Sting protein mutant, nucleic acid encoding sting protein mutant, and use of sting protein mutant

Abstract

Provided are a STING mutant protein, a nucleic acid encoding the protein, a vector, a composition or vaccine comprising the protein or nucleic acid or vector, and a use of the protein in stimulating an immune response or treating cancer.

Description

STING protein mutants, nucleic acids encoding same, and uses thereof Technical Field

[0001] The present application relates to the field of molecular biology technology, and specifically to the use of STING mutants to change the tumor microenvironment, thereby activating the body's immune system's immune surveillance of tumor cells; the present application also relates to the use of STING mutants in treating tumors. Background Art

[0002] The cGAS-STING signaling pathway is an important signaling pathway for monitoring abnormal cytoplasmic DNA in cells. When host cells are invaded by pathogens such as viruses, the pathogen DNA will be recognized by cGAS in the cytoplasm. The activated cGAS will catalyze the formation of cGAMP using ATP and GTP as substrates. cGAMP is a cyclic dinucleotide that can bind to and activate STING located on the endoplasmic reticulum. The activated STING will transfer to the Golgi apparatus and recruit and activate TBK1 in the process, further leading to the phosphorylation and nuclear translocation of IRF3 and NF-κB. The activation of IRF3 will induce the expression of type I interferon (Type I IFN) and inflammatory factors, activating the immune response.

[0003] The cGAS-STING signaling pathway not only recognizes exogenous pathogen DNA but also abnormally elevated levels of DNA in the cell's own cytoplasm. For example, in tumor cells, defects in DNA repair proteins can lead to genetic instability, resulting in elevated cytoplasmic DNA and activation of the cGAS-STING signaling pathway. To evade immune surveillance caused by endogenous cGAS-STING activation in tumor cells, many cancers have evolved defects in the STING signaling pathway. It is currently believed that tumors suppress the STING signaling pathway through epigenetic modifications such as DNA methylation.

[0004] Therefore, reactivating the STING signaling pathway in tumors may reactivate the immune system's immune surveillance of tumor cells. Activating STING in antigen-presenting cells, such as DCs, may alter the tumor microenvironment by releasing type I interferons (Type I IFNs) and inflammatory factors, thereby activating the immune system's immune surveillance of tumor cells.

[0005] SUMMARY OF THE INVENTION

[0006] This application is based on constitutive mutagenesis targeting STING (stimulator of interferon genes), resulting in a series of human STING protein mutants with excellent STING pathway activation activity. The amino acid positions of the human STING protein mutants described in this application are numbered as defined by the corresponding amino acid positions in SEQ ID NO: 1. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIG1 shows the detection results of the expression of downstream pathways activated by the STING gain-of-function mutation independently designed by the present invention in Example 2.

[0008] Detailed Description of the Invention

[0009] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide a general definition of many of the terms used in this invention for those skilled in the art: Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2 nd ed.1994); THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY(Walkered.,1988); THE GLOSSARY OF GENETICS,5 th ED., R.Rieger, et al. (eds.), Springer Verlag (1991); and Hale and Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY (1991).

[0010] Every publication, patent application, patent, and other reference cited herein is hereby incorporated by reference in its entirety to the extent not inconsistent with this disclosure.

[0011] STING: As used herein, the term "stimulator of interferon genes" or "STING" includes, but is not limited to, nucleic acids, polynucleotides, oligonucleotides, sense and antisense polynucleotide chains, complementary sequences, peptides, polypeptides, proteins, homologous and / or orthologous STING molecules, isoforms, precursors, mutants, variants, derivatives, splice variants, alleles, different species, and active fragments thereof.

[0012] STING gain-of-function mutants refer to STING protein mutants that result in constitutive STING activity. STING gain-of-function mutants are also STING variant polynucleotides that harbor mutations in wild-type STING proteins. Exemplary constitutively active mutations herein include, but are not limited to, V155M, V155M-K289R-K338R, L170E, A233E, I235E, D237R, D237K, S275Q, V155W, N188V, or L190V mutations of SEQ ID NO: 1. The STING V155M mutation is known in the art, while the other mutations are STING mutants independently designed and prepared by the present invention.

