Rna-coded antibody

Abstract

The present application describes an antibody-coding, non-modified or modified RNA and the use thereof for expression of this antibody, for the preparation of a pharmaceutical composition, in particular a passive vaccine, for treatment of tumours and cancer diseases, cardiovascular diseases, infectious diseases, autoimmune diseases, virus diseases and monogenetic diseases, e.g. also in gene therapy. The present invention furthermore describes an in vitro transcription method, in vitro methods for expression of this antibody using the RNA according to the invention and an in vivo method.

What is claimed is: 
     
         1 . An RNA for intracellular expression of an antibody, wherein the RNA contains at least one coding region, wherein at least one coding region codes for at least one antibody. 
     
     
         2 . An RNA according to  claim 1 , wherein the RNA is single-stranded or double-stranded, linear or circular, in the form of rRNA, tRNA or mRNA. 
     
     
         3 . An RNA according to  claim 1  or  2 , wherein the RNA is an mRNA. 
     
     
         4 . An RNA according to  claims 1  to  3 , wherein the antibody coded is chosen from monoclonal and polyclonal antibodies, chimeric antibodies, human antibodies, humanized antibodies, bispecific antibodies, intrabodies and fragments of these antibodies. 
     
     
         5 . An RNA according to  claims 1  to  4 , wherein the coded antibody fragments are chosen from Fab, Fab′, F(ab′)2, Fc, Facb, pFc′, Fd, and Fv or scFv fragments of these antibodies. 
     
     
         6 . An RNA according to  claims 1  to  5 , wherein the coded antibodies or antibody fragments specifically recognize and bind tumour-specific surface antigens chosen from (TSSA), 5T4, α5β1-integrin, 707-AP, AFP, ART-4, B7H4, BAGE, β-catenin/m, Bcr-abl, MN/C IX-antigen, CA125, CAMEL, CAP-I, CASP-8, β-catenin/m, CD4, CD19, CD20, CD22, CD25, CDC27/m, CD 30, CD33, CD52, CD56, CD80, CDK4/m, CEA, CT, Cyp-B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ETV6-AML1, G250, GAGE, GnT-V, GpIOO, HAGE, HER-2/neu, HLA-A*0201-R170I, HPV-E7, HSP70-2M, HAST-2, hTERT (or hTRT), iCE, IGF-IR, IL-2R, IL-5, KIAA0205, LAGE, LDLR/FUT, MAGE, MART-1/Melan-A, MART-2/Ski, MC1R, myosin/m, MUC1, MUM-I, -2, -3, NA88-A, PAP, proteinase-3, p190 minor bcr-abl, Pml/RARα, PRAME, PSA, PSM, PSMA, RAGE, RU1 or RU2, SAGE, SART-I or SART-3, survivin, TEL/AML1, TGFβ, TPI/m, TRP-I, TRP-2, TRP-2/INT2, VEGF and WT1, NY-Eso-1 and NY-Eso-B. 
     
     
         7 . An RNA according to one of  claims 1  to  6 , wherein the RNA is modified. 
     
     
         8 . An RNA according to one of the preceding claims, wherein the modification is chosen from modifications of the nucleotide sequence compared with a precursor RNA sequence by introduction of non-native nucleotides and/or by covalent coupling of the RNA with another group. 
     
     
         9 . An RNA according to  claim 8 , wherein the RNA has a G/C content in the coding region of the base-modified RNA which is greater than the G/C content of the coding region of the native RNA sequence, the coded amino acid sequence being unchanged with respect to the wild-type or, respectively, the precursor RNA. 
     
     
         10 . An RNA according to  claim 8  or  9 , wherein the coding region of the modified RNA is modified compared with the coding region of the native RNA such that at least one codon of the native RNA which codes for a tRNA which is relatively rare in the cell is exchanged for a codon which codes for a tRNA which is relatively frequent in the cell and which carries the same amino acid as the relatively rare tRNA. 
     
     
         11 . An RNA according to one of  claims 8  to  10 , wherein the RNA has a lipid modification. 
     
     
         12 . An RNA according to one of  claims 8  to  11 , wherein the RNA contains on at least one nucleotide of the RNA a modification of a nucleotide, wherein the nucleotides are chosen from 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5′-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-Oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), pseudouracil, 1-methyl-pseudouracil, queosine, β-D-mannosyl-queosine, wybutoxosine, and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine. 
     
