CN117947031B - Application of CGT-ODN in immunoregulation - Google Patents
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Abstract
The invention discloses application of CGT-ODN in immunoregulation, discloses a CGT-ODN nucleotide sequence, and simultaneously discloses a vector and a pharmaceutical composition containing the CGT-ODN nucleotide sequence, and also provides application of the GT-ODN nucleotide sequence, the vector and the pharmaceutical composition in immunoregulation, and application of the GT-ODN nucleotide sequence and the vector in preparation of pharmaceutical compositions for treating or preventing infectious diseases, allergic diseases, immunodeficiency diseases and tumors, and application of the GT-ODN nucleotide sequence and the vector in preparation of vaccines, antibody production or regulation of tRNA degradation degree.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to an application of CGT-ODN in immunoregulation.
Background
Nucleic acids are the major conserved pathogen determinants detected by Pattern Recognition Receptors (PRRs) that initiate the innate immune response. Extracellular bacterial single-stranded DNA (ssDNA) containing CpG motifs and synthetic mimetic CpG Oligonucleotides (ODNs) can be internalized by immune cells and recognized by Tolllike receptor 9 (TLR 9) in endosomes. CpG ODNs have shown great potential against cancer, infection and allergic diseases.
The first type of PRRs to be identified is Toll-like receptors (TLRs), which are membrane proteins that recognize ligands of extracellular or endosomal pathogens. However, some pathogens may evade detection of TLRs and enter the host cytoplasm or nucleus. Thus, most TLRs have intracellular complementary PRRs that sense similar ligands, such as TLR3 and RIG-I/MDA5 for RNA, and TLR4 and caspase for Lipopolysaccharide (LPS). Toll-like receptor 9 (TLR 9) is a specific receptor for CpG motifs in ssDNA that internalize into endosomes, but it is "blind" to cytoplasmic or nucleic acid ssDNA, expressed primarily in professional immune cells, indicating the presence of widely expressed intracellular ssDNA PRRs. However, to date, such TLR9 complementary receptors that specifically detect intracellular ssDNA have not been specifically identified.
Bacterial ssDNA was identified as a key active ingredient for Bacillus Calmette Guerin (BCG) immunotherapy several decades ago when TLR9 was cloned, a strategy closely related to the kohlrabi toxin. Unmethylated CpG motifs were subsequently found to be critical for the immunostimulatory activity of certain immunocytotic ssDNA and synthetic Oligonucleotides (ODNs), which led to various clinical trials of CpG-ODN based vaccine adjuvants, anti-tumor therapies, and treatments of allergic and infectious diseases. Notably, the delivery systems used in these applications, as well as antisense oligonucleotide (ASO) therapy, ssDNA donor-based gene editing and endocytosis, can also lead to accumulation of ssDNA in the cytoplasm and nucleus. However, it is currently unclear whether and how intracellular ssDNA stimulates the innate immune response.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention provides the following technical scheme:
the invention provides a CGT-ODN nucleotide sequence, which is shown as follows:
5’-(X)nCGT/A(X)m(G)a(X)b(G)c(X)d-3’,
Wherein X is any nucleotide base, n represents any integer number not less than 0, m represents any integer number not less than 0, a represents any integer number not less than 0, b represents any integer number not less than 0, C represents any integer number not less than 0, d represents any integer number not less than 0, C represents a member selected from cytosine and 5-methylcytosine, G represents a member selected from guanine, T represents thymine, A represents adenine, and at least one of a and C is an integer not less than 1;
further, at least one of the values a and c is an integer more than or equal to 2;
Further, the CGT-ODN nucleotide sequence flanking sequences are rich in consecutive G nucleotides;
further, the CGT-ODN nucleotide sequence has a phosphodiester linkage.
In some embodiments, the CGT-ODN nucleotide sequence is absent Phosphorothioate (PS) and 2' -O-methyl backbones, which would disrupt the immunostimulatory activity of the ODN. In some embodiments, the CGT-ODN nucleotide sequence may be a methylated sequence. In some embodiments, the CGT-ODN nucleotide sequence may be a palindromic sequence.
In some embodiments, the CGT-ODN nucleotide sequence requires at least one CGT/A motif, and the flanking sequence, i.e., the downstream sequence, has a contiguous at least 2G nucleotide sequences, which may be arranged directly after the CGT/A motif or may be arranged downstream of the CGT/A motif with additional sequences.
In some embodiments, the CGT-ODN nucleotide sequence does not limit the length of the nucleotide sequence, further, the length of the nucleotide sequence includes, but is not limited to 10 nt、11 nt、12 nt、13 nt、14 nt、15 nt、16 nt、17 nt、18 nt、19 nt、20 nt、21 nt、22 nt、23 nt、24 nt、25 nt、26 nt、27 nt、28 nt、29 nt、30 nt、31 nt、32 nt、33 nt、34 nt、35 nt、36 nt、37 nt、38 nt、39 nt、40 nt、41 nt、42 nt、43 nt、44 nt、45 nt、46 nt、47 nt、48 nt、49 nt、50 nt、51 nt、52 nt、53 nt、54 nt、55 nt、56 nt、57 nt、58 nt、59 nt、60 nt、61 nt、62 nt、63 nt、64 nt、65 nt、66 nt、67 nt、68 nt、69 nt、70 nt、71 nt、72 nt、73 nt、74 nt、75 nt、76 nt、77 nt、78 nt、79 nt、80 nt、81 nt、82 nt、83 nt、84 nt、85 nt、86 nt、87 nt、88 nt、89 nt、90 nt、91 nt、92 nt、93 nt、94 nt、95 nt、96 nt、97 nt、98 nt、99 nt、100 nt、200 nt、300 nt、400 nt、500 nt、1000 nt、10000 nt.
In the present invention, the term "ODN" refers to an oligodeoxynucleotide capable of activating an immune response, and the "ODN" is not particularly limited in nucleotide length in the present invention. In particular, the term "CGT-ODN" refers to an oligodeoxynucleotide having a CGT/A motif and a downstream continuous G nucleotide sequence, which exhibits an immune activation function. The term "continuous G nucleotide sequence" refers to a form in which at least 2 guanine deoxyribonucleotides are continuously coupled via phosphodiester bonds to a CGT-ODN, where the cytosine deoxyribonucleotides in the "CGT" may or may not be methylated, and where the CGT-ODN nucleotide sequence may or may not have a palindromic sequence.
Further, the CGT-ODN nucleotide sequence has at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% identity to the nucleotide sequence set forth in any of SEQ ID NOs 1-15.
The term "identity" as used herein refers to a value determined by comparing two optimally aligned sequences (e.g., nucleic acid sequences) in an alignment window, wherein for optimal alignment of the two sequences, the portion of the sequence in the alignment window may contain additions or deletions (i.e., gaps) as compared to the reference sequence (which does not contain additions or deletions). The percentage is calculated by determining the number of positions in the two sequences where the same nucleotide or amino acid residue is present to produce the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to produce the percentage of sequence identity. The sequence that is identical at each position compared to the reference sequence is said to be 100% identical to the reference sequence and vice versa.
Further, the nucleotide sequence of the CGT-ODN is shown as any one of SEQ ID NO. 1-15.
Further, the nucleotide sequence of the CGT-ODN is shown as any one of SEQ ID NO. 1-9.
Further, the nucleotide sequence of the CGT-ODN is shown as any one of SEQ ID NO 1, 3, 4, 6 and 8.