[0013] Coding sequence: This refers to the ribonucleotide sequence in mature mRNA that can be translated into protein, or the complementary sequence of the deoxyribonucleotide (DNA) sequence that serves as a template for transcription of the ribonucleotide (RNA) sequence. Furthermore, the term "coding sequence" herein may further include polynucleotide sequences encoding functional nucleic acids, such as miRNA, shRNA, dsRNA, and the like.

[0014] The term "5' cap" is located at the 5' end of the mRNA and contains methylated guanylate, which is linked to the 5' end of the mRNA via pyrophosphate to form a 5',5'-triphosphate linkage with its adjacent nucleotide. There are usually three types of 5' cap structures (m7G5'ppp5'Np, m7G5'ppp5'NmpNp, m7G5'ppp5'NmpNmpNp), which are called type O, type I and type II, respectively. Type O refers to the unmethylated ribose of the terminal nucleotide, type I refers to the methylation of the ribose of one terminal nucleotide, and type II refers to the methylation of the ribose of both terminal nucleotides. In this article, "CleanCap AG" is used to refer to the m7G(5')ppp(5')(2'-OMeA)pG cap.

[0015] The term "poly-A tail" or "poly-A sequence" refers to an uninterrupted or interrupted sequence of adenosine residues typically located at the 3'-end of an RNA molecule. Poly-A tails and poly-A sequences are known to those skilled in the art and can be selected based on practical needs. In mRNA, if a 3'-UTR is present, the poly-A sequence is attached to the 3' end of the 3'-UTR. An uninterrupted poly-A tail is characterized by a continuous sequence of adenosine residues. The poly-A tail can be of any length. In some embodiments, the poly-A tail comprises or consists of at least 20, at least 30, at least 40, at least 80, or at least 100, and at most 500, at most 400, at most 300, at most 200, or at most 150 adenosine residues (A), particularly about 120 A's. Typically, the majority of the nucleotides in the poly-A tail are adenosine, meaning at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%. or at least 99% of the nucleotides, but allowing the remaining nucleotides to be nucleotides other than A, such as U (uridylic acid), G (guanylic acid) or C (cytidylic acid).

[0016] As used herein, percentages of "identity," such as 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% identity, refer to a degree of similarity between amino acid sequences or nucleotide sequences determined by sequence alignment of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5%. For example, the percentage of positions with identical bases or amino acid residues is determined as a ratio of the total number of positions after two sequences have been aligned to have identical residues at as many positions as possible, such as by introducing gaps. Percentages of "identity" can be determined using software programs known in the art. Preferably, the alignment is performed using default parameters. A preferred alignment program is BLAST. Preferred programs are BLASTN and BLASTP. Details of these programs can be found on the Internet at the following address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0017] As used herein, "delivery body" refers to a structure that is packaged or wrapped to assist larger biomolecules such as polynucleotides and polypeptides to enter the cell and to form a structure with a higher affinity for the cell membrane and easier to transport across the membrane from the extracellular to the intracellular. Delivery bodies and their preparation methods are all known in the art, including but not limited to liposomes (such as lipid nanoparticles (LNP)), viruses (such as AAV, lentivirus), and quantum dots. The preparation method of LNP is known in the art, such as those disclosed in CN114901360A and CN113941011A. In some embodiments, the LNP comprises a PEG-modified lipid, a non-cationic lipid, a sterol, an ionizable lipid, or any combination thereof.

[0018] As used herein, the term "adjuvant" refers to an exogenous substance added to a pharmaceutical composition or formulation to enhance the response of an individual's immune system to an antigen, including but not limited to chemical adjuvants and bacterial antigens.

