCCCCACGTGAAGCGTTAGCTATCTTCCGACTACAAATACATGAACGTGCAGCGCAATTCTCTGCGTGAAAACACGCAGACGATCGAACGACTGACTGGGATATATCATAAGTGGTGATAACCAATGACTCGAAGGGTTGATGCATACATGAAACTGTGTCAGAGAGGATTTGGTATCTAGGTGGACATATCAACTCACTGCCCTACATGAGAAAACTCATGTATGAATGTCCAAACTAG GCCTAACTTACGTTTGATAACCACGACTCATTCGTCAGTCGCCTTGCTACGTATAGGTACACATTACTTCAAAGCACTTATGAGGAGTCACGTTCAATGTACTATCCAGAGTCGATTTCGCTAAACGAAGTGATCAAGTCTAGTGTTCG (SEQ ID NO: 4)

equipment

Brucker atomic force microscope, BD Rhapsody ^TM single cell analysis system, Guava easyCyte (Merck) flow cytometer, Bio Rad PCR instrument, Perkin Elmer intravital imager, ZEISS LSM 780 confocal microscope.

Design, preparation and characterization of RNA nanoadjuvants

The design and preparation of RNA nanoadjuvants with different side lengths includes the following steps:

(1) Design of RNA nanoadjuvants with different side lengths. Use Tiamat software to design single-stranded two-dimensional RNA nanostructures with different side lengths. The shape can be designed as a rectangle or rhombus. Use Tiamat software to automatically generate RNA sequences and control the GC base ratio below 60%;

(2) Preparation of RNA nanoadjuvant. Add the T7 transcription promoter before obtaining the RNA sequence, add the BamHI restriction endonuclease site after the sequence, and integrate it into the pUC57 plasmid. After the plasmid is amplified, linearized and transcribed into the target single-stranded RNA, the single-stranded RNA is purified and annealed in PBS solution (65°C to 15°C) to obtain the RNA nanoadjuvant.

To detect the stimulating effect of RNA nanoadjuvants with different side lengths on macrophages and to verify the anti-tumor effect, the main steps are as follows:

(1) Stability testing. The RNA adjuvant was incubated with RNase A and RNase T2 under different pH conditions in vitro, and its stability was verified by agarose gel diagram (Figure 2); the RNA adjuvant was labeled with a fluorescent molecule (Cy5), and the RNA adjuvant was labeled with a fluorescent molecule (Cy5), and the cells were analyzed by confocal microscopy. Flow cytometry was used to detect the uptake and intracellular stability of RNA adjuvants by macrophages (Figure 3).

(2) Macrophage activation function detection. RAW 264.7 macrophages were incubated with RNA adjuvants of different side lengths, and the ability of the adjuvants to activate macrophages was detected at the cellular level in vitro through flow cytometry, RT-qPCR and other means (Figure 4); single cell sequencing and tissue immunity were used Histochemistry technology was used to detect the macrophage activation ability of RNA adjuvant at the in vivo level (Figure 5).

(3) Verification of anti-tumor effect. Pancreatic cancer subcutaneous tumor model mice were used to verify the therapeutic effect of RNA adjuvant. Intratumoral injection of RNA adjuvant, combined with intraperitoneal injection of anti-PD-L1 antibody (50ug*3), was used to detect nuclear tumors in mice and verify the anti-tumor effect; finally, sections of the main organs of the mice were examined to verify the safety of the therapy.

The design and preparation of RNA nanoadjuvants with different side lengths are shown in Figure 7.

Among them, the shape of the RNA nanoadjuvant can be a single-layer two-dimensional structure such as a rhombus or a rectangle, with a thickness of about 2nm; the side length of the RNA nanoadjuvant can be 77bp, 143bp, 209bp, etc., and the width of the RNA nanostructure can be 5 to 10 RNA duplexes.

Preferably, in the synthetic preparation step (1), the proportion of RNA sequence may be 45%, 50%, 55%, or 58%.

Preferably, in the synthesis preparation step (2), the maximum annealing temperature of the RNA nanoadjuvant can be adjusted to 90°C, 85°C, 80°C, 75°C, and 70°C. The annealing cooling rate can be 30min/℃, 20min/℃, 10min/℃, 5min/℃.

Preferably, in the synthesis preparation step (2), the annealing concentration of the RNA nanoadjuvant can be 5 mg/mL, 2 mg/mL, 1 mg/mL, 0.5 mg/mL, 0.2 mg/mL, or 0.1 mg/mL.