Further, the nucleotide sequence of the CGT-ODN is shown as SEQ ID NO. 4.
Further, the CGT-ODN nucleotide sequence may be of natural origin or synthetic.
Further, the CGT-ODN nucleotide sequence has a CGT motif, a CGA motif and a CGT+CGA motif.
In some embodiments, the CGT-ODN nucleotide sequence is not limited to a particular biological source, which may include any organism that may have the general structure of the CGT-ODN nucleotide sequence, including, but not limited to, bacteria, viruses, plants, animals, and the like.
The term "nucleotide" or "nucleic acid" as used herein refers to a polydeoxynucleotide (DNA or analogue thereof) of base composition linked by a backbone. In DNA, common bases are adenine (a), guanine (G), thymine (T), and cytosine (C), although nucleic acids may contain base analogs (such as inosine) and abasic positions (e.g., a phosphodiester backbone lacking nucleotides at one or more read positions, U.S. patent No. 5585481).
The invention provides a vector comprising the CGT-ODN nucleotide sequence described above.
Further, the vector includes a plasmid vector, a lentiviral vector, an adenovirus vector, an adeno-associated virus vector, a piggyBac vector, or a sleep Beauty transposition vector.
The term "vector" or "expression vector" as used in the present invention includes any nucleic acid capable of directing the expression of a nucleic acid. The term "vector" includes viral vectors, plasmid vectors and the like. Expression vectors can be delivered to cells using two basic delivery protocols, a viral-based delivery system using viral vectors and a non-viral-based delivery system using, for example, plasmid vectors.
The invention provides a pharmaceutical composition comprising a CGT-ODN nucleotide sequence as hereinbefore described, a carrier as hereinbefore described, and a pharmaceutically acceptable adjuvant.
Further, the pharmaceutically acceptable auxiliary agents include auxiliary agents required for intravenous drip, subcutaneous injection, intravenous bolus injection, intravitreal injection, intramuscular injection.
In some embodiments, the pharmaceutically acceptable adjuvants include diluents, carriers, excipients, and stabilizers, which are non-toxic to the recipient at the dosages and concentrations employed. In some embodiments, the pharmaceutically acceptable adjuvants further include buffers, antioxidants, preservatives, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides, and other carbohydrates, chelating agents, sugars, salt-forming counterions, metal complexes, nonionic surfactants.
In some specific embodiments, the buffering agent comprises phosphates, citrates, and other organic acids. In some specific embodiments, the antioxidant includes ascorbic acid and methionine. In some specific embodiments, the preservative comprises octadecyldimethylbenzyl ammonium chloride, hexamethyl ammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl para-hydroxybenzoates, methyl or propyl para-hydroxybenzoates, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. In some specific embodiments, the protein comprises serum albumin, gelatin, or immunoglobulin. In some specific embodiments, the hydrophilic polymer comprises polyvinylpyrrolidone. In some specific embodiments, the amino acid comprises glycine, glutamine, asparagine, histidine, arginine, or lysine. In some specific embodiments, the monosaccharides, disaccharides, and other carbohydrates include glucose, mannose, or dextrins. In some specific embodiments, the chelator comprises EDTA. In some specific embodiments, the saccharide comprises sucrose, mannitol, trehalose, or sorbitol. In some specific embodiments, the salt-forming counterion comprises sodium. In some specific embodiments, the metal complex comprises a Zn-protein complex. In some specific embodiments, the nonionic surfactant comprises TWEENTM, PLURONICSTM or polyethylene glycol (PEG). In some embodiments, the active pharmaceutical ingredient may also be entrapped in prepared microcapsules, such as hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, such as by coacervation techniques or by interfacial polymerization, in colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions.
The invention provides the CGT-ODN nucleotide sequence, the vector and the pharmaceutical composition, and application of the pharmaceutical composition in immunoregulation.
Further, the immunomodulation includes activation of immune response, immunostimulatory activity, modulation of cytokine expression.
Further, the cytokines include cytokines of JNK, p38, nfkb signaling pathways.
Further, the cytokines include TNF、IFNB1、CXCL8、IL-11、IL-12A、IL-32、CCL2、CCL4、ANKRD1、TNFAIP3、JUN、FOS、FOSL1、FOSL2、NFKB1、NFKB2、CASP3、MAP3K14.
Further, the cytokines include TNF, IFNB1, CXCL8.
The invention provides the application of the CGT-ODN nucleotide sequence and the vector in preparing a pharmaceutical composition for treating or preventing infectious diseases, allergic diseases, immunodeficiency diseases and tumors.
In some embodiments of the present invention, in some embodiments, the immunodeficiency disease type includes, but is not limited to, inflammation, antiphospholipid syndrome, systemic lupus erythematosus, rheumatoid arthritis, autoimmune vasculitis, celiac disease, autoimmune thyroiditis, post transfusion immunity, maternal fetal incompatibility (maternal-fetal incompatibility), transfusion reactions, immunodeficiency such as IgA deficiency, common variant immunodeficiency, drug induced lupus, diabetes, type I diabetes, type II diabetes, juvenile rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, and, Immunodeficiency, allergy, asthma, psoriasis, atopic dermatitis, allergic contact dermatitis, chronic skin disease, amyotrophic lateral sclerosis, chemotherapy-induced injury, graft versus host disease (graft-vs-host diseases), bone marrow transplant rejection, ankylosing spondylitis, atopic eczema, pemphigus, behcet's disease, chronic fatigue syndrome fibromyalgia, chemotherapy-induced injury, myasthenia gravis, glomerulonephritis, allergic retinitis, systemic sclerosis, subacute cutaneous lupus erythematosus, cutaneous lupus erythematosus including chilblain-like lupus erythematosus, sjogren's syndrome, autoimmune nephritis, autoimmune vasculitis, Autoimmune hepatitis, autoimmune cardiotis, autoimmune encephalitis, autoimmune mediated hematopathy, lc-SSc (localized cutaneous scleroderma), dc-SSc (diffuse cutaneous scleroderma), autoimmune Thyroiditis (AT), graves' disease (GD), myasthenia gravis, multiple Sclerosis (MS), ankylosing spondylitis, graft rejection, immune aging, rheumatic/autoimmune diseases, mixed connective tissue disease, spinal arthropathy, psoriasis, psoriatic arthritis, myositis, scleroderma, dermatomyositis, autoimmune vasculitis, mixed connective tissue disease, idiopathic thrombocytopenic purpura, Crohn's disease, human adjuvant disease, osteoarthritis, juvenile chronic arthritis, spinal arthropathy, idiopathic inflammatory myopathy, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, thyroiditis, immune-mediated kidney disease, central or peripheral nervous system demyelinating disease, idiopathic demyelinating polyneuropathy, grin-Barlich syndrome, chronic inflammatory demyelinating polyneuropathy, hepatobiliary disease, infectious or autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, sclerosing cholangitis, inflammatory bowel disease, gluten-sensitive bowel disease, humulus' disease, autoimmune or immune-mediated skin disease, Bullous skin disease, polymorphous erythema, allergic rhinitis, atopic dermatitis, food allergy, urticaria, pulmonary immune