[0019] As used herein, an "open reading frame (ORF)" is a continuous DNA or RNA segment that begins with a start codon, such as a methionine codon (ATG or AUG), and ends with a stop codon, such as TAA, TAG, or TGA, or UAA, UAG, or UGA. An ORF typically encodes a protein.

[0020] "Variant" refers to a sequence or molecule that retains the same or substantially the same biological activity as the original sequence. The variant may be from the same or a different species, or may be a synthetic sequence based on a naturally occurring molecule or an existing molecule. In this application, "variant" may be used to refer to a variant of a protein, polypeptide, or amino acid sequence, and may also be used to refer to a variant of a nucleic acid molecule or polynucleotide sequence.

[0021] In some embodiments, a "variant" of the amino acid sequence has at least one amino acid difference relative to the amino acid sequence, for example, at least one amino acid addition, insertion, deletion, or substitution. For example, the amino acid substitution can be a conservative amino acid substitution, i.e., replacing the original corresponding amino acid with an amino acid having similar properties. "Conservative substitutions" can be polar to polar amino acids, such as glycine (G, Gly), serine (S, Ser), threonine (T, Thr), tyrosine (Y, Tyr), cysteine ​​(C, Cys), asparagine (N, Asn), and glutamine (Q, Gln); non-polar to non-polar amino acids, such as alanine (A, Ala), valine (V, Val), tryptophan (W, Trp), leucine (L, Leu), proline (P, Pro), methionine (M, Met), phenylalanine (F, Phe); acidic to acidic amino acids, such as aspartic acid (D, Asp), glutamic acid (E, Gln); u); basic to basic amino acids, such as arginine (R, Arg), histidine (H, His), lysine (K, Lys); charged amino acids to charged amino acids, such as aspartic acid (D, Asp), glutamic acid (E, Glu), histidine (H, His), lysine (K, Lys) and arginine (R, Arg); hydrophobic to hydrophobic amino acids, such as alanine (A, Ala), leucine (L, Leu), isoleucine (I, Ile), valine (V, Val), proline (P, Pro), phenylalanine (F, Phe), tryptophan (W, Trp) and methionine (M, Met). In some other embodiments, the variant may also comprise non-conservative substitutions. In some embodiments, the "variant" of the amino acid sequence may have at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity relative to the amino acid sequence. Compared to the amino acid sequence, the "variant" of the amino acid sequence may have an activity of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% or a range consisting of any two of the aforementioned values. As used herein, a "conservative substitution variant" of a protein, polypeptide or amino acid sequence refers to one or more amino acid residues that undergo amino acid substitution without changing the overall conformation and function of the protein or enzyme, including but not limited to replacing the amino acids in the amino acid sequence of the parent protein in the manner described by the aforementioned "conservative substitution". Therefore, the similarity of two proteins or amino acid sequences with similar functions may be different. For example, a similarity (identity) of 70% to 99% based on the MEGALIGN algorithm."Conservative substitution variants" also include polypeptides or enzymes having more than 60% amino acid identity as determined by BLAST or FASTA algorithms, preferably more than 75%, preferably more than 85%, and even more than 90%, and having the same or substantially similar properties or functions as the native or parent protein or enzyme.

[0022] Therefore, the STING protein mutants described in the present application should include the above variants.

[0023] Those skilled in the art will be aware that variants of protein-encoding nucleic acid molecules or polynucleotide sequences include "synonymous mutants", which refer to nucleic acid molecules or polynucleotide sequences obtained by replacing one or more codons in the nucleic acid molecule or polynucleotide sequence with other codons encoding the same amino acid as the codons.

[0024] The term "at least comprising" means that the polynucleotide sequence may consist of the coding sequence of the aforementioned STING protein mutant, or may further comprise other polynucleotide sequences in addition to comprising the coding sequence of the aforementioned STING protein mutant.

[0025] For example, sequences that regulate the expression of the aforementioned STING protein mutants, and sequences that make the polynucleotide more stable.