     
         13 . An RNA according to one of  claims 8  to  12 , wherein the RNA contains on at least one nucleotide of the RNA a modification of a nucleotide, wherein the nucleotides are base-modified nucleotides chosen from the group consisting of 2-amino-6-chloropurine riboside 5′-triphosphate, 2-aminoadenosine 5′-triphosphate, 2-thiocytidine 5′-triphosphate, 2-thiouridine 5′-triphosphate, 4-thiouridine 5′-triphosphate, 5-aminoallylcytidine 5′-triphosphate, 5-aminoallyluridine 5′-triphosphate, 5-bromocytidine 5′-triphosphate, 5-bromouridine 5′-triphosphate, 5-iodocytidine 5′-triphosphate, 5-iodouridine 5′-triphosphate, 5-methylcytidine 5′-triphosphate, 5-methyluridine 5′-triphosphate, 6-azacytidine 5′-triphosphate, 6-azauridine 5′-triphosphate, 6-chloropurine riboside 5′-triphosphate, 7-deazaadenosine 5′-triphosphate, 7-deazaguanosine 5′-triphosphate, 8-azaadenosine 5′-triphosphate, 8-azidoadenosine 5′-triphosphate, benzimidazole riboside 5′-triphosphate, N1-methyladenosine 5′-triphosphate, N1-methylguanosine 5′-triphosphate, N6-methyladenosine 5′-triphosphate, O6-methylguanosine 5′-triphosphate, pseudouridine 5′-triphosphate, puromycin 5′-triphosphate or xanthosine 5′-triphosphate. 
     
     
         14 . An RNA according to  claim 13 , wherein the base-modified nucleotides are chosen from the group consisting of 5-methylcytidine 5′-triphosphate and pseudouridine 5′-triphosphate. 
     
     
         15 . An RNA according to one of the preceding claims, wherein the RNA additionally has a 5′ cap structure chosen from the group consisting of m7G(5′)ppp (5′(A,G(5′)ppp(5′)A and G(5′)ppp(5′)G. 
     
     
         16 . An RNA according to one of the preceding claims, wherein the RNA additionally has a poly-A tail of from about 10 to 200 adenosine nucleotides. 
     
     
         17 . An RNA according to one of the preceding claims, wherein the RNA additionally has a poly-C tail of from about 10 to 200 cytosine nucleotides. 
     
     
         18 . An RNA according to one of the preceding claims, wherein the RNA additionally codes a tag for purification chosen from the group consisting of a hexahistidine tag (HIS tag, polyhistidine tag), a streptavidin tag (Strep tag), an SBP tag (streptavidin-binding tag) or a GST (glutathione S-transferase) tag, or codes for a tag for purification via an antibody epitope chosen from the group consisting of antibody-binding tags, a Myc tag, a Swal 1 epitope, a FLAG tag or an HA tag. 
     
     
         19 . An RNA according to one of the preceding claims, wherein the RNA additionally codes a signal peptide and/or a localization sequence, in particular a secretion sequence. 
     
     
         20 . An RNA according to  claim 19 , wherein the localization sequence is chosen from one of the sequences according to SEQ ID NO: 18 to 50. 
     
     
         21 . An RNA according to one of the preceding claims, wherein the RNA contains an antibody-coding sequence which codes for the heavy chains according to SEQ ID NO: 2 and the light chains according to SEQ ID NO: 4. 
     
     
         22 . An RNA according to one of the preceding  claims 1  to  20 , wherein the RNA contains an antibody-coding sequence according to SEQ ID NO: 5. 
     
     
         23 . An RNA according to one of the preceding  claims 1  to  20 , wherein the RNA contains an antibody-coding sequence which codes for the heavy chains according to SEQ ID NO: 7 and the light chains according to SEQ ID NO: 9. 
     
     
         24 . An RNA according to one of the preceding  claims 1  to  20 , wherein the RNA contains an antibody-coding sequence according to SEQ ID NO: 10. 
     
     
         25 . An RNA according to one of the preceding  claims 1  to  20 , wherein the modified RNA contains an antibody-coding sequence which codes for the heavy chains according to SEQ ID NO: 12 and the light chains according to SEQ ID NO: 14. 
     
     
         26 . An RNA according to one of the preceding  claims 1  to  20 , wherein the RNA contains an antibody-coding sequence according to SEQ ID NO: 15. 
     
     
         27 . An RNA according to one of the preceding  claims 1  to  20 , wherein the modified RNA contains an antibody-coding sequence which has a sequence identity of at least 70% to the sequence SEQ ID NO: 5, 10 or 15 over the total length of the nucleic acid sequence of SEQ ID NO: 5, 10 or 15. 
     
     
         28 . A pharmaceutical composition comprising an RNA according to one of  claims 1  to  27 . 
     
     
         29 . Use of an RNA sequence as defined in  claims 1  to  27  for the preparation of a pharmaceutical composition for treatment of cancer diseases, cardiovascular diseases, infectious diseases or autoimmune diseases. 
     
     
         30 . Use according to  claim 29 , wherein the pharmaceutical composition is in the form of a passive vaccine for treatment of tumour or infectious diseases. 
     