disorders, eosinophilic pneumonia, idiopathic pulmonary fibrosis, allergic pneumonia, graft-related disorders, graft rejection or graft versus host disease, psoriatic arthritis, psoriasis, dermatitis, polymyositis/dermatomyositis, toxic epidermolysis necrosis, systemic scleroderma and sclerosis, responses associated with inflammatory bowel disease, crohn's disease, ulcerative colitis, respiratory distress syndrome, adult Respiratory Distress Syndrome (ARDS), meningitis, encephalitis, uveitis, colitis, glomerulonephritis, allergic conditions, Eczema, asthma, conditions involving T-cell infiltration and chronic inflammatory responses, atherosclerosis, autoimmune myocarditis, leukocyte adhesion deficiency, allergic encephalomyelitis, immune responses associated with acute and delayed allergies mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis including Wegener granulomatosis, vasculitis (including ANCA), aplastic anemia, diamond Blackfan anemia, immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia, pure Red Cell Aplasia (PRCA), granulomatosis, vasculitis (including ANCA), aplastic anemia, diamond Blackfan anemia, autoimmune hemolytic anemia (AIHA), Factor VIII deficiency, hemophilia a, autoimmune neutropenia, whole blood cytopenia, leukopenia, diseases involving leukocyte exudation, central Nervous System (CNS) inflammatory disorders, multiple organ injury syndrome, myasthenia gravis, antigen-antibody complex mediated diseases, anti-glomerulonephropathy, anti-phospholipid antibody syndrome, allergic neuritis, behcet's disease, castleman's syndrome, goodpasture's syndrome, lambert-eaton's muscle weakness syndrome, raynaud's syndrome, sjogren's syndrome, bullous pemphigoid, autoimmune polycystic gland disease, Reiter disease, stiff person syndrome, giant cell arteritis, immune complex nephritis, igA nephropathy, igM polyneuropathy or IgM mediated neuropathy, idiopathic Thrombocytopenic Purpura (ITP), thrombotic Thrombocytopenic Purpura (TTP), autoimmune thrombocytopenia, testicular and ovarian autoimmune diseases including autoimmune orchitis and oophoritis, primary hypothyroidism, autoimmune endocrine diseases including autoimmune thyroiditis, chronic thyroiditis (hashimoto thyroiditis), subacute thyroiditis, idiopathic hypothyroidism, edison's disease, Graves ' disease, autoimmune multiple endocrine gland syndrome (or multiple endocrine disease syndrome), sheehan's syndrome, autoimmune hepatitis, lymphocytic interstitial pneumonia (HIV), occlusive bronchiolitis (non-transplant) pair NSIP, green-Barlich syndrome, macrovasculitis (including polymyalgia rheumatica and giant cell (high-safety) arteritis), medium vasculitis (including Kawasaki disease and polyarteritis nodosa), ankylosing spondylitis, berger's disease (IgA nephropathy), rapid progression of glomerulonephritis, primary biliary cirrhosis, sprue (Celiac sprue) (gluten enteropathy), Cryoglobulinemia, and Amyotrophic Lateral Sclerosis (ALS).
In some embodiments, the tumor type includes, but is not limited to, acute Lymphoblastic Leukemia (ALL), acute myelogenous leukemia, adrenocortical carcinoma, adult acute myelogenous leukemia, adult primary unknown carcinoma, adult malignant mesothelioma, AIDS-related cancer, AIDS-related lymphoma, anal carcinoma, appendicular carcinoma, astrocytoma, childhood cerebellum or brain cancer, basal cell carcinoma, cholangiocarcinoma, bladder carcinoma, bone tumor, osteosarcoma/malignant fibrous histiocytoma, brain cancer, brain stem glioma, breast cancer, bronchial adenoma/carcinoid, burkitt's lymphoma, carcinoid, primary unknown carcinoma, central nervous system lymphoma, Astrocytomas of the cerebellum, astrocytomas/glioblastomas of the brain, cervical cancer, childhood acute myelogenous leukemia, childhood cancers with unknown primary sites, childhood cancers, childhood astrocytomas of the brain, childhood mesothelioma, chondrosarcoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumors, endometrial cancer, endometrial uterine cancer, ependymoma, epithelial-like vascular endothelial tumors (EHE), esophageal cancer, ewing tumor sarcoma family, ewing sarcoma in Ewing tumor family, extracranial germ cell tumors, Extragonadal germ cell tumors, extrahepatic cholangiocarcinomas, ocular cancers, intraocular melanoma, gall bladder cancers, gastric (gastic) cancers, gastric carcinoid, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), gestational trophoblastomas, brain stem gliomas, hairy cell leukemias, head and neck cancers, heart cancers, hepatocellular (liver) cancers, hodgkin lymphomas, hypopharynx cancers, hypothalamic and vision pathway gliomas, islet cell carcinomas (endocrine pancreas), kaposi's sarcoma, renal cancers (renal cell carcinoma), laryngeal cancers, acute lymphoblastic leukemias (also known as acute lymphoblastic leukemias), and, Acute myelogenous leukemia (also known as acute myelogenous leukemia), chronic lymphocytic leukemia (also known as chronic lymphocytic leukemia), leukemia (leukaemia), chronic myelogenous leukemia (also known as chronic myelogenous leukemia), hairy cell leukemia (leukemia), lip and mouth cancer, liposarcoma, liver cancer (primary), non-small cell lung cancer, lymphoma (AIDS-related), lymphoma, macroglobulinemia, male breast cancer, bone malignant fibrous histiocytoma/osteosarcoma, medulloblastoma, melanoma, meeker cell carcinoma, primary focal hidden metastatic cervical squamous carcinoma, and, Oral cancer, multiple endocrine tumor syndrome, childhood multiple myeloma (bone marrow cancer), multiple myeloma/plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic/myeloproliferative diseases, chronic myelogenous leukemia, myxoma, nasal and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-hodgkin lymphoma, non-small cell lung cancer, oligodendroglioma, oral cancer, oropharyngeal cancer, osteosarcoma/osteomalignant fibrous histiocytoma, ovarian cancer, ovarian epithelial cancer (surface epithelial mesenchymal tumor), ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, islet cell carcinoma, paranasal and nasal cavity cancers, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germ cell tumor, pineal blastoma and supratentorial primitive neuroectodermal tumors, pituitary adenoma, plasmacytoid neoplasm/multiple myeloma, pleural pneumoblastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (renal cancer), transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, sme zary syndrome, skin carcinoma (melanoma), skin carcinoma (non-melanoma), merkel cell skin carcinoma, small cell lung cancer, small intestine cancer, lung cancer, Soft tissue sarcoma, squamous cell carcinoma, primary focal occult metastatic neck squamous carcinoma, gastric carcinoma, supratentorial primitive neuroectodermal tumors, cutaneous T-cell lymphomas, testicular cancer, laryngeal carcinoma, thymoma and thymus carcinoma, thymoma, thyroid carcinoma, renal pelvis and ureter transitional cell carcinoma, ureter and renal pelvis transitional cell carcinoma, urethral carcinoma, uterine sarcoma, vaginal carcinoma, visual pathway and hypothalamic glioma, childhood visual pathway and hypothalamic glioma, vulval carcinoma, macroglobulinemia and nephroblastoma (renal carcinoma).
The invention provides the CGT-ODN nucleotide sequence and the application of the vector in preparing vaccines.
Further, the vaccine comprises subunit vaccine and virus vector vaccine.
The term "subunit vaccine" refers to a vaccine prepared by cloning a CGT-ODN nucleotide sequence into a prokaryotic or eukaryotic expression system by using a genetic engineering method so as to enable the CGT-ODN nucleotide sequence to be efficiently expressed.