[0026] The polynucleotide sequence may be a DNA sequence, an RNA sequence, or a hybrid of a DNA sequence and an RNA sequence.

[0027] As used herein, the term "treating" refers to obtaining a desired pharmacological and / or physiological effect. The effect may be complete or partial prevention of the occurrence or onset of a disease or its symptoms, partial or complete alleviation of a disease and / or its symptoms, and / or partial or complete cure of a disease and / or its symptoms, including: (a) preventing the occurrence or onset of a disease in a subject who may have a predisposition to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., blocking its development; and (c) alleviating the disease and / or its symptoms, i.e., causing the disease and / or its symptoms to subside or disappear.

[0028] The term "subject" in this application refers to mammals, including but not limited to murines (rats, mice), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cows, sheep, pigs, goats), etc.

[0029] "Therapeutically effective amount" or "effective amount" refers to an amount sufficient to achieve the described prevention and / or treatment of the disease when administered to a mammal or other subject for the treatment of a disease. The "therapeutically effective amount" will vary depending on the drug used, the severity of the disease and / or its symptoms, and the age, weight, etc. of the subject to be treated. A person skilled in the art can readily determine the appropriate therapeutically effective amount and frequency of administration of the protein or composition of the present invention based on various parameters, particularly the age, weight and condition of the subject to be treated, the severity of the disease or condition, and the route of administration. Routes of administration include, but are not limited to, enteral, topical, suppository, inhalation, and parenteral administration, such as subcutaneous, intramuscular, or intravenous injection. DETAILED DESCRIPTION

[0030] The present application provides STING mutant proteins and nucleic acids encoding the same for reactivating the STING signaling pathway in tumors, delivery vehicles, cells, pharmaceutical compositions or pharmaceutical products comprising the STING mutant proteins or nucleic acids encoding the same, and uses of the nucleic acids or proteins.

[0031] Specifically, in the first aspect of the present application, an engineered human STING protein mutant is provided, wherein the mutation comprises (1) V155W; (2) a combination of V155M-K289R-K388R; (3) S275Q; (4) L170E; (5) A233E; (6) I235E; (7) D237R; (8) D237K; (9) L190V or (10) N188V; the amino acid position numbers are as defined by the corresponding amino acid positions shown in SEQ ID NO: 1.

[0032] In some embodiments, the amino acid sequence of the STING protein mutant comprises or consists of the following amino acid sequence: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12, or a sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0033] In a second aspect of the present application, a nucleic acid molecule encoding any of the aforementioned STING protein mutants is provided; preferably, the nucleic acid molecule is DNA or RNA.

[0034] In some embodiments, the nucleic acid molecule comprises a 5'UTR structure; preferably, the 5'UTR structure comprises at least the polynucleotide sequence shown in SEQ ID NO: 13, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity with SEQ ID NO: 13.

[0035] In some embodiments, the nucleic acid molecule further comprises a 3'UTR structure; preferably, the 3'UTR structure comprises at least the polynucleotide sequence shown in SEQ ID NO: 14, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity with SEQ ID NO: 14.

[0036] In some embodiments, the nucleic acid molecule is mRNA.

[0037] In some embodiments, the open reading frame (ORF) sequence of the mRNA is selected from any one of the sequences shown in SEQ ID NOs: 18-27, or a sequence having at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0038] In some embodiments, part or all of the uridine in the mRNA is chemically modified uridine; preferably, the chemically modified uridine is pseudouridine or N1-methyl-pseudouridine.

[0039] In some embodiments, the mRNA further comprises a 5' cap structure; preferably, the 5' cap structure is m7G(5')ppp(5')(2'-OMeA)pG.

[0040] In some embodiments, the mRNA further comprises a polyA tail; preferably, the polyA tail comprises at least 50, at least 60 or at least 100 A nucleotides; more preferably, the polyA tail comprises at least the polynucleotide sequence as shown in SEQ ID NO: 15, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity with SEQ ID NO: 15.