Preferably, in the anti-tumor effect verification step (3), the concentration of the RNA adjuvant can be 5 mg/mL, 2 mg/mL, 1 mg/mL, 0.5 mg/mL, 0.2 mg/mL, or 0.1 mg/mL. The dosage of a single RNA adjuvant can be 200ug, 100ug, 50ug, 25ug, 10ug, 5ug.

Example 1

Preparation of RNA nanoadjuvant:

The synthesis method is as follows:

(1) Use Tiamat to design and construct the target RNA adjuvant sequence, and construct a plasmid containing the T7 RNA polymerase binding site, RNA adjuvant sequence and restriction endonuclease (BamHI).

(2) Use NEB restriction endonuclease to linearize the plasmid and purify it.

(3) Take 1ug of linearized plasmid, use NEB HiScribe T7 High Yield RNA synthesis kit to synthesize single-stranded RNA, and purify it.

(4) Take 200 micrograms of single-stranded RNA and dissolve it in 200 microliters of 1X PBS solution. Heat the solution at 95 degrees Celsius for 5 minutes, then cool it from 65 degrees Celsius to 10 degrees Celsius. The cooling rate is one degree every 15 minutes. The cooling is completed. You can get the sample.

Quality and structural characterization of RNA nanoadjuvants. Brucker atomic force microscope was used for characterization. 20ng of the sample was placed on a mica sheet, and 30 microliters of 5mM nickel chloride solution was added dropwise. Then the Scan-Analyst mode was used for liquid phase characterization. The morphology characterization picture is shown in Figure 1.

Example 2

Verification of the innate immune activation effect of RNA adjuvant:

(1) Prepare mouse pancreatic cancer subcutaneous tumor model.

(2) Inject 50 micrograms of RNA adjuvant into the mouse tumor through intratumoral injection, and each group of experiments was set up with 3 mice.

(3) After 24 hours, sacrifice the above mice, remove the tumor tissue, monodisperse the tumor tissue into a cell suspension, and use CD45+ magnetic beads to screen immunological cells.

(4) Send the obtained CD45+ immune cells to the BD Rhapsody ^TM single cell analysis system for single cell sequencing analysis to obtain the transcriptome information of the innate immune cells of different experimental groups.

(5) Analyze the phenotypic and functional information of macrophage groups, and compare the anti-cancer functions of different adjuvants (experimental conditions) on macrophages in mouse tumors.

The results obtained in step (5) of this embodiment are shown in Figure 5. It can be seen from the figure that RNA nanostructures can activate the anti-cancer function of macrophages more than polyIC.

Example 3

Analysis of the enhanced anti-cancer effect of RNA adjuvants:

(1) Construct a mouse pancreatic cancer subcutaneous tumor model.

(2) Dosage according to the dosing plan shown in Figure 6a, in which RNA nano-adjuvant is 50 μg each time, polyIC is 50 μg each time, and PD-L1 antibody is 10 μg each time.

(3) According to the mouse tumor growth status, measure the mouse tumor volume every 2-3 days.

The mouse tumor volume and growth curves obtained in this example are shown in Figures 6b and 6c. RNA adjuvant inhibits tumor growth and can enhance the tumor therapeutic effect of aPD-L1 antibody.

Claims (10)

1. A single-stranded two-dimensional RNA nanostructure of a specific shape and size, the nanostructure being rectangular or diamond-shaped in shape, the nanostructure having a side length of greater than 80bp, more preferably greater than 100bp.

2. The RNA nanostructure of the specific shape and size of claim 1, which is rectangular with sides of 143bp and 209bp.

3. The RNA nanostructure of the specific shape and size of claim 1, which is diamond-shaped with a side length of 143bp.

4. Use of the RNA nanostructure of any one of claims 1-3 for the preparation of a macrophage activator.

5. Use of the RNA nanostructure of any one of claims 1-3 for the preparation of an immune activator.

6. Use of the RNA nanostructure of any one of claims 1-3 for the preparation of a cancer therapeutic.

7. A pharmaceutical composition or pharmaceutical combination comprising the RNA nanostructure of any one of claims 1-3 and an immune checkpoint inhibitor.

8. Use of the pharmaceutical composition or combination of claim 7 for the preparation of a cancer therapeutic.

9. The pharmaceutical use according to claim 6 or 7, wherein the cancer is a solid tumor, preferably pancreatic cancer.

10. The pharmaceutical composition or combination according to claim 7, or the pharmaceutical use according to any one of claims 8 or 9, wherein the immune checkpoint inhibitor is an aPD-L1 antibody.