The term "viral vector vaccine" refers to a viral vector having replication ability and capable of maintaining its own infectivity as a vector of foreign genes.
Further, the vectors in the viral vector vaccine self-assemble into virus-like particles.
Further, the vector includes a self-inactivating vector.
Further, the virus is selected from lentivirus, influenza virus, hepatitis virus, a virus, filovirus, adenovirus, adeno-associated virus and/or flavivirus.
In some embodiments, the vaccine is a formulation comprising a biomolecule, such as a protein, or a nucleic acid molecule encoding a protein, a carbohydrate, a lipid, or a combination of each of the foregoing. In some embodiments, both prophylactic and therapeutic vaccines are present. The term vaccine generally refers to a product that is administered to a subject, i.e., comprising an adjuvant (if present), a carrier (if present), an active ingredient (the present invention refers to a CGT-ODN nucleotide sequence), a stabilizer, or other excipient. The term vaccine as used herein does not imply that the formulation is effective in preventing or curing a disease.
The invention provides the CGT-ODN nucleotide sequence and the application of the vector in antibody production.
The term "antibody" as used in the present invention includes an iso-tetralin protein of about 150000 daltons having the same structural characteristics, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. One end of each heavy chain has a variable region (VH). Followed by a plurality of constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end, the constant region of the light chain being opposite the first constant region of the heavy chain and the variable region of the light chain being opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.
The invention provides the CGT-ODN nucleotide sequence and the application of the vector in regulating the degradation degree of tRNA.
Further, the tRNA comprises TYPE II TRNA.
The invention provides a method for preparing a CGT-ODN related antibody, which comprises the step of obtaining the antibody by immunization of a non-human animal by using the CGT-ODN nucleotide sequence and the vector.
Further, the non-human animal is a mammal.
Further, the mammal includes a mouse, rat, guinea pig, rabbit, sheep, cow, horse, non-human primate.
The terms "non-human animal" and "non-human mammal" are used interchangeably herein and include mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates.
Drawings
FIG. 1 is a graph of Ethidium Bromide (EB) staining and SYBR melting curve results for ssDNA;
FIG. 2 is a graph of TLR9 independent immunostimulatory activity results of ssDNA;
FIG. 3 is a graph of cell viability versus cytokine expression results for bacterial ssDNA and human ssDNA;
FIG. 4 is a flow chart of a human whole genome ssDNA screen;
FIG. 5 is a graph of gel detection results of an optimization method for ODN transfection of HEK293 and 293A cells;
FIG. 6 is a graph of absorbance detection results after ODN transfection of HEK293 and 293A cells;
FIG. 7 is a graph showing the results of crystal violet staining and cell morphology for detecting the dose-effect relationship of ODN-HEK 293;
FIG. 8 is a graph showing the results of time course analysis of cell morphology changes after transfection of 293A cells with ODN 60;
FIG. 9 is a graph showing the results of the immunostimulatory effect of ODNs on different cell types;
FIG. 10 is a graph showing morphological changes and ATP-based cell viability results of HEK293 cells transfected with different forms of ODN or ORN, wherein ODNrc reverse complement the ODN, ORN, oligoribonucleotides identical to the sequence of the ODN, dsODN, double-stranded ODN annealed by ODN and ODNrc;
FIG. 11 is a graph showing the effect of ODN sequence length on HEK293 cell viability;
FIG. 12 is a graph showing morphological results of ODN60 and ODN60rc electrotransformed 293A cells 12 h, and cell viability analysis results of different ODN transfected HEK293 or ODN direct extracellular incubation 48 h;
FIG. 13 is a diagram of Wen graph and KEGG Orthology database analysis after transfection of ODN60 or ODN60rc in 293A cells;
FIG. 14 is a thermal graph of cytokine expression profile after 293A cell treatment versus upregulated cytokine qPCR validation results;
FIG. 15 is a graph showing the results of qPCR detection after transfection of DU145 cells 24 h with ODNs;
FIG. 16 is a DEGs analysis result graph of IPA database analysis results related to TNFR pathway;
FIG. 17 is a graph showing time and dose effect results of immunoblot analysis of pathway activation following transfection of HEK293 with ODN60 and ODN60 rc;
FIG. 18 is a graph showing the results of antibody immunoblotting analysis after transfection of DU145 cells with designated top and bottom ODNs 24 h, wherein UT represents untreated cells;
FIG. 19 is a graph showing the results of antibody immunoblotting analysis of the nuclear fraction of 293A cells transfected with ODNs;
FIG. 20 is a graph showing the results of qPCR detection of IFNB1, CXCL8 expression of HEK293 cells transfected with different siRNAs after stimulation of 12 h with ODN 60;
FIG. 21 is a graph showing the results of transfection efficiency assays for different ODNs;
FIG. 22 is a graph showing the effect of exchange of adjacent nucleotides indicated in ODN60 on stimulatory activity;
FIG. 23 is a graph showing the effect of truncation at the 3' end of ODN60 on stimulatory activity;
FIG. 24 is a graph showing the results of the immunostimulatory activity assays after various mutations in the CGT motif of ODN60 and transfection of HEK293 cells;
FIG. 25 is a graph of the percent partitioning results for CGT/A with ODNs within the sequencing horizon shown in FIG. 6;
FIG. 26 is a graph showing the results of ATP cell viability assay, cell morphology and cytokine activation in HEK293 cells transfected with top ODNs harboring CGT/A motif mutations, wherein CGT, wild-type sequence, AGT, CGT→AGT mutation, CCG, CGT→CCG mutation, CGG, CGT (CGA for ODN 86) mutation to CGG mutation;
FIG. 27 is a statistical plot of the position of CGT/A motifs in the highest and lowest ODNs;
FIG. 28 is a graph of CGT motif flanking sequence characterization results versus different base percentage statistics for top and middle ODNs;
FIG. 29 is a graph showing specific sequences of ODN-H and ODN-L synthesized according to the sequence characteristics of ODN and cytokine activation results after HEK293 transfection;
FIG. 30 is a graph showing the effect of consecutive G or T base distributions of CGT/A motif flanking sequences in ODN sequences on cytokine activation potency in cells;
FIG. 31 is a graph of percent statistics for CGT/A and CGT/A flanking sequences comprising more than 3 Gs in ODN sequences;
FIG. 32 is a graph of statistics for more stimulatory ODNs of a viral genome;
FIG. 33 is a graph of the effect of 2' -O methylation modification, RNA base substitution, phosphorothioate (PS) linkage, and cytidine methylation on stimulatory activity in a CGT/A ODN versus a classical CpG ODN, wherein the uppercase letters, phosphodiester linkages, lowercase letters, phosphorothioate linkages, underlined, palindromic sequence;
FIG. 34 is a graph showing the results of qPCR detection of IFNB1 expression levels after transfection of WT and TLR9 knockout mouse lung fibroblasts with typical CpG-ODN and ODN-H, respectively;
FIG. 35 is a schematic of genome-wide CRISPR screening of ODN60 response essential genes;
FIG. 36 is a graph showing the gene abundance ranking of multiple gRNA average hits, fold change in abundance between ODN60 and ODN60rc treated cells, and gRNA enrichment ranking of ODN60 treated cells;