[0041] In some embodiments, the sequence of the nucleic acid molecule is shown in any one of SEQ ID NOs: 30-39.

[0042] The present application also relates to nucleic acid molecules complementary to the aforementioned nucleic acid molecules.

[0043] In the third aspect of the present application, an expression vector is provided, which comprises any one of the nucleic acid molecules described above; wherein the vector is a viral vector or a plasmid vector.

[0044] In some embodiments, the viral vector is selected from the group consisting of vesicular stomatitis virus (VSV), a lentivirus, an adenovirus, an adeno-associated virus, a vaccinia virus, and a modified vaccinia Ankara virus.

[0045] In the fourth aspect of the present application, a delivery vehicle is provided, comprising any one of the protein mutants or nucleic acid molecules described above.

[0046] In some embodiments, the delivery vehicle is a lipid nanoparticle (LNP) or a lipid complex (LPX).

[0047] In some embodiments, the LNP comprises ionizable lipids, phospholipids, cholesterol, and polyethylene glycol (PEG)-lipids.

[0048] In a fifth aspect of the present application, an isolated cell is provided, comprising any one of the protein mutants, nucleic acid molecules or vectors described above;

[0049] Preferably, the cell is selected from the group consisting of insect cells, mammalian cells, avian cells, bacteria and yeast cells; preferably the cell is an E. coli cell.

[0050] In the sixth aspect of the present application, a pharmaceutical composition is provided, comprising any one of the protein mutants, nucleic acid molecules, vectors, delivery bodies or cells described above, and a pharmaceutically acceptable excipient.

[0051] In some embodiments, the pharmaceutical composition is an mRNA vaccine.

[0052] In the seventh aspect of the present application, a method for activating STING in a subject in need is provided, comprising administering the engineered human STING protein mutant described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the delivery body described in the fourth aspect, the isolated cell described in the fifth aspect, or the pharmaceutical composition described in the sixth aspect, which induces STING signaling.

[0053] In some embodiments, the present application provides a method of stimulating an immune response in a subject in need thereof, comprising administering the engineered human STING protein mutant of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the delivery body of the fourth aspect, the isolated cell of the fifth aspect, or the pharmaceutical composition of the sixth aspect, which induces STING signaling.

[0054] In some embodiments, the subject has cancer or a microbial infection.

[0055] In some embodiments, the present application provides a method for treating cancer in a subject, comprising administering the engineered human STING protein mutant of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the delivery body of the fourth aspect, the isolated cell of the fifth aspect, or the pharmaceutical composition of the sixth aspect, which induces STING signaling.

[0056] In some embodiments, the cancer is ovarian cancer, colon cancer, melanoma, breast cancer, or lung cancer.

[0057] In some embodiments, the composition is administered intratumorally, intravenously, intraarterially, intraperitoneally, intranasally, intramuscularly, intradermally, or subcutaneously.

[0058] In some embodiments, the STING protein mutant or the composition induces immune cell infiltration into a tumor.

[0059] In the eighth aspect of the present application, an adjuvant is provided, which comprises the engineered human STING protein mutant described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the delivery body described in the fourth aspect, the isolated cell described in the fifth aspect or the pharmaceutical composition described in the sixth aspect, which has the effect of inducing STING signal transduction.

[0060] In some embodiments, the present application provides a method for enhancing the immune response to a vaccine in a subject receiving the vaccine, the method comprising administering an adjuvant as described above.

[0061] In the ninth aspect of the present application, provided is the use of the aforementioned STING protein mutant, nucleic acid molecule, vector, delivery vehicle, cell or pharmaceutical composition in the preparation of a drug for the treatment and / or prevention of cancer or microbial infection.