     
         31 . Use according to one of  claim 29  or  30 , wherein the cancer diseases or tumour diseases are chosen from the group consisting of melanomas, malignant melanomas, colon carcinomas, lymphomas, sarcomas, blastomas, kidney carcinomas, gastrointestinal tumours, gliomas, prostate tumours, bladder cancer, rectal tumours, stomach cancer, oesophageal cancer, pancreatic cancer, liver cancer, mammary carcinomas (=breast cancer), uterine cancer, cervical cancer, acute myeloid leukaemia (AML), acute lymphoid leukaemia (ALL), chronic myeloid leukaemia (CML), chronic lymphocytic leukaemia (CLL), hepatomas, diverse virus-induced tumours, such as e.g. papilloma virus-induced carcinomas (e.g. cervix carcinoma=cervical cancer), adenocarcinomas, herpes virus-induced tumours (e.g. Burkitt's lymphoma, EBV-induced B cell lymphoma), hepatitis B-induced tumours (hepatocell carcinomas), HTLV-I- and HTLV-2-induced lymphomas, acusticus neurinoma, lung carcinomas (=lung cancer=bronchial carcinoma), small cell lung carcinomas, throat cancer, anal carcinoma, glioblastoma, rectum carcinoma, astrocytoma, brain tumours, retinoblastoma, basalioma, brain metastases, medulloblastomas, vaginal cancer, testicular cancer, thyroid carcinoma, Hodgkin's syndrome, meningeomas, Schneeberger's disease, pituitary tumour, mycosis fungoides, carcinoids, neurinoma, spinalioma, Burkitt's lymphoma, laryngeal cancer, kidney cancer, thymoma, corpus carcinoma, bone cancer, non-Hodgkin's lymphomas, urethral cancer, CUP syndrome, head/neck tumours, oligodendroglioma, vulval cancer, intestinal cancer, colon carcinoma, oesophageal carcinoma (=oesophageal cancer), wart conditions, small intestine tumours, cra-niopharyngeomas, ovarian carcinoma, soft tissue tumours (sarcomas), ovarian cancer (=ovarian carcinoma), pancreatic carcinoma (=pancreatic cancer), endometrium carcinoma, liver metastases, penis cancer, tongue cancer, gallbladder cancer, leukaemia, plasmocytoma, lid tumour and prostate cancer (=prostate tumours). 
     
     
         32 . Use according to one of  claims 29  to  30 , wherein the infectious diseases are chosen from the group consisting of influenza, malaria, SARS, yellow fever, AIDS, Lyme borreliosis, leishmaniasis, anthrax, meningitis, viral infectious diseases, such as AIDS, condyloma acuminata, molluscum contagiosum, dengue fever, three-day fever, Ebola virus, colds, early summer meningoencephalitis (ESME), influenza, shingles, hepatitis, herpes simplex type I, herpes simplex type II, herpes zoster, influenza, Japanese encephalitis, Lassa fever, Marburg virus, measles, foot and mouth disease, mononucleosis, mumps, Norwalk virus infection, Pfeiffer's glandular fever, smallpox, polio (poliomyelitis), pseuodcroup, infectious erythema, rabies, warts, West Nile fever, chicken-pox, cytomegalovirus (CMV), bacterial infectious diseases, such as abortion (infectious, septic), prostatitis (prostate inflammation), anthrax, appendicitis (inflammation of the caecum), borreliosis, botulism,  Campylobacter, Chlamydia trachomatis  (inflammation of the urethra, conjunctiva), cholera, diphtheria, donavonosis, epiglottitis, louse-borne typhus, typhoid fever, gas gangrene, gonorrhoea, hare plague,  Helicobacter pylori , whooping-cough, climatic bubo, osteomyelitis, legionnaires' disease, leprosy, listeriosis, pneumonia, meningitis, bacterial meningitis, anthrax, inflammation of the middle ear,  Mycoplasma hominis , neonatal sepsis (chorioamnionitis), noma, paratyphoid fever, plague, Reiter's syndrome, Rocky Mountain spotted fever,  Salmonella  paratyphoid fever,  Salmonella  typhoid fever, scarlet fever, syphilis, tetanus, gonorrhoea, tsutsugamushi fever, tuberculosis, typhus, vaginitis (colpitis), soft chancre, and infectious diseases caused by parasites, protozoa or fungi, such as amoebic dysentery, bilharziosis, Chagas' disease,  Echinococcus , fish tapeworm, ichthyotoxism (ciguatera), fox tapeworm, mycosis pedis, dog tapeworm, candiosis, ptyriasis, the itch (scabies), cutaneous leishmaniasis, lamblian dysentery (giadiasis), lice, malaria, onchocercosis (river blindness), fungal diseases, beef tapeworm, schistosomiasis, sleeping sickness, pork tapeworm, toxoplasmosis, trichomoniasis, trypanosomiasis (sleeping sickness), visceral leishmaniasis, nappy dermatitis, or infections caused by the dwarf tapeworm. 
     