FIG. 37 is a graph of post-deletion ODN60 and other top-ranked ODN triggered cytokines, cell viability statistics of SLFN 11;
FIG. 38 is a graph showing the effect of immunoblotting detection SLFN on ODN 60-induced activation of p38 and JNK by complementary expression;
FIG. 39 is a graph showing the results of qPCR detection of TNF and CXCL8 expression after transfection of different ODNs into WT and SLFN11 -/- HEK293 cells;
FIG. 40 is a graph showing the results of immunoblot analysis of SLFN11 expression levels in cells;
FIG. 41 is a graph showing statistics of different types of nucleotide-induced cytokine expression in SLFN knockout DU145 cells;
FIG. 42 is a schematic of luminescent ODN immunoprecipitation (ODIP) and biotin-ODN pulldown assays;
FIG. 43 is a graph showing the results of ODIP determination of endogenous and re-expressed SLFN11 binding activity to ODN60 and ODN60 rc;
FIG. 44 is a graph showing the results of a ODIP and biotin-ODN pulldown assay for detecting the binding of SLFN and ODN in cells;
FIG. 45 is a graph showing the effect of "CGT" motifs obtained in biotin pull-down detection ODN60rc in HEK293 cells on binding to SLFN 11;
FIG. 46 is a schematic diagram of structure prediction of three subgroups of SLFN family;
FIG. 47 is a graph of the results of ODIP analysis of full length and truncated SLFN11 and ODN60 following ODN60 transfection using a schematic representation of full length and truncated SLFN11 and SLFN11 -/- HEK293 cells expressing full length and truncated SLFN 11;
FIG. 48 is a graph showing the results of in vitro experiments on ODN binding activity of SLFN, including Coomassie blue staining, electrophoresis mobility change experiments, fluorescence ODN GST pull-down experiments, BLI experiments;
FIG. 49 is a graph of immunoblot analysis results of truncated SLFN11 versus ODN-induced activation of the MAPK pathway;
FIG. 50 is a distribution diagram of SLFN subgroups of NCBI database among clusters;
FIG. 51 is a graph of comparison of gene trees for the third subgroup SLFN, AIM2 and cGAS in TreeFam database;
FIG. 52 is a graph showing the results of protein expression levels of the SLFN family and the SLFN homologous proteins from NCBI human and mouse redundant protein sequence databases and GTEx databases;
FIG. 53 is a graph of the results of biotin-ODN 60 experiments with SLFN members of subgroup III;
FIG. 54 is a graph showing the result of staining SLFN for copolymerization of ODN60 with SLFN in the nucleus;
FIG. 55 is a graph of immunoblot analysis of SLFN11 distributions in HEK293 cytoplasm and nucleus;
FIG. 56 is a graph of immunoblotting detection results of SLFN of the distribution of SLFN in cytoplasm, perinucleus, and nucleus before and after stimulation of HEK293 cells with ODN 60;
FIG. 57 is a graph of ESMA measurements of subcellular localization of SLFN11 bound by ODN versus SLFN cytoplasmic-perinuclear-nuclear fraction bound by ODN;
FIG. 58 is a graph of co-localization results between SLFN and the highest ranked ODNs in DU145 cytoplasma;
FIG. 59 is a graph showing the results of screening SLFN for the homolog of 11 in mice;
FIG. 60 is a schematic diagram of the construction of SLFN-KO mice;
FIG. 61 is a graph showing the results of cytokine qPCR detection in WT and SLFN-KO mice;
FIG. 62 is a graph showing the results of qPCR assay Ifnb1 mRNA expression levels and ELISA assay IFNα secretion after transfection of WT, SLFN9 -/- and tr19 -/- mouse primary cells with HIV6441 and classical TLR9 CpG ODN, respectively;
FIG. 63 is a graph showing the results of SLFN in CGT-ODN-induced mouse immune responses, wherein A-F, CGT-ODN induced neonatal (B, C) and adult (D-F) mice, schematic representations (A) of CpG ODNs encapsulated in LNPs and luciferase mRNA-LNPs in liver by intravenous injection, qPCR and immunoblotting to detect activation of cytokine (B) and immune pathway (C), qPCR and HE staining to detect expression level of cytokine 6H (D) after injection and histological changes of 3D (E) after injection, respectively, and to monitor 4 days of survival, H and I, AAV2 single-stranded DNA genome encapsulated in LNP, intravenous injection into mice, detection of 4 days of survival and expression of validation factor in liver, respectively, and J-L, injection of WT or SLFN-/-B16-F10 cells subcutaneously into C57/6 mice, respectively, and then injection of HIV6441 encapsulated with transfection reagent into tumors, as indicated by individual tumor volumes (average tumor volumes) at each time point shown in groups of K and individual tumor volumes (average volume of mice);
FIG. 64 is a graph showing the results of SLFN and TRL9 expression in the livers of B16-F10 and WT mice;
FIG. 65 is a graph showing the results of cell morphology changes, ATP-based cell viability, and cytokine expression in SLFN-/-and Trl 9-/-B16-F10 mice;
FIG. 66 is a graph showing the results of reduced tRNA cleavage by CGT ODN and pathogenic ssDNA such as bacteria or AAV, wherein A is PAGE gel electrophoresis staining, B is dose-response analysis, C-D is SLFN11 knockdown effect on tRNA cleavage by CGT-ODN stimulation, E-F is cell survival result, G is cytokine expression result, H is effect of other source ssDNA on tRNA degradation of SLFN11, I is cell survival result in other source ssDNA, J is degradation of tRNA by HeLa cells of wild type SLFN11 when receiving CGT ODN stimulation, K is cell survival result when exogenous SLFN is expressed by HeLa cells, L is inflammatory response result when exogenous SLFN11 is expressed by HeLa cells.
Detailed Description
EXAMPLE 1 intracellular ssDNA decreases cell viability in a sequence-specific manner
1. Acquisition of human and bacterial ssDNA and preliminary analysis of ssDNA to reduce cell viability
To obtain ssDNA, human and bacterial genomic DNA was heat denatured and identified using dsDNA-preferred ethidium bromide staining and SYBR green melting curves, the results are shown in fig. 1. To test TLR 9-independent immunostimulatory activity of ssDNA, ssDNA and dsDNA were transfected into HEK293 cells deficient in TLR9 pathway, the results are shown in fig. 2, which shows that bacterial ssDNA reduced cell viability to a greater extent than bacterial dsDNA or human ssDNA. Bacterial ssDNA activates greater expression of TNF and CXCL8 than bacterial dsDNA or human ssDNA, as shown in fig. 3, indicating that intracellular ssDNA can also activate TLR 9-independent immune responses in a sequence-dependent manner.
2. Screening of human and bacterial ssDNA and detailed analysis of ssDNA to reduce cell viability
To further investigate the stimulatory activity of ssDNA on the human genome, the stimulatory sequences were screened by preparing a full genome 24 nucleotide (nt) ODN library, the results of which are shown in fig. 4. Libraries were transfected into HEK293 and 293A cells using the optimized ODN transfection method as shown in fig. 5. The results are shown in fig. 6, which shows that 14 ODNs out of 200 were observed to reduce the average cell viability of these cells to below 40%. The first 11 active high ODNs were validated in concentration gradients and as shown in fig. 7, ODN60 was found to exhibit the highest immunostimulatory potency. Therefore, in the subsequent study, ODN60 was used as a representative of ODN. In addition, the immunostimulatory potency of ODN60 was as low as 24nM, while its reverse complement ODN (ODN 60 rc) did not decrease cell viability even at 120nM, as shown in fig. 8, indicating that immunostimulatory activity was determined by the sequence and not its targeted genomic position. The ODN sequences used in the experiments are shown in table 1.