[0062] It should be understood that the present application includes various aspects, embodiments and combinations of the aspects and / or embodiments described herein. The above description and subsequent examples are intended to illustrate rather than limit the scope of the present application. Other aspects, improvements and modifications within the scope of the present application will be apparent to those skilled in the art. Therefore, those of ordinary skill in the art will recognize that the scope of the present application also includes the improvements and modifications to the aspects and embodiments.

[0063] Example

[0064] Example 1: Construction and preparation of mRNA sequences encoding STING mutants

[0065] 1.1 Synthesis of STING mRNA sequence and construction of recombinant vector

[0066] From the 5' end, the following sequence is: T7 promoter with XbaI at the 5' end, 5'UTR, tPA-SP, Flt3L, STING mutant ORF, 3'UTR and / or poly A tail coding nucleotide sequence. They were double-digested with XbaI and NotI, and ligated with the pUC57-GW-Kan (Jinweizhi) vector backbone fragment digested with XbaI and NotI to construct a recombinant plasmid.

[0067] 1.2 mRNA preparation

[0068] 1.2.1 Plasmid linearization

[0069] The recombinant plasmid constructed in step 1 has a Sap I restriction site after the last A in the polyA tail sequence. Linearize the plasmid containing the target gene with the restriction endonuclease Sap I using the reaction system shown in Table 1. Incubate the enzyme digestion at 37°C for 3 hours.

[0070] Table 1. Plasmid linearization enzyme digestion system

[0071] 2 μL of the digested product was subjected to 1% agarose gel electrophoresis to check the linearization of the plasmid. The linearized plasmid was purified using a PCR product recovery kit (Comvison).

[0072] (2) In vitro transcription and purification

[0073] The linearized recombinant plasmid obtained in step (1) was used as a template for in vitro transcription using a high-yield T7 RNA transcription kit. The high-yield T7 RNA transcription kit, product name is High Yield T7 RNA Synthesis Kit, Shanghai Zhaowei Technology Development Co., Ltd., product catalog number is ON-040; 5× Reaction Buffer, 100mM ATP Solution, 100mM CTP Solution, 100mM GTP Solution, Enzyme mix, DNase I, Ammonium Acetate Stop Solution, Lithium Chloride (LiCl) Precipitation Solution are all components of the high-yield T7 RNA transcription kit. 100mM ΨUTP Solution (pseudouridine triphosphate), full name N1-Me-pUTP, 100mM, Shanghai Zhaowei Technology Development Co., Ltd., product catalog number is R5-027. Add each component according to the following system (Table 2) (taking 20μL reaction system as an example), mix well, and react at 37℃ for 3h.

[0074] Table 2. In vitro transcription system

[0075] Among them, CleanCap AG is m7G(5')ppp(5')(2'-OMeA)pG, product number is ON-134, Shanghai Zhaowei.

[0076] After the transcription reaction is completed, add 1 μL of DNase I and react at 37°C for 15 minutes. Add 15 μL of Ammonium Acetate Stop Solution and mix well. Then add 1 / 3 volume of 7.5M Lithium Chloride (LiCl) Precipitation Solution (to a final concentration of 2.5M) and incubate at -20°C for 30 minutes. Centrifuge at 12,000g for 15 minutes to allow the RNA to precipitate at the bottom. Discard the supernatant. Add 1 mL of 70% ethanol to wash the RNA. Centrifuge at 12,000g for 5 minutes and discard the supernatant. After drying, add 50 μL of RNase-free water to dissolve the precipitate and quantify the mRNA using a UV spectrophotometer to obtain capped in vitro transcribed mRNA.

[0077] Example 2: Detecting the activity of the STING gain-of-function mutant designed by the present invention by activating the STING downstream pathway

[0078] THP-1 cells were seeded in 12-well cell culture dishes at a density of 2*10^5 per well at 37°C and 5% CO 2 Cultured overnight in an incubator. TM 3000 cells / well were transfected with 200 ng of STING mRNA. 6 h later, total RNA was extracted using the FastPure Cell / Tissue Total RNA Isolation Kit V2, cDNA was synthesized using the Bio-Rad iScriptTM cDNAsynthesis kit, and the reaction system was prepared using ChamQ Universal SYBBR qPCR Master Mix according to the instructions for qPCR experiments to detect the transcription level of IFN-β in each group.