     
         33 . Use according to one of  claim 29  or  30 , wherein the cardiovascular diseases are chosen from the group consisting of coronary heart disease, arteriosclerosis, apoplexy and hypertension, and neuronal diseases chosen from Alzheimer's disease, amyotrophic lateral sclerosis, dystonia, epilepsy, multiple sclerosis and Parkinson's disease. 
     
     
         34 . Use according to one of  claim 29  or  30 , wherein the autoimmune diseases are chosen from the group consisting of autoimmune type I diseases or autoimmune type II diseases or auto-immune type III diseases or autoimmune type IV diseases, such as, for example, multiple sclerosis (MS), rheumatoid arthritis, diabetes, diabetes type I (diabetes mellitus), systemic lupus erythematosus (SLE), chronic polyarthritis, Basedow's disease, autoimmune forms of chronic hepatitis, colitis ulcerosa, allergy type I diseases, allergy type II diseases, allergy type III diseases, allergy type IV diseases, fibromyalgia, hair loss, Bechterew's disease, Crohn's disease, myasthenia gravis, neurodermatitis, polymyalgia rheumatica, progressive systemic sclerosis (PSS), psoriasis, Reiter's syndrome, rheumatic arthritis, psoriasis or vasculitis. 
     
     
         35 . An in vitro transcription method for the preparation of an antibody-coding, optionally modified RNA, comprising the following steps: a) provision of a nucleic acid which codes for an antibody, as defined in  claims 4  to  6 ; b) addition of the nucleic acid to an in vitro transcription medium comprising an RNA polymerase, a suitable buffer, a nucleic acid mix comprising one or more modified nucleotides in exchange for one or more of the naturally occurring nucleotides A, G, C or U, and optionally one or more naturally occurring nucleotides A, G, C or U, if not all the naturally occurring nucleotides A, G, C or U are to be exchanged, or optionally only naturally occurring nucleotides and optionally an RNase inhibitor; c) incubation of the nucleic acid in the in vitro transcription medium and in vitro transcription of the nucleic acid to give an antibody-coding, optionally modified RNA according to  claims 1  to  27 ; d) optionally purification of the antibody-coding, optionally modified RNA and removal of the non-incorporated nucleotides from the in vitro transcription medium. 
     
     
         36 . An in vitro transcription and translation method for expression of an antibody, comprising the following steps: a) provision of a nucleic acid which codes for an antibody, as defined in  claims 4  to  6 ; b) addition of the nucleic acid to an in vitro transcription medium comprising an RNA polymerase, a suitable buffer, a nucleic acid mix comprising one or more modified nucleotides in exchange for one or more of the naturally occurring nucleotides A, G, C or U, and optionally one or more naturally occurring nucleotides A, G, C or U, if not all the naturally occurring nucleotides A, G, C or U are to be exchanged, or optionally only naturally occurring nucleotides and optionally an RNase inhibitor; c) incubation of the nucleic acid in the in vitro transcription medium and in vitro transcription of the nucleic acid to give an antibody-coding, optionally modified RNA according to  claims 1  to  27 ; d) optionally purification of the antibody-coding, optionally modified RNA and removal of the non-incorporated nucleotides from the in vitro transcription medium, e) addition of the optionally modified RNA obtained in step c) (and optionally in step d) to an in vitro translation medium; f) incubation of the optionally modified RNA in the in vitro translation medium and in vitro translation of the antibody coded by the optionally modified RNA; g) optionally purification of the antibody translated in step f). 
     
     
         37 . An in vitro transcription and translation method for expression of an antibody in a host cell, comprising the following steps: a) provision of a nucleic acid which codes for an antibody, as defined in  claims 4  to  6 ; b) addition of the nucleic acid to an in vitro transcription medium comprising an RNA polymerase, a suitable buffer, one or more modified nucleotides in exchange for one or more of the naturally occurring nucleotides A, G, C or U and optionally one or more naturally occurring nucleotides A, G, C or U, if not all the naturally occurring nucleotides A, G, C or U are to be exchanged, or only naturally occurring nucleotides and optionally an RNase inhibitor; c) incubation of the nucleic acid in the in vitro transcription medium and in vitro transcription of the nucleic acid to give an antibody-coding, optionally modified RNA according to  claims 1  to  27 ; d) optionally purification of the antibody-coding, optionally modified RNA according to the invention and removal of the non-incorporated nucleotides from the in vitro transcription medium, e′) transfection of the optionally modified RNA obtained in step c) (and optionally d)) into a host cell; f) incubation of the optionally modified nucleic acid in the host cell and translation of the antibody coded by the optionally modified RNA in the host cell; g′) optionally isolation and/or purification of the antibody translated in step f).