TABLE 1
To explore whether the response to ssDNA was cell-type limiting, the top-ranked ODNs were transfected into various types of human cells, and as shown in fig. 9, ODN-responsive cell types were found to include DU145, T47D, U, 251 and a549 cells, and HUVECs, but not 293T cells. By observing the morphological features exhibited by these reactive cells, cell death after stimulation is clearly indicated. Furthermore, by the results shown in fig. 10, only ODN, not Oligonucleotide (ORN) or double-stranded ODN (dsODN), reduced cell viability. This immunological activity is not limited to a length of 24nt, since 96nt ODN containing the highest sequence also reduced cell viability, as shown in fig. 11. Finally, both electroporation and transfection (rather than incubation) made the top-ranked ODN active, and the results are shown in fig. 12, indicating that the presence of ssDNA in the cell is essential for its immunostimulatory activity.
Example 2 intracellular ssDNA activates cytokine expression and immune-related Signal pathway
In view of the fact that cell viability is not a specific indicator for assessing immune responses, to further determine the immunostimulatory activity of ODN, RNAseq assays were performed on 293A cells 10 and 24 hours after stimulation of the cells with ODN60 or ODN60 rc. As a result, as shown in fig. 13, only ODN60 triggered their expression, ODN60rc did not trigger expression, compared to the expression of immune-related genes in the untreated control group. Importantly, ODN60 stimulated the expression of a number of cytokines and chemokines (hereinafter "cytokines"), the specific cytokine results shown in fig. 14, further indicating the immunostimulatory activity of ssDNA, as activation of transcription of cytokines is a major component of the pathogen-associated molecular pattern (PAMP) induced innate immune response. Similarly, experiments were performed in DU145 cells, with all top-ranked ODNs activating cytokine expression, as shown in fig. 15, and also including IFNB1 that was not triggered in HEK293 cells, with specific results shown in fig. 14.
In addition, most of the apical enriched signaling pathways in ODN60 treated cells were associated with immune responses, except for the innate immune response revealed by cytokine activation, and the results are shown in fig. 16, which show that these genes contribute to the enrichment of TNFR1 and TNFR2 pathways. ODN60 also activated JNK, p38 and nuclear factor- κb (nfkb) signaling pathways, the results of which are shown in fig. 17. Consistently, all top-ranked ODNs activated JNK, p38 and nfkb pathways in DU145 cell lines, the results are shown in fig. 18. Whereas a decrease in iκbα levels indicates activation of the nfkb pathway, for which nuclear translocation of nfκb was further assessed, it was found that p65 and NF- κb1 translocation to the nucleus was significantly increased following top-ranked ODN stimulation, and the results are shown in fig. 19. Knocking down p65 and NF- κb1 partially reduced ODN 60-induced activation of IFNB1 and CXCL8 in DU145 cells, as shown in figure 20. In summary, decreased cell viability, increased cytokine expression and activation of immune pathways revealed immunostimulatory activity of intracellular ODNs.
Example 3 CGT/A with continuous G is the core motif of intracellular ODN immunostimulatory Activity
Before studying the immunostimulatory sequence pattern in the top-ranked ODN, we excluded the possibility that the difference in immune activity between the top-ranked and bottom-ranked ODNs was caused by different transfection efficiencies, the results being shown in fig. 21. Next, we designed a series of ODN60 mutant and truncated constructs. The use of position switches for three pairs of adjacent bases from the 5 'end region instead of the 3' end region deactivates the ODN60 as shown in FIG. 22. Consistently, the 3' -end truncation of about 8nt had little effect on ODN60 immunocompetence, and the results are shown in fig. 23. Importantly, switching of only the first pair of adjacent nucleotides was sufficient to significantly reduce the immune activity of ODN60, as shown in figure 22. These results indicate that the immune activity of ODN60 is primarily determined by its 5' end sequence and is sensitive to single nucleotide changes.
Next, the 1 st to 7 th nucleotides of the 5' end of ODN60 were systematically substituted with the other three types of nucleotides, and the resulting activities were evaluated by evaluating cell viability and activation of TNF cytokines and JNK pathways, and the results are shown in fig. 24. These results consistently indicate that nucleotides 2 and 3 are highly restricted to C and G, respectively, and that nucleotide 4, T, exhibits a certain tolerance to a for the immunostimulatory activity of ODN60, indicating that the CGT/a motif is not only extracellular ssDNA, but also is unexpectedly required for intracellular ssDNA to activate an immune response. Then, we further analyzed the presence of CGT/a motifs in the 200 screened ODNs, and found that the presence of CGT and CGA (CGT/a) motifs determines the stimulation level of the ODN, as shown in fig. 25. Any single nucleotide mutation in the CGT/a motif disrupted the immunostimulatory activity of the top-ranked ODN, as shown in figure 26. Thus, the presence of CGT/A is necessary for an immunostimulatory ODN.
We found that not all ODNs containing the CGT/A motif have strong immunostimulatory effects in the screening experiments of example 1. Thus, we speculate that flanking sequences, which are CGT/A motifs, cooperate with CGT/A to determine the immunostimulatory effect of intracellular ODNs. To determine the flanking sequence patterns that distinguish the efficacy of CGT/a motifs, we first analyzed the base composition of all ODNs and aligned the CGT/a flanking motifs of top-ranked (high-lived) and middle-ranked (mid-lived) ODNs. As shown in fig. 27, most ranking underlayment (low activity) CGT/a ODNs were excluded from this alignment because they lack downstream flanking sequences. We observed that the CGT/a flanking sequences in the top-ranked ODNs are G-rich, while the flanking sequences in the other ODNs are T-rich, and the results are shown in fig. 28. We then synthesized ODN-H and ODN-L with CGT/A motifs surrounded by high-and medium-activity flanking sequences, respectively. As expected, only ODN-H triggered TNF expression, whereas ODN-L did not, as shown in FIG. 29. Furthermore, substitution of G by T and disruption of G continuity downstream of the CGT/A motif impair the immunostimulatory activity of ODN-H, as shown in FIG. 30, indicating that the highly active CGT/A motif requires assistance from a continuous G downstream of CGT/A. This also explains why the CGT/a motif in the underlying ODN tends to lie within the last 10 nt of the 3' end, see in particular fig. 28.
To verify the universality of this sequence law in predicting the potential immunostimulatory capacity of single-stranded DNA, we analyzed the proportion of such immunostimulatory sequences present in the genomes of multiple species. We found that the proportion of this motif in the bacterial genome is much higher than in the human genome, and the results are shown in FIG. 31, which explains why bacterial ssDNA is more immunostimulatory than human ssDNA. Cas9 protein is a protein of bacterial origin that is widely used in gene editing, and therefore its coding sequence also contains a very high proportion of immunostimulatory sequences. Most single stranded DNA viruses and retroviruses contained fewer immunostimulatory sequences than double stranded DNA viruses, but the genomes of adeno-associated viruses were the exception, and according to this motif rule we tested the immunostimulatory capacity of single stranded DNA derived from bacterial, aids and type 2 adeno-associated virus genomes, respectively, while we also searched for, pooled and tested ODNs from other viral genomes, the test results being shown in figure 32. In summary, our results indicate that CGT/A with downstream continuous G is a stimulatory motif of intracellular ssDNA, independent of the species source of the ssDNA.