[0079] As shown in Figure 1, each group showed a certain degree of IFN-β level, indicating that each STING mutant has the activity of activating the downstream pathway, among which the V155W mutant, V155M-K289R-K338R combination mutant, V155M, and S275Q groups performed the best. Among the four groups, the IFN-β transcription level of the V155M-K289R-K338R combination mutant group was significantly different from that of the V155M mutant group (p < 0.01), and the IFN-β transcription level of the V155W mutant group was extremely significantly different from that of the V155M mutant group (p < 0.001). The above results show that the STING mutants independently designed by the present invention exhibit STING pathway activation levels that are comparable to or even significantly better than those of the prior art.

[0080] The sequences used in the above examples of the present application are shown in the following sequence listing. It should be understood that the following sequences are merely exemplary sequences of the embodiments of the present application and are not intended to limit the present application. The nucleic acid sequences in the following sequence listing may represent DNA sequences or RNA sequences. When representing RNA sequences, "T" represents uridine.

[0081] Sequence Listing

Claims

1. An engineered human STING protein mutant, wherein the mutation comprises (1) V155W; (2) a combination of V155M-K289R-K388R; (3) S275Q; (4) L170E; (5) A233E; (6) I235E; (7) D237R; (8) D237K; (9) L190V or (10) N188V, wherein the amino acid position numbers are as defined by the corresponding amino acid positions shown in SEQ ID NO:

1.

2. The STING protein mutant according to claim 1, wherein the amino acid sequence of the mutant comprises or consists of the following amino acid sequence: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12, or a sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical thereto.

3. A nucleic acid molecule encoding the STING protein mutant according to claim 1 or 2, preferably, the nucleic acid molecule is DNA or RNA.

4. The nucleic acid molecule according to claim 3, comprising a 5'UTR structure, Preferably, the 5'UTR structure comprises at least the polynucleotide sequence shown in SEQ ID NO: 13, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity with SEQ ID NO:

13.

5. The nucleic acid molecule according to claim 3 or 4, further comprising a 3'UTR structure, Preferably, the 3'UTR structure comprises at least the polynucleotide sequence shown in SEQ ID NO: 14, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity with SEQ ID NO:

14. The nucleic acid molecule according to any one of claims 3 to 5 , which is mRNA.

7. The nucleic acid molecule according to any one of claims 3 to 6, wherein the open reading frame (ORF) sequence is selected from any one of the sequences shown in SEQ ID NOs: 18 to 27, or a sequence having at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

8. The nucleic acid molecule according to any one of claims 3 to 7, wherein some or all of the uridines in the mRNA are chemically modified uridines; Preferably, the chemically modified uridine is pseudouridine or N1-methyl-pseudouridine.

9. The nucleic acid molecule according to any one of claims 3 to 8, wherein the mRNA further comprises a 5' cap structure; preferably, the 5' cap structure is m7G(5')ppp(5')(2'-OMeA)pG.

10. The nucleic acid molecule according to any one of claims 3 to 9, wherein the mRNA further comprises a polyA tail; preferably, the polyA tail comprises at least 50, at least 60 or at least 100 A nucleotides; More preferably, the polyA tail comprises at least the polynucleotide sequence shown in SEQ ID NO: 15, or a polynucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% sequence identity with SEQ ID NO:

15. The nucleic acid molecule according to claim 3 , wherein the sequence of the nucleic acid molecule is shown in any one of SEQ ID NOs: 30-39.

12. A nucleic acid molecule complementary to the nucleic acid molecule of any one of claims 3 to 11.

13. An expression vector comprising the nucleic acid molecule according to any one of claims 3 to 12; wherein the vector is a viral vector or a plasmid vector.