Although intracellular active ODNs share some sequence similarity with TLR 9-sensed a class CpG ODNs, TLR9 is not able to be activated by 5-methylation modified CpG ODNs, whereas ODNs with Phosphorothioate (PS) backbones would enhance activation of TLR 9. As shown in FIG. 33, we found that the deoxyribose backbone of CGT-ODN was only natural, and that replacement with Phosphorothioate (PS) backbone, 2' -O methylated backbone and Ribonucleotide (RNA) backbone all destroyed the immunostimulatory activity of intracellular ODNs, while 5-methylated cytosine modification in the CGT/A motif had little effect on the efficacy of intracellular ODNs. Thus, we have found that such ODNs are not a class of ODNs that recognize CpG with TLR 9. These elements define a class of intracellular active ODNs where the CGT/A motif with natural Phosphodiester (PO) linkage and methylation tolerance is poly G3' downstream and palindromic sequences are optional. For brevity, we will refer to ODNs with these elements as CGT ODNs. Thus, a typical CpG-ODN is not as active as ODN-H when delivered into cells. Meanwhile, the lack of TLR9 did not affect the stimulatory activity of ODN-H, as shown in FIG. 34. These results further demonstrate that intracellular CGT-ODNs are not activated by TLR9 to activate immune responses, and further suggest the existence of a novel intracellular CGT-ODNs receptor.
Example 4 SLFN11 is critical for the ODN-induced immune response
To identify potential receptors for intracellular ssDNA, we performed a whole genome CRISPR-Cas9 screen, a schematic diagram as shown in figure 35. We focused on knockout of genes to eliminate ODN 60-induced cell depletion related genes. Interestingly, SLFN protein family member SLFN was most significantly enriched in single and three rounds of screening, whereas other identified DNA genes, such as TLR9, AIM2 and cGAS, were not highly enriched, and the results are shown in figure 36. SLFN11 has been previously identified as an interferon-stimulated gene (ISG) that plays a role in inhibiting HIV translation and tumor response to DNA damaging agents. To verify the role of SLFN11 in intracellular CGT-ODN-induced immune responses, we generated SLFN knockout clones from HEK293, 293A and DU145 cells. The deletion of SLFN11 completely blocked all previously defined immune responses, including cytokine activation, cell viability decline triggered by ODN60 and other top-ranked ODNs, as shown in figure 37. We failed to establish stable SLFN re-expression in SLFN11 -/- HEK293 cells using lentivirus, as overexpression SLFN11 from the vector strongly inhibited lentivirus production. Transient SLFN complementary expression made SLFN11 -/- HEK293 cells responsive to ODN60, although this re-expression of SLFN itself also triggered partially an immune response, as shown in figure 38. In addition, the virus-derived CGT/A ODN and bacterial ssDNA activated TNF and CXCL8 expression only in WT 293A cells and not in SLFN-/-293A cells, and the results are shown in FIGS. 32 and 39. Consistent with FIG. 9, only cells carrying SLFN expression reacted to the top-ranked ODN, and the results are shown in FIG. 40. Finally, SLFN11 was necessary for CGT-ODNs to activate IFNB1 and CXCL8 in DU145 cells, and the results are shown in FIG. 41.
Example 5 SLFN11 is a sequence-specific ssDNA binding protein
To determine whether SLFN11 was able to bind and recognize ssDNA in the form of ODN, we developed a biotin pull-down complementation method called luminescent ODN immunoprecipitation (ODIP) to quantify the intracellular ODN binding activity of SLFNs, a schematic of ODIP is shown in fig. 42. ODN60 and SLFN11 were specifically immunoprecipitated, while ODN60rc was unable, and the ODIP signal of ODN60 was SLFN dependent, and the results are shown in fig. 43. Furthermore, binding of ODN to SLFN11 is CGT motif dependent, since any single mutation in the CGT motif of ODN60 reduces its binding to SLFN11 (fig. 44), whereas the availability of CGT motif in ODN60rc enhances its binding to SLFN11 (fig. 45). As expected, SLFN11 specifically bound the top-ranked ODN, but not the bottom-ranked ODN (fig. 44), indicating that high affinity for SLFN11 is necessary for the immunostimulatory activity of ODN.
SLFN11 member of subgroup III belonging to the SLFN family, whose members comprise three predicted domains, the N-terminal AAA domain, the SWADL domain and the C-terminal helicase domain, is schematically shown in FIG. 46. Truncated analysis showed that what was able to bind to ssDNA was the SLFN C-terminal fragment containing residues 349 to 901, and any indication deletion between residues 349 to 901 reduced the ssDNA binding activity of SLFN11 (fig. 47). The direct ssDNA binding activity of SLFN11 was also determined with purified GST-and His-fused C-terminal SLFN11 (fig. 48). Notably, in SLFN11-/-HEK293 cells, only full length SLFN11 was able to restore reactivity to ODN60 (fig. 49). This suggests that the C-terminal helicase domain is responsible for ssDNA binding, while the N-terminal domain is necessary for signaling of the presence of ssDNA within the cell. In summary, the need for a CGT-DNA induced immune response and the specific binding activity for CGT/A motif-containing cells suggests that SLFN is an intracellular CGT-DNA immunosensor.
In contrast to the other SLFN subgroups, only SLFN in subgroup III contained the entire C-terminal ssDNA binding domain and was conserved from trunk fish to humans (fig. 50). In addition, subgroup III SLFNs showed a similar evolutionary distribution in the taxa with dsDNA sensors (fig. 51), indicating that full length subgroup III, SLFNs, was under potential selection pressure. However, of all subgroups III SLFNs expressed in humans (fig. 52), only SLFN11 had detectable ssDNA binding activity (fig. 53).
Example 6 Co-localization and binding of SLFN11 to CGT-ODNs in the cytoplasm
SLFN11 was exported out of the nucleus under stimulation of ODN60 and accumulated in spots co-localized with ODN60 in the cytoplasm. This is in contrast to the diffuse distribution of SLFN11 in the nucleus observed under basal conditions (fig. 54). When we analyzed cytoplasmic and nuclear extracts by immunoblotting, we found that the distribution of endogenous SLFN11 was sensitive to different extraction protocols (fig. 55). To further investigate this observation, we divided cells into nucleus, perinucleus and cytosol, and found SLFN that was predominantly stable in perinuclear fraction under basal conditions, but transferred into the cytoplasm under ODN60 stimulation (fig. 56). Consistent with immunofluorescence results, biotin pulldown experiments and EMSAs assays showed that ODNs-bound SLFN was predominantly distributed in the cytoplasmic portion (fig. 57). Finally, we confirmed co-localization between SLFN and the highest ranked ODN in DU145 cytoplasm (fig. 58).
EXAMPLE 7 SLFN9 (SLFN homolog) is critical to CGT-ODN-induced mouse immune response
Since mice do not have SLFN a 11, we screened a mouse SLFN that could functionally complement human SLFN in order to further investigate the role of SLFNs in the in vivo response to CGT-ODNs. In all mouse subgroups III SLFNs, which were highly homologous to human SLFN11, only SLFN9 was found to be able to bind ssDNA and resume ssDNA signaling in SLFN11 -/- HEK293 cells (fig. 59). Thus, we established Slfn knockout mice (fig. 60). Preliminary evaluation of Slfn9 -/- mice showed that they were healthy and fertile in our population under specific pathogen-free conditions.
We first analyzed Slfn in primary cells after CGT-ODNs stimulation. The lack of Slfn in primary Bone Marrow Derived Macrophages (BMDMs) blocked activation of Ifnb, il6 and Cxcl2 by ODN-H and HIV6441, a CGT ODN from HIV, but did not block poly (dA: dT), poly (I: C) or LPS (fig. 61), indicating that Slfn9 is specifically required for intracellular CGT-ODNs induction. HIV6441 was found to be more active than ODN-H in mouse cells, and therefore we used HIV6441 to represent intracellular ODN for the following mouse experiments. To explore the role of SLFN and TLR9 in response to different types of ODN, we delivered HIV6441 and all three types of typical CpG ODN, ODN1585 (class a), ODN1826 (class B) and ODN2395 (class C) into primary mouse fibroblasts (MAFs), lung Fibroblasts (LFs), primary BMDM and plasmacytoid dendritic cells (pDC), respectively.
TLR9 is reported to play a major role in pDCs and B cells. In agreement, all types of ODNs stimulated ifnα production in WT and Slfn9-/-pDCs, but not in tlir 9-/-pDCs, suggesting that transfected ODNs could also activate tlir 9 in endosomes due to endocytosis when tlir 9 is highly expressed. However, only the Slfn knockout, but not the tlir 9 knockout blocked Ifnb1 expression induced by HIV6441 and ODN1585 in MAFs, LFs and BMDM (fig. 62). Thus, the intracellular stimulatory activity of both typical CpG ODN and CGT ODN is dependent on SLFN9, and SLNF9 functions in a wider tissue type than TLR9.
We extended our findings to primary mouse cells by stimulating WT and Slfn9 -/- mice with intracellular delivered ODN of the Lipid Nanoparticle (LNP) system, which is also used for severe acute respiratory syndrome coronavirus type 2 mrna vaccine. Since intravenous injection of LNP resulted mainly in accumulation of nucleotides in the liver (fig. 63A), we mainly evaluated the role of Slfn in liver response to ODNs. HIV6441 activates expression of Ifnb1, tnf, il6, cxcl2 and Cxcl10, as well as STAT1, JNK and nfkb pathways in the liver (fig. 63B, C). Slfn9 lack almost completely abrogated HIV 6441-induced response. Similarly, slfn was also necessary for HIV 6441-induced cytokine expression in adult mouse livers (fig. 63D), except Ifnb1. Consistent with the morphological features of cell death in human cells of example 1, HIV6441 even triggered histological necrosis of the liver and acute hepatitis (fig. 63E, F), which is also SLFN9 dependent. Notably, cpG-ODNs induced fatal toxic shock was reported only in D-galactosamine sensitized mice, and only injection of CpG-DNA into mice was reported to be nontoxic even at relatively high doses. However, we found that intravenous injection of 60 μg of LNP-packaged HIV6441 was sufficient to cause WT mice to die (fig. 63G). In contrast, all Slfn -/- mice survived the same challenge. In addition, we encapsulated single-stranded DNA genome of adenovirus type 2 (AAV 2) into LNP for intravenous injection into mice, which also stimulated SLFN dependent immune responses (fig. 63-H, I). Taken together, these in vivo results confirm previous cellular studies, indicating that SLFN and SLFN are critical for intracellular ODN-induced immune responses in humans and mice, respectively.
Previous basic and clinical studies have shown intratumoral single agent therapeutic activity of classical CpG-ODNs, which are generally regarded as TLR9 agonists. And we speculate that CGT ODN should also have good tumor therapeutic effect by activating SLFN. To elucidate the role of SLFN and TLR9 in ODN anti-tumor monotherapy we produced SLFN9 -/- and TLR9 -/- B16-F10 cells, respectively, although WT B16-F10 cells hardly expressed TLR9 (fig. 64). We found Slfn cells -/-, but not Tlr9 -/-, were completely resistant to ODN-induced decrease in cell viability and increase in cytokine activation (fig. 65). To further confirm these findings, WT and Slfn -/- B16-F10 cells were subcutaneously transplanted into mice (FIG. 63 g). Consistently, intratumoral delivery of HIV6441 significantly reduced the growth of WT tumors, but had no effect on the growth of Slfn9 -/- tumors (fig. 63h, i), suggesting that Slfn9 is a key target for ODN monotherapy in tumor cells.
Example 8 CGT ODN and pathogenic ssDNA such as bacteria or AAV result in reduced tRNA cleavage
After 293A cells were stimulated with CGT ODN, RNA was extracted from the cells, and analyzed by PAGE gel electrophoresis staining, it was found that TYPE II TRNA was significantly decreased in abundance (FIG. 66A), and that the amount of CGT ODN used and the degree of tRNA degradation had a dose-response relationship (FIG. 66B). In HUVEC cells, CGT ODNs can also lead to tRNA cleavage. To verify whether the TYPE II TRNA decrease in CGT ODN was SLFN11 tRNA cleavage activity dependent, we mutated the tRNA cleavage active site E209 of endogenous SLFN in HEK293 cells in situ to 209A, disabling tRNA cleavage of endogenous SLFN expressed by HEK 293. When we stimulated HEK293 cells with CGT ODN, only WT cells and E209A heterozygous cells (one allele expressed WT SLFN11 and the other allele expressed E209A SLFN) were able to produce tRNA cleavage in response to CGT ODN stimulation, whereas SLFN11 knockdown cells and cells expressing only E209A SLFN11 were completely resistant to CGT ODN stimulation-induced tRNA cleavage (fig. 66C-D). And E209A SLFN11 was also unable to mediate downstream cell death (FIGS. 66E-F) and cytokine expression (FIG. 66G), suggesting that tRNA cleavage activity of SLFN11 is necessary to elicit a downstream natural immune response. in addition to CGT ODN, we also demonstrated that bacterial, AAV, HIV genome-derived ssDNA, bacterial and AAV-derived ssDNA were able to cause SLFN-dependent tRNA degradation (fig. 66H), as well as tRNA cleavage activity of SLFN11 was necessary for cell death by these pathogenic ssDNA (fig. 66I). HeLa cells hardly expressed endogenous SLFN11 and were also insensitive to CGT ODN stimulation. Thus, we artificially constructed cell lines stably expressed by exogenous wild-type SLFN11 and cleavage activity mutant SLFN11 (E209A, E214A, E209/214A) in HeLa cells. When HeLa cells expressing wild-type SLFN11 were stimulated with CGT ODN, we could see significant tRNA degradation (fig. 66J), whereas HeLa cells expressing SLFN with lost tRNA cleavage activity were unable to respond to stimulation with CGT ODN. As a result of the endogenous SLFN tRNA activity point mutation in HEK293 cells, exogenous SLFN11 was also required for the tRNA cleavage activity of SLFN in response to stimulation of CGT ODN to cause downstream cell death (FIG. 66K) and inflammatory response (FIG. 66L) upon HeLa cell expression. In combination with our work SLFN that can directly recognize data binding to CGT ODN, we identified SLFN as an RNase activated by ssDNA. This is also the first ssDNA-activated RNA nucleic acid hydrolase found in eukaryotic cells. The TYPE II TRNA level is directly predicted to be used as a clinical detection and diagnosis index of ssDNA induced immune response.
The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.
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