14. The expression vector according to claim 13, wherein the viral vector is selected from the group consisting of vesicular stomatitis virus (VSV), lentivirus, adenovirus, adeno-associated virus, vaccinia virus and modified vaccinia Ankara virus.

15. A delivery system comprising the protein mutant according to claim 1 or 2 or the nucleic acid molecule according to any one of claims 3 to 12.

16. The delivery body of claim 15, wherein the delivery body is a lipid nanoparticle (LNP) or a lipid complex (LPX).

17. The delivery system of claim 16, wherein the LNP comprises ionizable lipids, phospholipids, cholesterol and polyethylene glycol (PEG)-lipids; preferably, in the LNP, the molar ratio of ionizable lipids, phospholipids, cholesterol and polyethylene glycol (PEG)-lipids is (40-55):(10-15):(35-45):(0.5-2.5).

18. An isolated cell comprising the protein mutant according to claim 1 or 2, the nucleic acid molecule according to any one of claims 3 to 12, or the vector according to claim 13 or 14; Preferably, the cell is selected from the group consisting of insect cells, mammalian cells, avian cells, bacteria and yeast cells; preferably the cell is an E. coli cell.

19. A pharmaceutical composition comprising the protein mutant according to claim 1 or 2, the nucleic acid molecule according to any one of claims 3-12, the vector according to claim 13 or 14, the delivery body according to any one of claims 15-17 or the cell according to claim 18, and a pharmaceutically acceptable excipient.

20. The pharmaceutical composition according to claim 19, which is an mRNA vaccine.

21. A method for activating STING in a subject in need thereof, comprising administering the protein mutant of claim 1 or 2, the nucleic acid molecule of any one of claims 3-12, the vector of claim 13 or 14, the delivery vehicle of any one of claims 15-17, the cell of claim 18, or the pharmaceutical composition of claim 19 or 20, which induces STING signaling.

22. A method of stimulating an immune response in a subject in need thereof, comprising administering the protein mutant of claim 1 or 2, the nucleic acid molecule of any one of claims 3-12, the vector of claim 13 or 14, the delivery body of any one of claims 15-17, the cell of claim 18, or the pharmaceutical composition of claim 19 or 20, which induces STING signaling.

23. The method of claim 21, wherein the subject has cancer or a microbial infection.

24. A method of treating cancer in a subject, comprising administering the protein mutant of claim 1 or 2, the nucleic acid molecule of any one of claims 3-12, the vector of claim 13 or 14, the delivery body of any one of claims 15-17, the cell of claim 18, or the pharmaceutical composition of claim 19 or 20, which induces STING signaling.

25. The method of claim 24, wherein the cancer is ovarian cancer, colon cancer, melanoma, breast cancer, or lung cancer.

26. The method of any one of claims 21-25, wherein the composition is administered intratumorally, intravenously, intraarterially, intraperitoneally, intranasally, intramuscularly, intradermally, or subcutaneously.

27. The method according to any one of claims 21-26, wherein the STING protein mutant or the composition induces immune cell infiltration into the tumor.

28. An adjuvant comprising the protein mutant according to claim 1 or 2, the nucleic acid molecule according to any one of claims 3-12, the vector according to claim 13 or 14, the delivery body according to any one of claims 15-17, the cell according to claim 18, or the pharmaceutical composition according to claim 19 or 20, which has the function of making the STING protein constitutively active.

29. A method of enhancing the immune response to a vaccine in a subject receiving the vaccine, the method comprising administering the adjuvant of claim 28.

30. Use of the protein mutant according to claim 1 or 2, the nucleic acid molecule according to any one of claims 3-12, the vector according to claim 13 or 14, the delivery body according to any one of claims 15-17, the cell according to claim 18 or the pharmaceutical composition according to claim 19 or 20 in the preparation of a medicament for the treatment and / or prevention of cancer or microbial infection.

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